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British Journal of Nutrition (1998), 80, Suppl. 1, S1
This concerted action �Functional Food Science in Europe’
(FUFOSE) has been funded within the FAIR RTD
programme, which is part of the Commission’s Fourth
Framework Programme for research and technological
This programme aims at promoting trans-European
research in the primary production sectors of agriculture,
horticulture, forestry, fisheries, and aquaculture, linking
these with the input and processing industries, particularly
food processing and renewable biomaterials.
The food area is important within this programme and is
covered by the theme �Generic Science and Advanced
Technologies for Nutritious Foods’.
There is growing interest in Europe in the concept of
�Functional Foods’ and this concerted action, bringing
together Europe’s scientists and industry, is fundamental
to establishing a science-based approach to such foods.
Liam Breslin
European Commission
DG XII - FAIR Programme
British Journal of Nutrition (1998), 80, Suppl. 1, S3–S4
We stand today at the threshold of a new frontier in
nutritional sciences. The concepts of food are changing
from a past emphasis on survival, hunger satisfaction,
absence of adverse effect on health, and health maintenance
to an emphasis on the promising use of foods to promote
better health and well-being, thus helping to reduce the risk
of chronic illnesses such as cardiovascular disease, some
cancers and obesity.
These new concepts are of particular importance in view
of the benefits for health, consumer demand, the demand of
the elderly population for an improved quality of their late
life, the continuous increase in life expectancy, the increasing cost of health care, technical advances in the food
industry, and the changing regulatory environment.
There is already a recognition that European research
expertise must be at the forefront in understanding the role
of food in the maintenance and improvement of human
health and well-being, in the reduction of risk of major
diseases and in improving the competitive position of the
European food industry. The number of major research
programmes designed to investigate and clarify the healthpromoting value of foods and food components is forecast to
continue to grow, particularly where serious debilitating
diseases are concerned, e.g. heart disease, cancers and
The most recent knowledge in biochemistry, cell biology
and physiology, but also in pathology, supports the hypothesis
that diet also controls and modulates various functions in
the body, and, in doing so, participates in the maintenance of
the state of good health necessary to reduce the risk of some of
the diseases. It is such an hypothesis which is at the origin
both of the concept of �functional food’ and the development
of a new scientific discipline �functional food science’.
Functional food science aims to (1) identify beneficial
interactions between the presence or absence of a food
component (whether a macronutrient, micronutrient or socalled non-nutrient) and a specific function or functions in
the body, and (2) understand their mechanisms, so as to
support hypotheses to be tested in protocols relevant for
human studies. The demonstration, in human subjects, of a
specific interaction with one or a limited number of functions in the body will support a specific, often well-defined,
claim of functional effects or disease risk reduction. Functional food science is indeed part of nutrition science, where
the objectives are to maintain health and improve wellbeing and to create the conditions for disease risk reduction,
and it is, in this respect, quite distinct from the medical or
pharmaceutical sciences, where the objectives are mainly to
cure diseases.
A food is said to be �functional’ if it contains �a food
component (whether a nutrient or not) which affects one or
more targeted functions in the body in a positive way’. It can
also include foods in which a potentially harmful component
has (or components have) been removed by technological
European Commission objectives
An important objective is to improve the understanding of
the role of food in the general health and well-being of the
European consumer. Food can play a major role in maintaining and improving human health and well-being and in
reducing the risk of major diseases. This will also lead to the
design of special or tailored foodstuffs and ingredients for
specific population groups or for specific health benefits.
This will be an expanding area for the food industry in the
future, and European industry, building on the considerable
European research expertise, must be at the forefront here.
This will involve multidisciplinary research projects combining the expertise of scientific partners, such as biochemists, nutritionists, the medical professionals and process
The food and drink industry ranks as a major European
industry processing raw materials from agriculture, horticulture, fisheries and aqua-culture into the diverse range of
quality foodstuffs which are produced throughout Europe.
Research in this sector has the major objective to improve
the competitive position of the food industry which is
composed of leading multinationals and a wide range of
small and medium-sized enterprises specializing in food
throughout Europe.
ILSI Europe’s role
In response to these critical developments, ILSI Europe has
elaborated a project proposal for a European Commission
Concerted Action aimed at establishing a science-based
approach for concepts in functional food science. The goal
of this concerted action is to establish a multidisciplinary
European network to (1) critically assess the science base
required to provide evidence that specific nutrients positively affect functions, (2) examine the available science
from a function-driven point of view rather than a nutrientdriven one, and (3) reach consensus on targeted modifications of food and food constituents, and options for their
application. This approach aims to provide key actors from
Europe’s food and agricultural industry, governmental and
inter-governmental bodies and the scientific community
with an opportunity to exchange ideas and interact on a
neutral platform.
The project
The Functional Food Science in Europe (FUFOSE) project
was submitted in March 1995, approved in November
1995 and was expected to attain its objectives over a period
of 3 years. Project management and coordination was
especially provided by ILSI Europe. Overall guidance on
scientific and organizational issues was ensured through a
steering committee, comprising members from both industry
and academia.
To attain the project objectives, the steering committee
established individual theme groups (ITG) and organized a
series of plenary meetings.
The project started with a first plenary meeting, Functional
Food Science in Europe: State of the Art, held 2–4 April 1996
in Nice, France. Based on the results of this meeting, six areas
in human physiology were identified to be reviewed by the
ITG responsible for producing theme papers to critically
review the science base of the concept. The final composition
of the ITG included industry and non-industry scientists. A
draft brief was prepared by the steering committee to be
addressed by each ITG while reviewing the literature data:
(1) characterize specific body systems, including state-ofthe-art;
(2) critically assess methodologies to characterize and
quantify specific related functions;
(3) identify and critically assess nutritional options modulating these functions;
(4) evaluate potential safety implications related to these
nutritional options;
(5) identify the role of food technology in nutritional and
safety aspects;
(6) critically assess the science base required for providing
evidence that specific nutrients positively affect functions;
(7) identify areas where further research is required.
The resulting documents were scrutinized in a Second
Plenary Meeting held in July 1997 in Helsinki, Finland, and
revised by the ITG chairs to include the comments made.
The final reports of the six ITG are published in this issue of
the British Journal of Nutrition.
The papers need to be considered in the context of the
entire project. They are not individual contributions and
they form, all together, the reference to the FUFOSE
project. Some repetitions, overlaps and contradictions may
still appear. Only by reviewing all six papers will the reader
have a balanced overview of both primary and secondary
effects of functional foods.
(5) Research needs.
(6) Communication of the health benefits of functional
(7) Conclusions.
The expert group that undertook the elaboration of the
text was composed of two ITG chairs and four members of
the steering committee. The document is currently under
review. The goal is to publish this consensus document also
in the British Journal of Nutrition.
Food technology
A group of experts on food technology have also been
selected to examine the impact and feasibility of food
technology on functional food development. Relevant viewpoints are: safety, nutrition and consumer acceptance/
sensory quality. This expert group has identified areas to
concentrate on and terms of reference to be followed. Once
the report is completed, it will be reviewed by the participants in the third plenary meeting. As soon as the comments
are all taken into consideration, the paper will be published
in a scientific journal.
We wish to thank, especially, all of the individual contributors to this FUFOSE project for devoting their time and
efforts within such a tight timeframe. Their commitment
and dedication will be remembered as exceptional and
highly enthusiastic. Authors and contributors can be assured
of ILSI Europe’s recognition and they will be paid tribute,
as often as possible. Through their collaborative work they
have participated in the making of ILSI Europe’s history
and the Institute is extremely grateful to all.
Dr Berry Danse,
ILSI Europe,
83 Avenue E. Mounier, Box 6,
B-1200 Brussels, Belgium.
Scientific coordinator:
Prof. Marcel Roberfroid,
Catholic University of Louvain,
Ecole de Pharmacie,
Tour Van Helmont,
73 Avenue E. Mounier,
B-1200 Brussels, Belgium.
EC responsible:
Dr Liam Breslin,
Agro-industrial Research, Food,
Commission of the European
Directorate-General XII, Science,
Research and Development,
200 Rue de la Loi,
B-1049 Brussels, Belgium.
Project manager;
Dr Laura Contor,
ILSI Europe,
83 Avenue E. Mounier, Box 6,
B-1200 Brussels, Belgium.
Consensus document
These ITG papers provided the building blocks for a
more general consensus document Concepts in Functional
Food Science and Options for their Application. The outline
of this consensus document was prepared based on the
recommendations of the ITG and the steering committee
members. This outline was also reviewed by the participants
in the second plenary meeting who provided comments to be
taken into consideration. The topics that will be addressed in
the consensus document include the following.
(1) Introduction.
(2) Scientific basis for functional food science.
(3) Target functions in relation to health outcome.
(4) Food technology.
б­§ Nutrition Society 1998
British Journal of Nutrition (1998), 80, Suppl. 1, S5–S45
Growth, development and differentiation: a functional food science approach
B. Koletzko1 *, P. J. Aggett2 , J. G. Bindels3 , P. Bung4 , P. FerreВґ5 , A. Gil6 , M. J. Lentze7 , M. Roberfroid8
and S. Strobel9
Kinderpoliklinik, Klinikum Innenstadt der Ludwig-Maximilians-UniversitaВЁt, Pettenkoferstr. 8a, D-80336 MuВЁnchen, Germany
Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK
Nutricia Research, Verenigde Bedrijven Nutricia NV, PO Box 1, NL-2700 MA Zoetermeer, The Netherlands
University of Bonn, Women’s Hospital, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
INSERM, Unite´ 465, Centre Biome´dical des Cordeliers, 15, rue de l’Ecole de Me´decine, F-75270 Paris, France
University of Granada, School of Pharmacy, Department of Biochemistry and Molecular Biology, Campus of Cartuja,
E-18071 Granada, Spain
University of Bonn, Children’s Hospital, Adenauerallee 119, D-53113 Bonn, Germany
UCL, Ecole de Pharmacie, Tour Van Helmont, Avenue E. Mounier, B-1200 Brussels, Belgium
Clinical Sub-Dean’s Office, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
1. Introduction
2. Nutrient–gene interaction, genetic regulation
2.1. Introduction
2.2. Modulation of gene expression participates in the
adaptations of energy metabolism
2.3. Examples of gene regulation by nutrients
2.3.1. Carbohydrates
2.3.2. Fatty acids
2.3.3. Cholesterol
2.3.4. Amino acids
2.4. Nutrients and cell differentiation
2.4.1. Fatty acids
2.4.2. Retinoic acid
2.5. Concluding remarks
3. An overview of programmed cell death (apoptosis)
4. Supply of food ingredients before and during
4.1. Physiological aspects of nutritional requirements
in pregnancy
4.1.1. Energy
4.1.2. Protein
4.1.3. Carbohydrates
4.1.4. Lipids
4.1.5. Vitamins, minerals and trace elements
5. Modulation of growth
5.1. Introduction
5.2. Methods for the determination of growth
5.3. Growth factors in human milk and their influence
on infant growth
5.4. Potential roles of non-protein nitrogen compounds
as growth modulators
Human milk oligosaccharides and growth
Free amino acids and tissue growth
Polyamines and tissue growth
Dietary nucleotides and tissue growth
5.8.1. Nucleotides and small intestine growth
5.8.2. Nucleotides and liver growth
5.9. Long-chain polyunsaturated fatty acids and cell
5.10. Early growth and later obesity
6. Maturation of the gastrointestinal tract
6.1. Introduction
6.2. Development of sugar hydrolases and transporters
6.3. Biosynthesis of intestinal brush–border membrane
6.4. Intestinal absorption of glucose and fructose
6.5. Oligosaccharides and mucins
6.6 Probiotic substances in milk or milk substitutes
6.7. Dietary regulation of xenobiotic metabolism
7. Development of the immune system
7.1. Introduction
7.1.1. Which constituents of the immune system to
7.1.2. Special considerations for the immune
system of the developing child
7.2. Antioxidants and vitamins
7.2.1. In general
7.2.2. Vitamin A
7.2.3. Vitamin C
7.2.4. Vitamin B complex
7.2.5. Vitamin E
7.2.6. Vitamin D
Abbreviations: APRT, adenosine phosphoribosyltransferase; cDNA, complementary DNA; DEXA dual-energy X-ray absorptiometry; DHA,
docosahexaenoic acid; EFA, essential fatty acids; EGF, epidermal growth factor; GLUT, glucose transporter; hGH, human growth hormone; HGPRT,
hypoxanthine phosphoribosyltransferase; HPA, hyperphenylalaninaemia; IDDM, insulin-dependent diabetes mellitus; Ig, immunoglobulin; IGF, insulinlike growth factor; LPH, lactase–phlorizin-hydrolase; MUC1, high-molecular mass glycoprotein; NPN, non-protein nitrogen; PAH, phenylalanine
hydroxylase; PCD, programmed cell death; PKU, phenylketonuria; PPAR, peroxisome proliferator activated receptors; PUFA, polyunsaturated fatty acids;
SGLT1, sodium-dependent glucose transporter 1; SI, sucrase–isomaltase; SPA, single-photon absorptiometry; SREBP, sterol regulatory element binding
protein; XME, xenobiotic-metabolizing enzymes.
*Corresponding author: Professor B. Koletzko, fax +49 89 5160 3336, email: [email protected]
B. Koletzko et al.
Multiple micronutrient supplementation studies
Fatty acids
Maturation of the immune system in formula-fed v.
breast-fed infants
7.7.1. Effects of antigen transfer via breast milk
on the infant’s immunity
7.7.2. Maternal diet during pregnancy and effects
on the infant’s immunity
7.7.3. Maternal diet during pregnancy and
7.8. Role of the gut flora and probiotic bacteria in the
infant’s immunity and gut defence
7.8.1. Immune exclusion and elimination
7.9. Effects of formulas with protein hydrolysates on
the infant’s immune responses
7.10. Insulin-dependent type 1 diabetes mellitus and
cow’s milk exposure in infancy
8. Bone growth and mineralization
8.1. Cell biology of bone growth
8.2. Methodological aspects in bone-mass-related studies
8.3. Peak bone mass and relative risk of osteoporosis
Bone growth and mineralization in infants and
young children
8.5. Calcium supplementation in children and
adolescents and bone health
8.6. Nutrients other than calcium and environmental
factors involved in bone growth
9. Nutrient effects on development of neural functions
and behaviour
9.1. Introduction
9.2. Physiology of neural development
9.3. Nutrition and neural development
9.3.1. Protein
9.3.2. Iodine
9.3.3. Iron
9.3.4. Zinc
9.3.5. Polyunsaturated fatty acids
9.4. Early nutrition and development of taste
9.5. Methodological aspects
10. Production of bioactive factors for inclusion into
food products
11. Commentary on biomarkers
12. Conclusions
Few other aspects of food supply and metabolism are of greater biological importance than the
feeding of mothers during pregnancy and lactation, and of their infants and young children.
Nutritional factors during early development not only have short-term effects on growth, body
composition and body functions but also exert long-term effects on health, disease and mortality
risks in adulthood, as well as development of neural functions and behaviour, a phenomenon
called �metabolic programming’. The interaction of nutrients and gene expression may form the
basis of many of these programming effects and needs to be investigated in more detail. The
relation between availability of food ingredients and cell and tissue differentiation and its
possible uses for promoting health and development requires further exploration. The course of
pregnancy, childbirth and lactation as well as human milk composition and the short- and longterm outcome of the child are influenced by the intake of foods and particularly micronutrients,
e.g. polyunsaturated fatty acids, Fe, Zn and I. Folic acid supplementation from before conception
through the first weeks of pregnancy can markedly reduce the occurrence of severe embryonic
malformations; other potential benefits of modulating nutrient supply on maternal and child
health should be further evaluated. The evaluation of dietary effects on child growth requires
epidemiological and field studies as well as evaluation of specific cell and tissue growth. Novel
substrates, growth factors and conditionally essential nutrients (e.g. growth factors, amino acids,
polyunsaturated fatty acids) may be potentially useful as ingredients in functional foods and need
to be assessed carefully. Intestinal growth, maturation, and adaptation as well as long-term
function may be influenced by food ingredients such as oligosaccharides, gangliosides, highmolecular-mass glycoproteins, bile salt-activated lipase, pre- and probiotics. There are indications for some beneficial effects of functional foods on the developing immune response, for
example induced by antioxidant vitamins, trace elements, fatty acids, arginine, nucleotides, and
altered antigen contents in infant foods. Peak bone mass at the end of adolescence can be
increased by dietary means, which is expected to be of long-term importance for the prevention of
osteoporosis at older ages. Future studies should be directed to the combined effects of Ca and
other constituents of growing bone, such as P, Mg and Zn, as well as vitamins D and K, and the
trace elements F and B. Pregnancy and the first postnatal months are critical time periods for the
growth and development of the human nervous system, processes for which adequate substrate
supplies are essential. Early diet seems to have long-term effects on sensory and cognitive
abilities as well as behaviour. The potential beneficial effects of a balanced supply of nutrients
such as I, Fe, Zn and polyunsaturated fatty acids should be further evaluated. Possible long-term
effects of early exposure to tastes and flavours on later food choice preferences may have a major
impact on public health and need to be further elucidated. The use of biotechnology and
recombinant techniques may offer the opportunity to include various bioactive substances in
special dietary products, such as human milk proteins, peptides, growth factors, which may have
beneficial physiological effects, particularly in infancy and early childhood.
Growth: Development: Differentiation
Growth, development and differentiation
1. Introduction
There are few other aspects of food supply and the metabolism of food ingredients that are of greater biological
importance than the feeding of mothers during pregnancy
and lactation, and of their children. The rapid growth of
fetuses, infants and children, which double their body
weights within only 6 weeks in utero and 4–5 months
after birth respectively, depends on the supply of very
large amounts of nutrients per kg body weight through the
placenta, human milk and children’s diets. Marginal nutrient supplies are usually more critical in a developing and
growing organism than in steady-state situations during
adulthood. The ability to effectively utilize and compensate
for unbalanced supplies is severely limited in the fetus and
in young children due to small endogenous stores of a
number of relevant substrates, and in many cases also due
to immature metabolic pathways (e.g. amino acid metabolism) and physiological functions (e.g. renal conservation of
substrates). Nutritional factors during early development
have important immediate and short-term effects on growth,
body composition and body functions. In addition, accumulating data indicate long-term effects of nutritional and
metabolic factors during critical time periods of development on later physiological and metabolic processes, a
phenomenon referred to as �metabolic programming’
(Barker, 1994). For example, epidemiological studies have
suggested long-term effects of intrauterine and postnatal
nutrient supply on the prevalence of obesity in adulthood
(Ravelli et al. 1976) and on the risk of developing diabetes,
hypertension, hypercholesterolaemia, CHD, and other disorders during adult life (Barker, 1994). The rate of death
from all cardiovascular disease and from CHD (Fig. 1) in
adulthood was found to be significantly related to body
weight at birth (Osmond et al. 1993; Barker, 1994). Standardized mortality from CHD was also closely related to
weight at 1 year in males but not in females (Fig. 2). These
findings indicate the potential of influencing long-term
health and life expectancy by modulations of maternal
diet in pregnancy and of postnatal infant feeding. Moreover,
the type of postnatal infant feeding has been related to longterm outcomes such as the later incidence of insulin-dependent diabetes mellitus (IDDM) (Virtanen et al. 1991;
Gerstein, 1994) and cognitive development (Lucas et al.
1992; Lanting et al. 1994), and a lasting effect of Ca intake
during childhood and adolescence on bone mineral density
and the risk of fractures in old age has been proposed (Ribot
et al. 1995). This programming of permanent effects of the
physiology and function of the organism during critical time
periods of early development appears to be of major
importance in preventive health strategies. Improved
knowledge on the cellular and molecular mechanisms of
programming and of the complex physiological and
nutritional factors relevant to the health and well-being
during the periods of reproduction and childhood is
required. A better definition of the optimal supply of
relevant nutrients and other food ingredients is expected
to help optimize dietary intakes during these critical periods
of life which should improve the chances that infants and
children have to utilize fully their genetic potential, as well
as maintain maternal health.
2. Nutrient–gene interaction, genetic regulation
2.1. Introduction
The perinatal period is attended by important modifications
in energy metabolism (Girard et al. 1992). In utero, the fetus
receives a continuous intravenous supply of substrates for
growth and oxidative metabolism. Immediately after birth,
the maternal supply of substrates ceases abruptly and the
newborn has to withstand a brief period of starvation before
being fed at intervals with milk, a high-fat and low-carbohydrate diet. The sucking–weaning transition is also characterized by profound changes of nutrition (Girard et al.
1992). Towards the end of the sucking period, the milk is
progressively replaced by the solid food diet of the adult, the
composition of which is usually lower in fat and higher in
carbohydrates. The successful adaptation of neonates to
these changes in nutrition requires important modifications
Fig. 1. Standardized mortality ratios for CHD below age 65 years, showing a statistically significant relationship with birth weight in 5585 women
and 10 141 men born between 1911 and 1930. (Data from Barker, 1994.)
B. Koletzko et al.
Fig. 2. Standardized mortality ratios for CHD below age 65 years, showing a statistically significant relationship with weight at age 1 year in 10 141
men, but not in 5585 women born between 1911 and 1930. (Data from Barker, 1994.)
of energy metabolism in the vast majority of organs,
intestine, liver, muscle, adipose tissue and brain. Postnatal
development is also associated with differentiation processes and a high growth rate, involving specific requirements of nutrients such as amino acids (protein synthesis) or
fatty acids (e.g. brain growth). Thus, nutrients can be
considered to cause energy metabolism modifications but
also to play a major role in organ growth and/or functional
2.2. Modulation of gene expression participates in the
adaptations of energy metabolism
The adaptations of energy metabolism to the nutritional
environment imply the modulation and/or emergence of
metabolic pathways. This can be achieved through changes
in the efficiency of a given step (specific transporters,
enzymes) by allosteric or phosphorylation–dephosphorylation mechanisms, or by translocation of a protein into a
different cellular compartment. However, many of these
adaptations also imply a change in the amount of a given
protein. This phenomenon is usually related to a change of
the transcription rate of the corresponding gene.
A good example of such a mechanism is the appearance at
birth in mammals of phosphoenolpyruvate carboxykinase
(EC, an enzyme of the gluconeogenic pathway
allowing the de novo production of glucose by the liver
(Girard et al. 1992). This pathway is essential for the
survival of the newborn mammal, which undergoes a brief
period of starvation followed by the ingestion of a diet low
in carbohydrates, the milk. The transcription rate of this
gene is extremely low during the fetal period and increases
abruptly in the first hours after birth, allowing the newborn
to maintain glucose homeostasis.
Another example is the lipogenic pathway in the rat
species. This pathway allows synthesis of fatty acids from
glucose when this substrate is ingested in excess of energy
requirements. During the sucking period, the capacity of
this pathway is kept very low because the small quantity
of glucose ingested is essentially directed towards
oxidative processes. When the rat is weaned onto the
high-carbohydrate diet, this pathway is switched on and
excess glucose will be converted into fatty acids, ultimately
stored as triacylglycerols in the adipose tissue. The expression of one of the key enzymes of this pathway, fatty acid
synthase (EC is extremely low during the sucking
period but increases when the rat is weaned onto the adult
high-carbohydrate diet, but not if weaning occurs onto a
high-fat diet, clearly underlining the importance of the
nutritional environment in this process. This phenomenon
is due to the activation of the gene transcription process at
weaning (Foufelle et al. 1996). Thus, modulation of gene
expression must be considered as an integral part of the
adaptations occurring during development.
Although it has been known for a long time that nutrients
can regulate the expression of specific genes in prokaryotes
(the lactose operon in Escherichia coli for instance) or in
primitive eukaryotes such as yeasts, the demonstration that a
similar phenomenon occurs in higher eukaryotes is recent.
The regulation of specific gene expression in mammals in
response to changes in nutrition has become a major aspect
of modern nutrition, due to the emergence of molecular
biology that has allowed the cloning of most of the genes
involved in the regulation of energy metabolism. Recently,
it has been demonstrated that major (glucose, fatty acids,
amino acids) or minor (Fe, vitamins) dietary constituents
participate, in concert with hormones, in the regulation of
gene expression in response to nutritional changes (see for
instance Clarke & Abraham, 1992).
2.3. Examples of gene regulation by nutrients
2.3.1. Carbohydrates. In the liver and adipose tissue,
excess glucose, after its metabolism into pyruvate through
glycolysis is converted into fatty acids by the lipogenic
pathway. The expression of three enzymes of the combined
glycolytic–lipogenic pathway has been shown to respond to
an increased glucose concentration: L-pyruvate kinase (EC; liver), fatty acid synthase and acetyl-coA carboxylase (EC; liver and adipose tissue) (Foufelle et
al. 1992; Vaulont & Kahn, 1994). In vivo, it has been shown
in rats that high-carbohydrate diets induce the transcription
Growth, development and differentiation
of these genes, whereas transcription is inhibited by starvation
or a high-fat diet. During the sucking period in rats, the
expression of these enzymes is kept low and dramatically
increases at weaning on to a high-carbohydrate diet. In vitro
studies have shown that glucose is the primary inducer of the
gene transcription. This effect requires that glucose is
metabolized at least into glucose-6-phosphate which might
be the signal metabolite. The response of transcription to high
glucose is a very rapid phenomenon (less than 1 h). Glucose
response elements, which bind specific transcription factors of
the USF/MLTF family have been characterized on these
genes although the mechanism linking glucose-6-phosphate to
transcription factors is presently unknown (Foufelle et al.
1996). In the b-cells of the islets of Langerhans, glucose
induces the transcription of the insulin gene on which glucose
response elements have also been characterized (Docherty &
Clark, 1994). This feature is obviously relevant to the diabetic
2.3.2. Fatty acids. In vitro studies have shown that the
transcription of a number of genes of adipocytes is increased
in the presence of fatty acids. This is the case, for
instance, for phosphoenolpyruvate carboxykinase, an
enzyme involved in this tissue in the provision of
a-glycerophosphate necessary for the esterification of
fatty acids (Antras-Ferry et al. 1995). Similarly, the
expression of the fatty acid binding protein ap2, which
binds fatty acids into the cell, is strongly stimulated by fatty
acids (Grimaldi et al. 1992). Fatty acid response elements
have been characterized in the promoter of these genes.
They bind a transcription factor called peroxisome proliferator activated receptors (PPAR) which can be activated by
the binding of fatty acids or a metabolite of fatty acids
(prostaglandin for instance) (Schoonjans et al. 1996).
Fatty acids have also been shown to inhibit gene expression in rats. The addition of a small amount (20–30 g/kg) of
polyunsaturated fatty acids (PUFA) of the n-3 or n-6
families to a high-carbohydrate fat-free diet decreases
markedly the lipogenic capacity and the activity of lipogenic enzymes (Clarke, 1994). In contrast, monounsaturated
and saturated fatty acids have no effects. Interestingly
enough, this effect seems to be specific to the liver since
lipogenesis is not affected in the adipose tissue. The
decrease induced by PUFA of the activity of lipogenic
enzymes such as fatty acid synthase, acetyl-CoA carboxylase or glucose-6-phosphate dehydrogenase (EC,
is clearly linked to an inhibition of gene transcription
as shown in studies using primary cultures of rat hepatocytes (Clarke, 1994). At the present time the cellular and
molecular mechanisms involved in the inhibitory effect of
PUFA on gene transcription have not been elucidated.
2.2.3. Cholesterol. A very interesting series of studies
has been performed by the group of Brown and Goldstein on
the effect of cholesterol on gene expression (Wang et al.
1994). Cholesterol represses the expression of genes
involved either in the synthesis of cholesterol (cytoplasmic
hydroxymethylglutaryl-CoA synthase, EC or in its
uptake from external sources, the LDL receptor (LDL are
lipoproteins rich in cholesterol). In the absence of
cholesterol, the transcription of these genes is activated by
a transcription factor called sterol regulatory element
binding protein (SREBP). SREBP is usually hooked onto
the endoplasmic reticulum where it can be cleaved by a
protease. SREBP can then be transferred into the nucleus
and activates the transcription of relevant genes. In the
presence of cholesterol, the protease is inhibited and SREBP
can no longer enter into the nucleus and stimulate gene
transcription (see Fig. 3).
2.3.4. Amino acids. In yeast, amino acid starvation
results in the activation of several genes involved in N
metabolism and the mechanisms involved are now known
(Kilberg et al. 1994). In mammalian cells, amino acid
availability also modulates the expression of some genes as
shown, for instance, for asparagine synthetase (EC
which is responsible for the biosynthesis of asparagine from
aspartate and glutamine. When cultured cells are transferred
to a medium lacking asparagine, the concentration of
asparagine synthetase mRNA increases. It must be underlined, however, that the signalling mechanism exhibits a
broad substrate specificity since availability of other
amino acids controls the asparagine synthetase mRNA
Fig. 3. Model for the cholesterol-dependent control of gene transcription. SREBP, sterol regulatory element binding protein.
B. Koletzko et al.
concentration as well. The mechanisms for the increase in
asparagine synthetase mRNA concentration could involve
both cis-acting elements contained in the mRNA itself and
affecting its stability, as well as in the genomic promoter
sequence (Kilberg et al. 1994). The signal metabolite could
be, in fact, the degree of occupancy of the transfer RNA as
in yeast, although evidence is clearly lacking.
2.4. Nutrients and cell differentiation
The fetal–neonatal period is also characterized by the
differentiation of a number of organs. One important question, which is presently totally unaddressed, is whether
nutrients could affect differentiation per se and thus
modulate physiological functions on a long-term basis.
Two examples are listed here.
2.4.1. Fatty acids. One example of such a mechanism
stems from in vitro studies on pre-adipocyte cell lines. It has
been shown that fatty acids are not only able to modulate the
transcription rate of specific genes in differentiated
adipocytes, but are also able to induce preadipocyte
differentiation into adipocytes through their action on a
specific isoform of a PPAR transcription factor (Schoonjans
et al. 1996). Obviously, if this happens also in vivo, it would
imply that perinatal nutrition could modulate the number of
adipocytes and, thus, be a crucial determinant of a possible
expansion of this tissue, in relation to the obesity syndrome.
2.4.2. Retinoic acid. Vitamin A or retinol can be
oxidized to retinoic acid in cells. In addition to retinol,
b-carotene may also be a source of retinoic acid. Retinoic
acid and retinoid X receptors which belong to the family of
steroid–thyroid hormone receptors have been cloned (De
Luca, 1991). They are, in fact, ligand-activated transcription
factors (of the same family as the PPAR, see p.S9). It has
been shown in numerous studies that retinoic acid is a potent
morphogen and that it can affect fetal development (De
Luca, 1991). Thus, vitamin A or b-carotene deficiency or
overload might have major consequences on tissue
differentiation and fetal development.
2.5. Concluding remarks
These examples underline the idea that gene regulation by
nutrients, a process we induce each time we eat a food of
specific composition, includes a very wide range of mechanisms involved in the regulation of crucial pathways as well
as in cell differentiation. Since the fetal–neonatal period
is concomitant with marked changes in the nutritional
environment, it represents a period in which these
mechanisms are particularly important.
As for other components of cell functions, it is very likely
that gene regulation by nutrients is subject to variations
linked to genetic polymorphisms among individuals, of
which some could ultimately lead to pathologies. This
obviously opens new fields for genetic studies on nutrientrelated pathologies (obesity, for instance).
3. An overview of programmed cell death (apoptosis)
A type of cell death which does not involve primary cell
membrane disintegration and tissue inflammatory responses
was identified some 70 years ago, and the term apoptosis
was applied by analogy with the deciduous loss of leaves by
trees. The process was identified by characteristic histomorphological changes in the cell nuclei (Kroemer et al.
1995). Although apoptosis is sometimes used as a synonym
for programmed cell death (PCD), it has become apparent
that apoptosis can be caused by noxious or toxic events
which might affect several cell components and not just
primarily the nucleus. PCD applies to the entire process and
phenomenology of this form of cell death and thus embraces
the intrinsic mechanisms and regulatory processes involved.
PCD might not necessarily be induced by external toxic
stimuli, but rather by signals which are a fundamental
component of cellular and tissue physiology. The distinction
between the two terms PCD and apoptosis is subtle, and not
invariably respected. Clearly it is misleading to characterize
PCD on the basis of nuclear morphological changes since
these are not always the primary events in PCD (Kroemer et
al. 1995; Hale et al. 1996; Vaux & Strasser, 1996; Nagata,
1997) but one should not be distracted overmuch by the
distinction: rather interest should focus on the processes
involved in the induction, mediation, regulation and process
of the cellular events, and the ways in which dietary components might affect these mechanisms which are fundamental
to tissue differentiation, development and function.
PCD is a more appropriate description of a variety of
intrinsic cellular events, involving cytoplasmic as well as
nuclear processes which precede loss of intracellular and
cellular membrane integrity, should these occur at all.
However, non-physiological stimuli can effect mechanisms
involved with PCD, and the process or its dysfunction can
be an integral part of oncogenesis and autoimmune disorders (Hale et al. 1996).
All multicellular organisms use PCD to remove superfluous and damaged cells in tissues and organs, particularly,
but not exclusively, in proliferating tissues. There is a strong
evolutionary conservation of the mechanisms of PCD and
the nematode Caenorhabditis elegans is a valuable model
for the system in higher species, including man, in whom
several genes and effectors of PCD are homologous to cell
death genes and products in C. elegans (e.g. proto-oncogene
bcl-2 is homologous to the nematode �cell-suicide gene’
ced3) (Kroemer et al. 1995; Vaux & Strasser, 1996).
The induction mechanisms of PCD are more complex in
higher animals in whom co-operativity between the different tissues of organs plays an important part in determining
PCD. For example, the extracellular matrix exerts some
control over differentiation and morphogenesis of organs by
specific effects on constituent cells, tissue-specific gene
expression, and cell death (Roskelley et al. 1995). This is
important for the differentiation of tissues and organs (e.g.
the gastrointestinal tract) throughout life as well as during
PCD can be envisaged to comprise three stages: induction, the effector stage, and degradation (Fig. 4) (Kroemer et
al. 1995). There are at least two principal routes of inducing
PCD. One involves genotoxic events damaging DNA; the
other involves receptor-mediated stimuli such as specific
death signals, the absence of rescue signals such as growth
factors, and contradictory or conflicting signals (Kroemer et
al. 1995; Nagata, 1997).
Growth, development and differentiation
Fig. 4. The stages of programmed cell death and apoptosis. (From Kroemer et al : 1995.)
The origin of signals can be systemic or local (e.g.
extracellular matrix) (Hakomori & Igarashi, 1995) humoral
cytokines (Nagata, 1997), or cellular such as cytotoxic T
cells. The signal and transduction systems stimulating PCD
include steroids (Evans-Storms & Cidlowski, 1995), cytokines such as tumour necrosis factor and nerve growth factor
and interleukins, all of which operate through their cognate
receptors (Cosman, 1994; Nagata, 1997).
The responsiveness to such stimuli may also be determined by the nature of the cell surface receptors which
might change during ontogenesis (and, for that matter,
oncogenesis). The surface receptors include glycosphingolipids, lectins and sphingosines and their respective roles
have not been totally clarified. It is noteworthy that inherited
defects of cell-surface CD40 cause a human X-linked
immunodeficiency syndrome associated with defective
PCD, and analogous syndromes in mice are associated
with congenital abnormalities of Fas receptors and ligands
(Cosman, 1994; Nagata, 1997). Changes in structural components of cellular membranes (e.g. ceramide formation)
can induce signal transduction cascades (Ballou et al. 1996)
leading to cell death.
The heterogeneity of signals is matched by the variety of
effector mechanisms which are stimulated. These involve
tyrosine kinase, protein kinase C, and other kinases, Ca
permeability routes and intracellular proteases. These initiate an amplification cascade affecting a number of genes,
several of which are known proto-oncogenes (e.g. c-myc) or
tumour suppressors (e.g. p53) and which are common both
to PCD and to the regulation of normal cell differentiation,
function and intermediate metabolism (Kroemer et al. 1995;
Hale et al. 1996; Nagata, 1997). This highlights the central
conundrum of PCD, namely what is the ultimate determinant of whether a cell undergoes PCD or not? Is there a
threshold event? It has been suggested that one basic
determinant is the stage of the cell cycle when the cell
receives the relevant signals (Meikrantz & Schlegel, 1995)
or the availability of an appropriate nutrient supply (that is,
do some stressors such as a specific nutrient excess or
deficiency induce cell death by more direct means than,
say, oxidant damage or simple starvation?).
Intrinsic effector systems for cell death programmes
involve cytoplasmic and nuclear metabolic and functional
disintegration with ultimate morphological damage. Within
the mitochondria there is a decrease in transmembrane
potential, energy uncoupling of the respiratory chain, and
increased production of reactive O species. These increase
oxidative damage and increase leakage of mitochondrial Ca.
In the cytoplasm there is a loss of anabolic activities with a
corresponding activation of proteases, disruption of the
cytoskeleton and of the endoplasmic reticulum: similarly
in the nucleus the endonucleases are activated and there is
nucleolysis with some activation of enzymes usually associated with repair activity.
The mechanisms and regulation of PCD and apoptosis in
abnormal cell proliferation have focused on their role in
cancer and the possibility that external events or compounds
might induce, suppress, regress, or protect against cancer by
affecting these processes. The potential role of dietary
components influencing these processes is being investigated actively in relation to functional food science. In
other physiological areas there might exist other opportunities to explore and exploit a better understanding of these
processes in cellular proliferation and differentiation. These
are outlined in the following paragraphs.
PCD of uterine cells enables blastocyst implantation and
placentation (Welsh, 1993). Prostaglandins, leukotrienes,
platelet-activating factor, and transforming growth factor
are thought to induce this process. In embryogenesis there is
extensive mesodermal PCD to eliminate undifferentiated
B. Koletzko et al.
cells (Sanders & Wride, 1995; Wride & Sanders, 1995). The
extracellular matrix and the regulation of cell adhesion
molecules and integrins, perhaps involving tumour necrosis
factor-a as a cellular growth and differentiation factor, have
been shown in the regulatory processes in the morphogenesis of the limb bud (Hurle et al. 1995).
Again extracellular matrix and specified cell adhesion
molecules control endothelial cell position and behaviour
and enable the definition of mechanisms directing endothelial cell differentiation, commitment, migration and organization into a tube as the fundamental components of
vasculogenesis (Baldwin, 1996). PCD has also been
shown to be integral to the development of the conduction
processes and to be involved in the pathogenesis of
congenital cardiac structural and electrical conduction
abnormalities as well as acquired cardiovascular disease
(James, 1993). The role of PCD in intestinal mucosal
development and differentiation is discussed elsewhere: a
similar dependence on PCD has been demonstrated in renal
organogenesis (Igarashi, 1994).
It is in haematopoiesis and in the development of the
immune system that there might be some particular aspects
of relevance to functional food science. Haematopoiesis and
PCD involve an extensive multigene cytokine network
which has positive regulators such as colony-stimulating
factors and interleukins and negative regulators such as
transforming growth factor-b and tumour necrosis factor
(Krammer et al. 1994; Cidlowski et al. 1996; Sachs, 1996).
Apoptosis in T and B lymphocytes is involved in all
fundamental processes in the immune system and PCD
appears to be vital in selecting or favouring development
of specific lymphocytes and eliminating cells which are
sensitized to autoantigens. Factors which would interfere
with this process would be undesirable and there exists a
possibility that similar selective processes might underlie
the acquisition of immunotolerance to ingested antigens in
foods or the development of adverse immune reactions to
food constituents.
In the central nervous system neurones and glia synthesize
and secrete cytokines which affect the differentiation and
function of nerve cells (Dragunow & Preston, 1995; Kawata,
1995; Sei et al. 1995). Disturbance of these cytokinemediated interactions may lead to neuronal dysfunction
and/or cell death and contribute to the pathogenesis of
central nervous system diseases. The functional and cellular
differentiation underlying sexual dimorphism in the central
nervous system might depend on the effects of steroids on
PCD in the relevant regions of the brain.
Many of the phenomena regarded as inevitable
consequences of ageing, including nerve cell death, involve
processes of PCD and apoptosis, and a better understanding
of these events might in due course lead to a more sophisticated and rational scientific basis on which to base
functional food science and its application to avoid the
depredations of ageing (Zakeri & Lockshin, 1994).
4. Supply of food ingredients before and during
The nutritional status of the mother before and during
pregnancy may influence fertility, the course of pregnancy
and the incidence and severity of complications during
gestation and birth, lactation (Rasmussen 1992), and the
short- and long-term health and development of the baby.
During recent years scientific understanding of the physiology of energy metabolism, weight gain and particularly
the effects of micronutrient status and their clinical consequences have considerably improved. The dietary supply
of certain nutrients may have beneficial or preventive
aspects both for the course of gestation and for the offspring.
4.1. Physiological aspects of nutritional requirements in
The range of weight changes that occurs in pregnant women
is wide and ranges from a loss of weight to a gain of 25 kg or
more. A normal weight gain during pregnancy for most
women with normal prepregnancy weight is in the order of
11–15 kg (Institute of Medicine, 1990). In addition to food
intake, weight gain is also influenced by medical disorders
such as development of oedema associated with preeclampsia. The weight increase and substrate deposition in
maternal and fetal tissues is not only due to increased
dietary intake during pregnancy, but also supported by a
variety of complex adaptations of gastrointestinal, endocrine and metabolic functions. The effects of food ingredients on these adaptations as well as on placental function
and the efficacy of materno-fetal substrate transfer are not
well understood and require further exploration. Substrate
needs during pregnancy are not only met by the food
consumed during this period of time, but food intake
before or between pregnancies may be of major importance
for providing adequate availability of a number of nutrients,
e.g. Ca and Fe. Also, it is increasingly appreciated that
substrate needs during pregnancy may show marked interindividual variability, partly due to genetic heterogeneity in
a population as is the case for metabolic pathways involving
folic acid.
4.1.1. Energy. Based on a factorial approach calculating the additional energy requirements for the deposition of
protein and fat as well as the energetic cost of tissue
synthesis and enhanced maintenance metabolism, the total
energy cost of pregnancy is assumed to be in the order of
334.72 MJ (80 000 kcal) (Food and Agriculture Organization/World Health Organization/United Nations University
(FAO/WHO/UNU), 1985). In women who maintain their
previous physical activity, this results in an additional
energy allowance of about 837 kJ/d (200 kcal/d) equivalent
to an increase of 9 % over the energy needs of 9.2 MJ (2200
kcal) of a non-pregnant, moderately active woman, or if
adapted to an estimated increase of energy needs during the
course of pregnancy, about 628 kJ/d (150 kcal/d) during the
first and about 1464 kJ/d (350 kcal/d) during the second and
third trimesters of pregnancy (FAO/WHO/UNU, 1985).
However, energy needs may be lower with reduced physical
activity, particularly towards the end of pregnancy.
While moderately reduced energy intakes have little
effect on pregnancy outcomes, severe energy deprivation
during pregnancy results in reduced infantile birth weight.
During the 6-month period of the Dutch famine with an
available energy supply below 4184 kJ/d (1000 kcal/d) and
a protein intake of no more than 30–40 g/d, average birth
Growth, development and differentiation
weight fell by 200 g (Ravelli et al. 1976). The effect was
most marked if the hunger period coincided with the latter
part of pregnancy. Later studies have confirmed these
observations. In underweight women, a close relationship
between maternal and neonatal body weights has been
observed, while the correlation is much less close in
pregnant women with a normal body weight (Luke &
Petrie, 1980). Not only total body weight, but also the
organ weights of liver, spleen, heart, adrenal gland, kidneys,
skeleton and thymus are reduced in newborn infants of
mothers who are underweight (Naeye et al. 1969), and
placental size and number of cells by 15–20 % in pregnancy
with intrauterine infantile growth failure or even up to 50 %
in severe maternal malnutrition (Winnick, 1970). In malnourished South American women, average placental
weights were reduced by about 15 %, and there was a
marked reduction of the placental peripheral villi surface,
the site of materno-fetal nutrient transfer, which may
aggravate the restricted nutrient supply to the fetus in
malnourished mothers. These findings are of concern,
since neonatal birth weight is the strongest predictor of
infant morbidity and mortality, and epidemiological studies
have found strong associations of low birth weights with
increased risks for cardiovascular disease and total mortality
rates in adulthood (Barker, 1994).
Several studies on energy supplementation during pregnancy in malnourished women found an increase in average
birth weight and a reduction of the rate of low-birth-weight
infants (Lechtig et al. 1975; Prentice et al. 1987), while
intervention studies with an increased energy intake in
apparently healthy women in Europe did not have significant effects on neonatal weight. However, dietary supplementation in Canadian women with twin pregnancies
resulted in an increase of average birth weight by 80 g,
and reductions of preterm deliveries and the rate of verylow-birth-weight infants by 30 and 50 % respectively
(Dubois et al. 1991). In conclusion, energy supplements
during pregnancy appear to be beneficial in selected populations at risk of very low intakes or increased demands.
4.1.2. Protein. With a factorial approach again based
on calculated materno–fetal accretion, the additional
protein requirements for the four successive 10-week
periods of a normal pregnancy have been estimated as 0.6,
1.8, 4.8 and 6.1 g/d, respectively (Hytten & Leitch, 1971),
and an increase of recommended protein intakes by about
6 g/d (expressed as milk or egg protein) over non-pregnancy
values has been recommended (FAO/WHO/UNU, 1985).
Even though this represents an increase of about 30 % over
recommended intakes in non-pregnant women, and hence
the recommended relative increment of protein intake is
clearly higher than that of energy, the protein intakes of
most pregnant women in Europe tend to exceed minimal
requirements by far, with the possible exception of small
subgroups consuming low-protein diets. It has been
proposed that protein-enriched diets during pregnancy
may be beneficial for the prevention of pregnancy-induced
hypertension and pre-eclampsia, but conclusive evidence to
support these hypotheses is missing (Roberts et al. 1974;
Williams et al. 1981).
4.1.3. Carbohydrates. Glucose serves as the major
energy source for the fetus, comprising about 90 % of the
energy supply. Hence, maternal carbohydrate metabolism
during gestation is of potential relevance to the optimal
supply for the fetus. Little is known about the effects of
dietary habits, particularly the total amount and relative
composition of sugars and starches on gestation, and
potential implications for clinically relevant outcome
variables such as macrosomia, postpartal hypoglycaemia,
or increased risk of developing glucose intolerance later in
4.1.4. Lipids. Although there is a tendency in some
pregnant women to aim at a low consumption of dietary fat
(Hachey, 1994), there are no conclusive data to demonstrate
the safety of maternal low-fat diets or immediate benefits for
pregnant women and the fetus and infant. On the other hand,
pregnant women appear to have high requirements for lipidsoluble vitamins and PUFA. During pregnancy, the
concentrations of blood lipids and their constituent fatty
acids increase considerably. Amounts (mg/l) of plasma
phospholipid-associated essential fatty acids (EFA) were
reported to increase during the course of pregnancy by about
40 % and those of the long-chain PUFA arachidonic acid
(20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3) by
about 23 and 52 % respectively (Al et al. 1995). It has been
proposed that a high supply of long-chain n-3 fatty acids
may be beneficial for fetal development because of the
importance of these compounds for neural tissue development (Koletzko, 1992), and that they may improve some
obstetric complications, particularly lessen the severity of
pregnancy-induced hypertension (Secher & Olsen, 1990).
Moreover, observations in the population of the Faroe
islands which consumes a diet rich in fish suggested that this
high intake of n-3 fatty acids may increase average birth
weight by prolonging gestation (Olsen et al. 1986).
However, in this population there were also apparent
adverse effects of n-3 fatty acids, including higher rates of
blood loss on delivery, which may be explained by fish-oilinduced suppression of platelet aggregation, and a higher
perinatal mortality. An intervention with supplementation
of n-3 long-chain PUFA was reported to prolong pregnancy
without any detrimental effects on growth of the fetus or the
course of delivery (Olsen et al. 1992). Supplementation of
fish oil with vitamins and minerals has been considered to
reduce the frequency of pre-eclampsia, but a double-blind
placebo-controlled trial did not find any effect of fish-oil
supplementation on the occurrence of pregnancy-induced
hypertension (Onwude et al. 1995).
Pregnancy may be associated with DHA mobilization
from maternal stores. Under the present dietary conditions,
pregnancy is associated with a reduction of the EFA status
and particularly of the DHA status. After delivery normalization takes place, but recovery of the DHA status appears
to be still incomplete after 6 months (Al et al. 1995).
Throughout pregnancy, the DHA content of plasma phospholipids of primigravida is significantly higher than that of
multigravida, and a negative relationship was observed
between DHA content and gravida number, which is reflected
in neonatal DHA status and may have functional consequences for infant growth and development. Since increased
supplies of selected PUFA during pregnancy may well have
some benefits, the possible benefits as well as the potential
risks of such strategies should be carefully evaluated.
B. Koletzko et al.
4.1.5. Vitamins, minerals and trace elements. Relative
to the increased energy needs (about 10 %), the recommended relative increase of some other nutrients is
markedly higher, for example, for folate the reference
intake increases by 100 % in pregnancy (Scientific Committee for Food, 1993). Based on these considerations, the
potential for an inadequate intake of one or more of these
specific nutrients during pregnancy is greater than for total
Calcium. During the latter part of pregnancy, the fetus
has a high rate of Ca accretion in the order of 250–300 mg/
d. This appears to be partly accounted for by extensive
adjustments in Ca metabolism during pregnancy, particularly the inhibitory effects of placental oestrogen on
maternal bone resorption that result in an enhanced release
of parathyroid hormone and, hence, increased maternal
Ca absorption, decreased urinary excretion and enhanced Ca
retention (Cole et al. 1987). Although low dietary Ca intakes
of malnourished pregnant women have been associated with
reduced bone mineral densities of the newborn infants
(Ramam et al. 1978), in well-nourished mothers no
enhancement of neonatal Ca accretion by increased
maternal intakes has been demonstrated. However, at
relatively low intakes, maternal body stores of Ca may be
utilized and depleted to meet fetal needs (Duggin et al. 1974).
In women with multiple pregnancies, in particular, low Ca
intakes may increase the risk of osteomalacia at a later age.
Although dental caries is relatively common during
pregnancy, there is no evidence for a causal link of its rate
of occurrence with dietary Ca intakes.
Low Ca intakes during pregnancy have also been
associated with the occurrence of EPH-gestosis (oedema,
proteinuria and hypertension) as well as eclampsia (Burke
et al. 1943). In a controlled study, supplementation of the
diets of pregnant women with Ca was associated with a
reduced systolic blood pressure at term and a lower incidence of pregnancy-induced hypertension (Villar et al.
1987). In contrast, another study found no effect of a Ca
supply on the incidence of pregnancy-induced hypertension,
although mean blood pressure was lowered.
Magnesium. Mg status is related to neuromuscular
excitability, and it has been proposed that an additional
Mg supply may contribute to prevention of eclampsia and
other complications of pregnancy. However, controlled
trials have not provided conclusive evidence for an
improved course of pregnancy or delivery (Spatling &
Spatling, 1988; Sibai et al. 1989).
Iron. The total Fe needs for pregnancy have been
estimated to be in the order of 300–850 mg (Bothwell et al.
1979; Hallberg, 1988). Although the efficacy of Fe
absorption is markedly enhanced in pregnancy, the
incidence of Fe deficiency and Fe-deficient anaemia
during pregnancy remains a sizeable problem that may
increase maternal and fetal morbidity and mortality
(Llewellyn-Jones, 1965), cardiovascular stress associated
with increased complication rates before and at birth (Banks
& Beutler, 1988) and an elevated risk of delivering lowbirth-weight and premature infants (Scholl & Hediger,
1994). At particular risk of poor Fe status in pregnancy are
women with vegetarian or predominantly vegetarian diets,
because of the relatively low absorption of non-haem Fe.
Thus, foods that promote net Fe absorption may be
beneficial for some pregnant women.
Zinc. Maternal Zn deficiency is highly teratogenic in
rodents, and in monkeys it induces abnormal fetal brain
development (Worthington-Roberts 1985). Studies in
human pregnancies found that maternal leucocyte Zn
levels during pregnancy were correlated with later infantile
birth weight (Wells et al. 1987) and inversely related to the
rate of low-birth-weight infants (Neggers et al. 1991).
Moreover, a better maternal Zn status was associated with
lower rates of pregnancy-induced hypertension, protracted
delivery and maternal complications at birth and the rate of
premature rupture of membranes (Sikorski et al. 1990). In
contrast, another controlled study evaluating Zn supplementation in the second and third trimesters of pregnancy in
Europe did not find any effect. To what extent these
differing results were influenced by variations in Zn status
of the respective populations studied, and may be
reproduced in other populations, remains to be clarified.
Iodine. In parts of Europe, the I status of many pregnant
women is inadequate with a sizeable prevalence of
increased thyroid size in mothers and newborn infants
(World Health Organization, 1993). Poor I status is
associated with an increased risk of miscarriage in early
gestation and preterm delivery as well as compromised
mental development of the infant (Xue-Yi et al. 1994).
Fluoride. Since postnatal F В№ supply has a strong
preventive effect on the incidence of dental caries, the
question was raised whether an increased F В№ supply to
pregnant women may contribute to protecting pre-eruptive
teeth of the unborn child, but conclusive evidence to answer
this question is not available.
Folic acid. Folic acid is essential for the synthesis of
pyrimidines, purines and hence of DNA and RNA, as well
as amino acids and neurotransmitters; thus, an adequate
folate status is of importance to allow undisturbed cell
multiplication and growth during pregnancy. Following
observations of a relation between folic acid status and the
occurrence of neural tube defects in the offspring, several
controlled clinical trials have demonstrated that folic acid
supplied before conception and during the first weeks of
pregnancy can markedly reduce the incidence of neural tube
defects, such as spina bifida and anencephaly, by 40–70 %
(Butterworth & Bendich, 1996). This important preventive
effect is apparently associated with a folic acid-responsive
derangement in homocysteine metabolism in a genetically
determined subgroup of the population (SteegersTheunissen et al. 1991). Poor folic acid status of pregnant
women has also been associated with miscarriages, repeated
abortions, pregnancy length and neonatal outcome as well
as unexplained sterility (Pietrzik et al. 1992; Bung et al.
1993, 1995). It has been recommended that all pregnant
women should be supplemented with a daily dose of 0.4 mg
folic acid from before conception to at least four completed
weeks after conception (Scientific Committee for Food,
1993). In view of the postulated key role of folic acid status
in the aetiology of atherosclerosis by means of modulating
homocysteine metabolism, the question of a potential
programming effect of intrauterine folic acid supply on
the later risk of cardiovascular diseases has been raised
(Pietrzik et al. 1992).
Growth, development and differentiation
5. Modulation of growth
5.1. Introduction
The term �growth’ expresses the increase in number and size
of cells of a particular species and it refers to changes in
body dimensions. Growth is a phenomenon usually associated with increase in length and weight whereas the term
�development’ is a physiological concept indicating the
progressive differentiation of tissues and organs with acquisition of their specific functions. All mammals start life as a
single cell; during the early part of the gestation the
fertilized ovum divides many times and different kinds of
cells develop during the process of differentiation and
arrange themselves to form part of the various organs of
the body. The general principles of growth apply to all
species but the rate of cell division is genetically determined
and depends on nutrient supply and utilization. Regardless
of the exact time that differentiation occurs it always results
in the transformation of the parental cell into a large number
of morphologically different progeny cell types. The rapidity of physical growth is regulated during the life cycle and
is modulated by genetics, a variety of growth factors that
interact with target cells, as well as environment and diet
(HernaВґndez & Argente, 1992; Philips, 1995).
Human growth hormone (hGH) or somatotropin is essential for normal postnatal growth. It is released from the
anterior pituitary gland on stimulation by growth hormonereleasing hormone or somatocrinin, a factor produced by the
hypothalamic region of the brain. Like other pituitary
hormones, hGH acts on target tissues, primarily the liver,
to cause synthesis and release of a second hormone
mediator, insulin-like growth factor (IGF)-I, also called
somatomedin C, into the systemic circulation. IGF-I is a
growth-accelerating peptide that acts directly on cartilage to
promote bone growth. Deficiency of hGH production causes
metabolic alteration and growth failure (Philips, 1995).
Different requirements for growth of different cell types
have been established (Sato et al. 1982) but despite
considerable progress in tissue culture systems we are still
a long way from identifying all the stimulatory and inhibitory macromolecules that regulate the growth of all the
human cell types in vivo. Most growth factors are polypeptides or small proteins with molecular masses that vary from
1 to 40 kDa and bind to specific cell surface receptors, with
pleiotropic effects on cells including changes in gene
expression (Watson et al. 1987). By binding to their
receptors, growth factors modify the activity of the membrane-bound enzyme adenylate cyclase (EC, using
the GTP-binding protein as an intermediate so that the level
of cAMP in the cell is altered. This, in turn affects the
activity of cAMP-dependent protein kinases which phosphorylate specific target proteins, regulating their activity.
Other possible second messengers to carry signals from
growth receptors to the cell include Ca, inositol triphosphate
and diacylglycerol (Lodish et al. 1995).
In addition to genetic factors, neurohormonal and tissuespecific growth factors, growth is also affected by a number
of metabolic and environmental factors which include the
availability of nutrients. Recommended daily nutrient
intakes have been established for all periods of life and
both sexes to support an adequate growth of the human
being (National Research Council, 1989). However, there is
a lack of information about how semi-essential nutrients can
affect growth in specific periods of life and in particular
situations including disease states.
The rapidity of physical growth in the normal infant
during the first months of life is remarkable and unmatched
during later times of life. Moreover, physiological and
developmental changes during infancy are as notable as
the speed of physical growth. Changes in the rates of
physical growth and in the allocation of dietary intake of
energy and protein for growth and maintenance occur as a
continuum rather than in discrete stages, but the progression
of changes during the early months of life is very rapid.
Human milk provides all nutrients necessary to support
adequate growth of the term infant during the first 4–6
months of life. Furthermore, in addition to universally
recognized nutrients, human milk contains a number of
semi-essential nutrients, enzymes, hormones and growth
factors which appear to have a role in supporting infantile
growth (Koldovsky & Strbak, 1995). However, there is a
lack of information about how those nutrients and factors
interact with the growth process and how they affect specific
tissue growth.
5.2. Methods for the determination of growth
Anthropometric measurements such as weight, length, head
circumference, skinfold thickness, limb circumference,
mid-arm cross-sectional area, BMI etc. at various ages
during infancy have been published in many developed
countries and are useful in evaluating the size of an infant
in relation to the size of his or her peers (Fomon & Nelson,
1994). In the infant the Quetelet index is difficult to interpret
since the body mass increases rapidly from birth to 4 months
of age and the percentage of body weight contributed by
body fat also increases rapidly during that period. Reference
data for increments in size and other indices of nutritional
status are more sensitive in children beyond infancy,
particularly in alerting to the possibility of illness or nutritional inadequacy (Fomon, 1991).
Data on body composition at various ages are one of the
bases for the factorial approach in estimating the requirements for various nutrients for growth. Chemical composition of animals is determined by direct whole-body
analysis. The most extensive data on the composition of
the term infant are those published by Widdowson (1982),
but data beyond infancy are scanty. The most useful indirect
methods of estimating various aspects of body composition
in the infant are determination of total body water from the
concentration of a suitable tracer in body fluid, i.e. heavy
water, extracellular water, and determination of natural
abundance of 40K in the whole body (Forbes, 1987).
Measurements of both bioelectric impedance and of total
body electrical conductivity can estimate non-invasively the
fat-free body mass of infants and children (Mayfield et al.
1991; Houtkooper et al. 1992). Many new techniques are
available for measurement of total body fat, although only a
few can be used in general practice or in epidemiological
research (Deurenberg, 1992).
The urinary excretion rates of endogenous creatinine and
3-methyl-histidine have been proposed as indices of muscle
B. Koletzko et al.
mass, and the urinary excretion rates of endogenous hydroxyproline and type I and type III procollagen propeptides as
indices of growth rates (Trivedi et al. 1991). Measuring cell
kinetics represents a new approach in evaluating cell and
tissue growth. Cell kinetics can be used to measure the
proliferation rate of cells, the phases of the cell cycle and the
percentage of cells in cycle, to plot the position of dividing
cells and determine the size of the proliferation compartment
and to follow the movements of labelled cells. Cell kinetic
studies received a boost when [3H]thymidine was introduced
in the 1950s. Recently, several new techniques have been
used in cell kinetics, i.e. incorporation of bromodesoxyuridine
and labelling of cell antibodies (Kember, 1993). There is a
need for new methods to evaluate the specific organ and
tissue growth applicable in a wide range of conditions.
5.3. Growth factors in human milk and their influence on
infant growth
A large number of hormones and growth factors are present
in human and bovine milks (Koldovsky & Thornburg, 1987;
Strbak, 1991). Non-peptide hormones (thyroid hormones,
cortisol, progesterone, pregnanediol, oestrogens and artificial contraceptives) and peptide hormones and growth
factors (erythropoietin, hGH, growth hormone releasing
factor, gonadotropin-releasing hormone, epidermal growth
factor (EGF), insulin, IGF-I, nerve growth factor, gastrointestinal regulatory peptides and thyroid–parathyroid hormones) have been isolated and quantitated in human milk
(Koldovsky & Strbak, 1995). The orogastric effects of
hormones and growth factors on infant growth require
further elucidation. However, there is some evidence that
a number of hormones in human milk may contribute to the
intestinal maturation of sucking infants. For example, oral
administration of EGF to 10-d-old sucking rats resulted in
changes in protein and DNA content of colonic mucosa
(Koldovsky & Thornburg, 1987).
5.4. Potential roles of non-protein nitrogen compounds as
growth modulators
Non-protein N (NPN) compounds are present in most foods
mainly as nucleic acids, free amino acids, small peptides
and other minor compounds. The concentration of nucleic
acids in foods depends on the number of cells of the original
biological tissue. Thus, meat, fish and vegetal seeds are rich
in nucleic acids whereas fruits have a low content (Gil &
Uauy, 1995b). NPN accounts for 18–30 % of the total N in
human milk. Some of this N, namely urea, contributes to the
pool available for synthesis of non-essential amino acids in
infants. Other NPN components may have particular roles in
tissue growth. Among the NPN known to have specialized
roles in the ontogeny of the human newborn are the growth
factors, namely EGF, amino sugar oligosaccharides, free
amino acids like taurine, arginine and glutamine, amino
alcohols of phospholipids i.e. choline, nucleotides and
nucleic acids and polyamines.
5.5. Human milk oligosaccharides and growth
In addition to lactose, the carbohydrates of human milk
include nucleotide sugars, glycolipids, glycoproteins, and
oligosaccharides. Viverge et al. (1990) have isolated three
oligosaccharide fractions representing 13–18 g/l; the concentration varied with the mother’s genetic ability to
synthesize specific fucosyl linkages. Approximately eighty
neutral and sialic acidic oligosaccharides have been isolated
and identified (Newburg & Neubauer, 1995).
Human milk oligosaccharides appear to be synthesized
by some of the same glycosyltransferases that participate in
the synthesis of glycoprotein and glycolipid cell surface
components. Thus, it is reasonable to postulate that some of
those compounds can act as analogues to host cell surface
receptors for pathogens. Anderson et al. (1986) reported that
specific oligosaccharides can inhibit binding of Streptococcus pneumoniae and Hemophilus influenzae to their receptors and Cravioto et al. (1991) described an oligosaccharide
that inhibits adherence of enteropathogenic E. coli to their
receptors. Other authors have reported that specific fucosylated oligosaccharides inhibit binding of invasive strains of
Campylobacter jejuni (Ruiz-Palacios et al. 1992) and the
toxicity of E.coli in vivo (Newburg et al. 1990).
Gangliosides are glycosphingolipids that contain sialic
acid (N-acetylneuraminic acid) as part of their carbohydrate
moiety. GM1, a milk ganglioside present in human milk,
binds to E. coli and Vibrio cholerae toxins and may
contribute to infant protection against infection by those
enteropathogens (Laegrid et al. 1986).
Since lactating mothers differ genetically in their ability
to produce various oligosaccharides, this variability might
influence the susceptibility of breast-fed infants to enteric
disease. The influence of supplementing infant milk formulas with oligosaccharides on the susceptibility of infants
to gastrointestinal diseases, namely acute diarrhoea, is one
of the current fields of intense investigation.
5.6. Free amino acids and tissue growth
The free amino acid pool of human milk is small compared
with the total amount of milk amino acids in protein. Free
amino acids contribute only 10 % of the total NPN in human
milk. Glutamine and taurine are the free amino acids found in
higher concentrations in human milk. While clinical effects
of low dietary intakes of taurine have not been demonstrated,
there is a concern about the possibility of subclinical deficiency, particularly in the premature infant since its ability to
synthestize taurine may be limited (Gaull et al. 1977).
Free glutamine accounts for about 20 % of the total
glutamine pool in human milk. Glutamine is currently
extensively investigated because of its importance in cell
and tissue culture and because it serves as a preferred
respiratory fuel for rapidly proliferating and growing cells,
such as enterocytes and lymphocytes. Moreover, it is a
regulator of acid–base balance through the production of
urinary NH 3, a carrier of N between tissues and an important
precursor of nucleotides, amino sugars and proteins (Lacey
& Wilmore, 1990). There is increasing evidence that glutamine may become a conditionally essential amino acid in
critically ill patients, and glutamine appears to be important
for the maintenance of small-intestinal structure and functionality (Newsholme & CarrieВґ, 1994).
Arginine and ornithine are also present in human milk as
free amino acids, although the first is found in higher
Growth, development and differentiation
concentrations in milk proteins. Arginine has multiple
biological properties, including the ability to stimulate
anabolic hormone secretion: intravenous and enteral administration of arginine increases both insulin and hGH secretion (Cynober, 1994). Several studies show that arginine
given to patients, as well as in various experimental stress
models, acts by improving N balance, accelerating wound
healing, and restoring depressed immunity. Dietary supplements of arginine have been shown to inhibit tumour growth
in animals, probably by activating the immune system.
However, in cancer patients arginine stimulates tumour
protein synthesis, suggesting that arginine might have
separate stimulatory effects on the tumour and on the
immune system, the outcome depending on which effect
predominates (Garlick & McNurlan, 1994). Arginine has
also been shown to enhance the growth-hormone-releasinghormone-induced hGH rise in patients with anorexia nervosa (Ghigo et al. 1994). Moreover, oral administration of
arginine enhances the hGH response to growth hormone
releasing hormone in short children (Loche et al. 1993).
Ornithine shares with arginine the ability to stimulate
hGH secretion. In addition, ornithine as its a-ketoglutarate
salt generates various molecules, i.e. glutamine. Ornithine
ketoglutarate has been shown to improve N balance in
various acute and chronic malnutrition states. It increases
muscle protein anabolism in moderate catabolic states and
reduces protein catabolism in hypercatabolic states
(Cynober, 1994).
Arginine and ornithine are precursors of NO and
polyamines respectively. These metabolites participate intimately in permeability and adaptive responses of the gut.
Recent animal studies showed improved morphology after
ornithine ketoglutarate administration, acting perhaps
through increased polyamine synthesis (Cynober, 1994). It
is controversial whether exogenous arginine can be a
relevant precursor of polyamines.
5.7. Polyamines and tissue growth
Polyamines are detectable in relatively high quantities in
human and rat milk. Artificial infant formulas do not contain
appreciable amounts of polyamines, specifically putrescine
and spermidine, and spermine is undetectable. Thus,
formula-fed infants are not exposed to polyamines nor to
any potential effects that these compounds may have on the
developing intestine. It is noteworthy that food contains
polyamines and that polyamines are produced by the gastrointestinal microflora. Thus, the direct uptake by enterocytes
of preformed polyamines could contribute to the polyamine
cellular pool. Indeed, putrescine and spermidine uptake
has been shown in isolated rat enterocytes (Cynober,
5.8. Dietary nucleotides and tissue growth
Human milk is the exclusive source of dietary nucleotides
for infants during the first months of life, and its nucleotide
profile (Gil & SaВґnchez-Medina, 1982; Gil & Uauy, 1995a)
differs markedly from that of cow’s milk and most infant
formulas (Gil & SaВґnchez-Medina, 1981; Gil & Uauy, 1989,
1995a). Preformed nucleotides may be of importance for the
growth of tissues with a rapid turnover (Van Buren et al.
1985; NunЛњez et al. 1990; Uauy et al. 1990; Gil & Uauy,
1995b), particularly bone marrow, leucocytes and the intestinal mucosa which preferentially use the nucleotide salvage
pathway to fulfil their purine and pyrimidine nucleotide
requirements (Mackinnon & Deller, 1973; Savaiano &
Clifford, 1981; LeLeiko et al. 1983; Cohen et al. 1984).
Dietary nucleotides may modulate lipoprotein and fatty
acid metabolism in human early life (Gil et al. 1986b, 1988;
De-Lucchi et al. 1987; Pita et al. 1988; Morillas et al. 1994;
SaВґnchez-Pozo et al. 1994), and they may enhance the
growth of bifidobacteria and limit that of enterobacteria in
the gut of newborn infants (Gil et al. 1986a; Gil & Uauy,
1995). Dietary nucleotides may affect small-intestinal
growth in experimental animals (Gil & Uauy, 1995) and
may have a role in the maintenance of the immune response
both in animals (Van Buren et al. 1983, 1985; Pizzini et al.
1990; Kulkarni et al. 1992) and in human subjects (Carver et
al. 1991; Gil & Uauy, 1995).
5.8.1. Nucleotides and small intestine growth. A
number of factors are involved in the regulation of the
renewal of the absorptive epithelium and in the repair of the
epithelium under pathological conditions (Shiner et al.
1990). N-containing nutrients appear to be important for gut
growth. At weaning, protein modulates the ontogenic
changes in tissue DNA synthesis and plays a role in
completing the growth of the rat’s gastrointestinal tract
(Buts & Nyakabasa, 1985). Dietary nucleotides have been
shown to influence gut development and repair after injury.
Uauy et al. (1990) reported that intestinal disaccharidase
activities are increased in rats during development by
dietary nucleotides, and DNA, lactase (EC,
sucrase (EC and maltase (EC activities
increase with a nucleotide-supplemented diet in animals
after chronic diarrhoea (NunЛњez et al. 1990; Bueno et al.
1994). Dietary nucleotides promote the enterocyte growth in
tissue culture (He et al. 1993; Sanderson & He, 1994) and
improve the intestinal repair in an animal model of radiation
injury (Uauy et al. 1994). Recent studies on the potential
roles of exogenous nucleotides on proliferation, differentiation and apoptosis of human small-intestinal epithelium
have shown that AMP may have an important role in
controlling the dynamic balance of cellular turnover in the
developing human small intestine (Tanaka et al. 1996).
Moreover, dietary nucleotides influence the gene transcription in the intestine (LeLeiko et al. 1995). Animals
receiving a purine and/or pyrimidine-free diet have a
decreased protein synthesis and RNA throughout the
intestine and specific mRNA for the enzymes hypoxanthine
phosphoribosyltransferase (HGPRT) and adenosine phosphoribosyltransferase (APRT) (LeLeiko et al. 1987). A
35-base pair region identified in the HGPRT promoter is
necessary to confer sensitivity to exogenous purines as a site
for binding to trans-acting regulatory proteins (Walsh et al.
1990, 1992).
5.8.2. Nucleotides and liver growth. Extracellular
nucleotides and nucleosides have been reported to modulate
hepatocyte growth (Ohyanagi, 1989; Gil & Uauy, 1995b)
and regeneration and to play an important role in the
synthesis of glycogen (Buxton et al. 1986). Ogoshi et al.
(1985, 1988) reported that parenterally administered
B. Koletzko et al.
nucleotides improved the hepatic function and promoted
earlier restoration of the N balance after liver injury or partial
hepatectomy. Moreover, it has been observed that adenosine
administration partially prevents cirrhosis induced by CCl 4 in
rats due to a stimulation of total hepatic collagenase activity
and is able to counteract the drastic decrease in adenine
nucleotides (HernaВґndez-MunЛњoz et al. 1990).
Deprivation of dietary nucleotides results in a transient
decrease in acid-soluble nucleotides and RNA content in rat
liver as well as in a decreased protein synthesis rate (LoВґpezNavarro et al. 1995). Dietary nucleotides have improved
liver structural recovery and binuclearity in experimental
cirrhosis induced by thioacetamide (Torres et al. 1996). In
that model dietary nucleotides led to a lower number of
stellate cells and to a lower collagen deposition.
5.9. Long-chain polyunsaturated fatty acids and cell growth
Despite recent advances in neonatal care, low-birth-weight
infants do not achieve first year growth equivalent to that of
infants born at term. It has not been clarified how the
administration of long-chain fatty acids to infants may
affect growth and specific tissue growth and differentiation.
Direct evidence that normalized growth might relate to
arachidonic acid status came from the observation that
formula supplemented with marine oil but no arachidonic
acid decreased the concentrations of plasma phosphatidylcholine arachidonic acid (Carlson et al. 1991) and reduced
weights compared with standard formula without marine oil
(Carlson et al. 1992). Phosphatidylcholine arachidonic acid
declined in preterm infants fed on non-supplemented formulas, and weight fell progressively beginning at 2 months
of age. The nadir of plasma phosphatidylcholine arachidonic acid and growth was further reduced by formula containing marine oil compared with the non-supplemented
formulas (Carlson et al. 1993).
Koletzko & Braun (1991) have investigated whether birth
weight correlates with the postnatal EFA status in premature
infants. A significant and positive correlation between body
weight and plasma triacylglycerol content of arachidonic
acid and total n-6 long-chain-PUFA was found as well as a
negative correlation with a-linolenic acid.
There is only limited information about how intakes of
n-6 and n-3 long-chain-PUFA may affect specific tissue
growth. Animal studies using diets supplemented with both
types of fatty acids have shown that in addition to plasma
and erythrocyte cell membranes, small intestine, liver,
kidney, lung and heart are affected in their fatty acid
composition. Depending on the composition of the diet,
susceptibility to oxidation may be affected, which might
influence tissue growth and function (Suarez et al. 1996a,b).
5.10. Early growth and later obesity
In animal studies, early overfeeding may have lasting
effects on nutrient utilization and body composition
(Davis et al. 1973; Lewis et al. 1986). In male infants of
pregnant women who suffered from starvation, obesity in
young adulthood was more prevalent if the mothers were
exposed to the famine during the first half of gestation,
while the incidence of later obesity was reduced if starvation
occurred during late gestation and the early postnatal period
(Ravelli et al. 1976). Also, food composition, and particularly protein intake, in early childhood has been suggested
as a predictor of later risk of obesity. In view of the high
prevalence of obesity in Europe and its major importance
for public health and health-care costs, the potential
modulation of later obesity by early food choice needs to
be further explored.
6. Maturation of the gastrointestinal tract
6.1. Introduction
Digestion and hydrolysis of macro- and micronutrients by
the gastrointestinal tract are essential prerequisites for longterm survival of mammals including man. Proteins, fats and
carbohydrates are digested and hydrolysed by a variety of
potent excretory glands and by the brush-border enzymes of
the small intestine as well as by bacterial breakdown within
the large intestine. As for carbohydrates, a cascade of
hydrolytic events finally leads to the presence of monosaccharides within the lumen of the gastrointestinal tract
which are transported across the microvillus membrane by
highly specialized transporters. Carbohydrates with high
molecular mass in the form of amylose and amylopectin
are hydrolysed by a-amylase (EC of the saliva and
the pancreas. a-Amylase can hydrolyse 1-4-a-glycosidic
bonds which are present in both amylose and amylopectins.
The branches of 1-6-a-glycosidic side chains in amylopectins remain after the action of a-amylase as a-limitdextrins, and are further hydrolysed by sucrase–isomaltase
(SI) (Gray, 1967). Hydrolysis of starches is dependent on
the age of the infant. In the first 6 months of life, activity of
a-amylase is low and reaches full activity at the end of the
first year of life (Lentze, 1986). Defects in sugar digestion
occur because of disturbances within the combined action of
pancreatic a-amylase and that of intestinal brush-border
enzymes. Decreased digestion and hydrolysis of carbohydrates will induce either osmotic diarrhoea and/or bacterial
overgrowth within the small intestine as well as bacterial
breakdown of carbohydrates within the colon.
Malnutrition is very often combined with chronic
diarrhoea and damage of the gastrointestinal mucosa as a
consequence of lack of protein and energy. Key factors in
this devastating cascade are the brush–border membrane of
the small intestine and its hydrolysing and absorptive capacity. Important observations with regard to the hydrolytic
capacity of intestinal disaccharidases, which are responsible
for sugar hydrolysis, have come through the study of their
intracellular pathways and processing of enzyme molecules
in normal and altered human as well as animal mucosa using
various techniques of molecular biology. The knowledge of
these investigations has considerably increased our understanding as to how carbohydrates are hydrolysed and
absorbed from the intestinal epithelial cell.
6.2. Development of sugar hydrolases and transporters
The morphological development of the small intestine starts
in the 9th week of gestation from the proximal to the distal
part of the gut (Hauri, 1986). Small villi develop over a
Growth, development and differentiation
stratified epithelium of several layers. The first crypts are
seen at the age of 10–11 weeks gestation within the
duodenum and jejunum, and at 11–12 weeks in the ileum
and colon. The changes into a columnar epithelium occur
together with the appearance of secondary lumina visible by
electronmicroscopy and parallel invagination of mesenchymal cells and extrusion of surface cells (Naim et al. 1988).
The development of a brush-border membrane is seen
together with crypt development in the 10–12th week of
gestation. At the same time brush-border membrane hydrolases start to appear. Lactase–phlorizin-hydrolase (LPH), SI
and maltase–glucoamylase are first detectable at the 10th
week of gestation (Dahlqvist & Lindberg, 1965). Their
enzymic activities increase during gestation. As SI and
maltase–glucoamylase reach their full activities by the
25th week of gestation (Jirsova et al. 1965–6), the activity
of LPH remains low until the 28th week of gestation and
increases slowly between the 32nd and 34th weeks of
gestation (Dahlqvist & Lindberg, 1966). For the nutrition
of very immature premature babies with very low birth
weight between the 26th and 28th weeks of gestation this
could play a role in the digestion and hydrolysis of lactose
given in breast milk or infant formula. After introduction of
lactose-containing milk the activity of LPH matures quickly
to normal enzymic activities. The glucose transporters in the
small intestine develop during gestation at about the same
time as the sugar hydrolases. Sodium-dependent glucose
transporter 1 (SGLT 1), glucose transporter (GLUT) 5 and
GLUT 2 appear at the 11th week of gestation as seen by the
expression of specific mRNA in human fetal intestine
(Davidson et al. 1992).
6.3. Biosynthesis of intestinal brush-border membrane
Mature intestinal epithelial cells are highly polarized and
are composed of two main membranous regions: the apical
cell membrane, with its unique feature of a brush border,
and the basolateral membrane. The microvillar membrane is
characterized by a network of microvilli which contain the
important glycoproteins responsible for the hydrolysis and
absorption of micronutrients and minerals. For the degradation of various sugar and peptide molecules of different
composition and chain length, the intestinal disaccharidases
SI, maltase–glucoamylase, LPH, trehalase and a variety of
peptide hydrolases are present within the microvillar region
of the columnar epithelia in order to digest carbohydrate
molecules and oligopeptides from nutritional intakes. The
disaccharidases are the best studied brush-border hydrolases. Their enzymic activities and their distribution
throughout the gastrointestinal tract as well as their age
dependency have been investigated by many groups of
researchers. The biogenesis of the disaccharidases produced
and processed by the mature enterocyte has been elucidated
in mammals as well as in man, demonstrating common
pathways within the translational and post-translational
SI and LPH are the best studied brush-border membrane
hydrolases in all species including man. The data accumulated from these studies have led to a general understanding
as to how these hydrolases are synthesized and processed
within the small-intestinal enterocyte. After transcription a
single-chain precursor (pro-SIh) rich in mannose (high
mannose precursor) is produced in the rough endoplasmic
reticulum. This contains carbohydrate residues which are
N-glycosylated and has an apparent molecular mass of
210 kDa in human subjects (Hauri et al. 1980; Ghersa et
al. 1986). From the rough endoplasmic reticulum the proSIh is transported to the Golgi apparatus where trimming of
the mannose residues and addition of complex carbohydrates occur to yield pro-SIc (molecular mass 245 kDa)
(Hauri et al. 1982). The complete primary structure of the
pro-SI from rabbit is composed of 1827 amino acid residues
containing the two active catalytic subunits isomaltase
(140 kDa) and sucrase (120 kDa) which are associated by
oncovalent, ionic interactions (SjoВЁstroВЁm et al. 1980). After
complex glycosylation in the Golgi the pro-SIc is translocated and inserted into the microvillus membrane by
vesicular transport directly into the apical microvillus
membrane. The exact route for the transportation of glycoproteins from the Golgi to the microvillar membrane
remains to be established. The time course of transport of
the pro-SIc from the Golgi into the brush-border membrane
in a human colon carcinoma cell line (CaCo-2 cells) is
rather slow (Herskovics et al. 1981). Similar transport
kinetics were also obtained when the biosynthesis of SI
was investigated in the organ culture of human intestinal
explants (Naim et al. 1988). Insertion of pro-SIc into the
microvillus membranes is obtained by anchoring a single
hydrophobic segment of the molecule which is located at the
N-terminus of isomaltase (Hauri et al. 1986). After insertion
into the microvillar membrane pro-SIc is cleaved into
sucrase and isomaltase by pancreatic proteases (Naim et
al. 1988). The mature catalytic enzymes sucrase and
isomaltase cleave various substrates including sucrose, isomaltose, maltose, maltotriose and amylose as well as a-limit
dextrins which are derived from the hydrolysis of amylopectins. SI, together with maltase–glucoamylase, plays a
major role in starch digestion during the first month of life as
a-amylase in human infants is not developed during the first
6 months of life (Danielsen et al. 1981). Striking structural
and functional similarities suggest that intestinal SI, human
lysosomal a-glucosidase and Schwanniomyces occidentalis
glucoamylase are derived from a common ancestral gene
(Naim et al. 1991).
LPH, as the only b-glycosidase of the brush-border
membrane, has been reported in earlier work to be synthesized also as a single-chain precursor with a molecular mass
of 150 kDa (SjoВЁstroВЁm et al. 1983). However, conflicting
results were obtained on the structure and identification of
the precursor molecules. In the pig a precursor protein of
200 kDa was observed (Danielsen et al. 1984). The same
group reported in a more recent publication that the precursor molecule of LPH in the pig small intestine is a
membrane-bound polypeptide of 225 kDa which is intracellularly cleaved after complex glycosylation (Mantei et al.
1988). Similar data were obtained in CaCo-2 cells
(Herskovics et al. 1981). In human intestinal epithelial
cells a high-mannose precursor of 215 kDa was demonstrated in intestinal explants maintained in organ culture
(Hauri, 1986). Here the intracellular cleavage of the highmannose precursor occurs during the translocation of the
B. Koletzko et al.
molecule across the Golgi before complex glycosylation
takes place. The mature form of LPH is then inserted into
the membrane with a molecular mass of 160 kDa. The
primary structure of the human lactase molecule is known
and comprises 1927 amino acids in man and 1926 amino
acids in the rabbit (Messer & Kerry 1967). The place at
which the mature form of lactase is cleaved from its
precursor is position 866 of the whole molecule. In contrast
to most other brush-border membrane hydrolases the mature
lactase is anchored within the lipid bilayer from its carboxyl
end of the protein chain (Messer & Kerry, 1967).
Maltase–glucoamylase hydrolyses 1-4-a-glycosidiclinked glucose polymers including maltose and maltotriose
(Naim et al. 1989). The enzyme is developed early in
gestation and contributes to the digestion of starch after
birth. The biosynthesis of maltase–glucoamylase is similar
to that of SI as a precursor molecule of 225 and 245 kDa.
The former represents the high mannose and the latter the
complex glycosylated precursor of maltase–glucoamylase
in the pig small intestine (Danielsen et al. 1984). The
biosynthesis and processing of maltase–glucoamylase in
human intestinal biopsy specimens does not involve intracellular or extracellular proteolytic modifications, in
contrast to SI and LPH (Pfeffer & Rothman, 1987).
6.4. Intestinal absorption of glucose and fructose
Our current understanding of glucose (galactose) and fructose is that the monosaccharides are transported by different
methods into the intestinal absorptive epithelial cells.
Whereas SGLT 1 is responsible for the active transport of
glucose or galactose with equimolar amounts of Na against
a concentration gradient into the cytoplasm of the enterocyte (Crane 1975), fructose undergoes facilitated transport
by the GLUT 5 transporter which is also located on the
brush-border membrane (Davidson et al. 1992). Once taken
up into the enterocyte, Na Гѕ is exchanged with K Гѕ by the
Na Гѕ, K Гѕ-ATPase (EC which is located in the
basolateral membrane and glucose is pumped into the
intracellular space by another glucose transporter protein,
GLUT 2. GLUT 2 has also been shown to be localized
within the basolateral membrane. The function of SGLT 1 is
essential for survival of a given species such as man. When
absent or deficient, as in congenital glucose–galactose
malabsorption, the malfunction of SGLT 1 is a lethal factor.
The absorption of these simple monosaccharides when
present in the intestinal lumen is dependent on a variety of
factors contributing to the rate of absorption. It is dependent
on age, composition of food and species. In mice a relationship between the type of diet and sugar uptake was demonstrated. When fed on a high-carbohydrate, low-protein
chow, the uptake of glucose remained high, and dropped
considerably when the mice were put on a low-carbohydrate, high-protein diet (Karasov et al. 1983). Moreover, it
was shown that a strong correlation exists between glucose
uptake and the type of natural diet within various vertebrate
species. The more herbivore the species is, the more glucose
is absorbed; the more carnivore the species is, the less
glucose is taken up (Riby et al. 1993). Fructose absorption
depends strongly on the presence of other carbohydrates
within the intestinal lumen. Fructose given together with
glucose, galactose, sucrose or starch is better absorbed than
fructose alone (Fujisawa et al. 1991) whereas the presence
of sorbitol or dextrin leads to fructose absorption as with
fructose alone. Species differences have a considerable
influence on fructose absorption which depends entirely
on the composition of the natural diet. Whereas the carnivores (cats) have a low fructose absorption after weaning,
the rat and the rabbit increase their fructose absorption
considerably after weaning (Buddington & Diamond,
The complete mechanism for fructose absorption from
the human intestine remains to be elucidated. A model of
fructose absorption can be deduced from animal studies as
well as from human studies. The rate of fructose absorption
is influenced by glucose, but also by the amino acid
glycine. Two mechanisms have been proposed to explain
this effect. Fujisawa et al. (1991) speculate that a mechanism in the brush-border membrane exists which they call the
disaccharidase-related transport system. This speculation is
based on their findings that the specific inhibitor of SI,
acarbazone, decreases fructose absorption when given with
sucrose, whereas absorption increases without the inhibitor.
Another explanation could well account for this effect:
glucose absorption as well as glycine absorption increases
the water flow from the lumen into the intercellular space by
osmosis. Therefore, a solvent drag occurs leading to an
enhanced uptake of fructose. The same mechanism applies
when starches or sucrose are present at the same time as
fructose because of the rapid hydrolysis of these carbohydrates to glucose and/or fructose by the action of maltase–
glucoamylase and SI. In order to elucidate this effect
fructose absorption should be studied in individuals with
SI deficiency which would be the equivalent model to the rat
intestinal fructose uptake studies with acarbazone. Except
for LPH, the activities of other sugar hydrolases as well as
SGLT 1 can be influenced by substrates (Buddington et al.
1991; Quan & Gray, 1993). The close presence of sugar
hydrolases and sugar transporters within the microvillar
membrane of the small-intestinal enterocyte as well as the
transporters for amino acids is the guarantee of a steady
uptake of sugars derived from various sources.
6.5. Oligosaccharides and mucins
Besides the most abundant sugar lactose, human milk
contains more than 130 different oligosaccharides and is
unique among all mammalian species for its content of
higher oligosaccharides, i.e. larger than lactose. For a long
time the oligosaccharide fraction in human milk has been
overlooked although it is the third largest solute (up to 18.5
g/l) and present in higher amounts than protein (Egge et al.
1983). As oligosaccharides escape the hydrolysis in the
small intestine, two possible functions are discussed. One
function would be the intact absorption of these components
serving as substrates for organ maturation such as the
brain, where rapid synthesis of sialoglycoproteins and
gangliosides occurs (Sabharwal et al. 1991). Their role in
the large bowel as �dietary fibre’ and their fermentation
would be of significant value in nutrition. The oligosaccharides in human milk are based on five monosaccharide
residues: sialic acid, N-acetylglucosamine, fucose, glucose
Growth, development and differentiation
and galactose. All oligosaccharides possess a lactose moiety
at their reducing end, with sialic acid (when present) and
fucose at the non-reducing end. The chain length varies
between three and eleven units. The oligosaccharide composition of human milk shows temporal and individual
variations (Miller et al. 1994). At the same stage of lactation
the variation in oligosaccharide content was shown to be
Recent findings on the chemical structure of oligosaccharides in milk have demonstrated structural homologies to carbohydrates carried by glycoproteins and
glycolipids on cell surfaces. Such oligosaccharides are
very antigenic and were targets of monoclonal antibodies
in the search for specific binding to human cancer cells.
Similar novel oligosaccharides which can inhibit antigen–
antibody reactions have been detected in human milk
(Fievre et al. 1991; Kitagawa et al. 1991). They are useful
hapten inhibitors to study the binding specificities of anticarbohydrate antibodies produced as mucins on cancer cells.
This observation has great clinical implications with respect
to breast cancer. High-molecular-mass glycoproteins
(MUC1) in milk and lactating tissue have been found to
contain up to 80 % of carbohydrates. The low level of
expression of MUC1 in healthy, undifferentiated (nonlactating) breast tissue, and its presence in many, particularly metastasizing, breast tumours has established a very
useful marker in breast cancer screening. The exact nature
of MUC1, its biological role and expression, has been
studied in milk and mammary tissue (Patton et al. 1995).
MUC1 as expressed in tumours activates B- and T-lymphocytes. These epitopes represent underglycosylated forms of
MUC1 characteristic of breast and pancreatic cancer. It is
also responsible for keeping ducts and lumens, such as the
mammary ducts, open. Here it binds also to L-selectin
which is expressed on the surface of leucocytes. By this
action leucocytes are bound to the lumen and excreted
into the milk (Welply et al. 1994). It also escapes digestion
and is excreted in the stools of breast-fed infants. In the
colon it binds to micro-organisms, particularly to the
fimbrins of E. coli, contributing to the host defence of
the breast-fed infant (Cravioto et al. 1991; Schroten et al.
6.6. Probiotic substances in milk or milk substitutes
There are indications that certain ingested micro-organisms
added to milk or milk products may exert some physiological effects and promote health in human infants. Such an
example is the use of Lactobacillus GG added to the milk
formula for premature infants. After administration of these
bacteria to premature infants it was noted that the bowel was
colonized by Lactobacillus GG, and no clinical side-effects
were seen. However, no clinical benefit was noted either
(Millar et al. 1993). As far as potential effects on fermentation are concerned, Lactobacillus GG given to premature
infants had no effect on production of short-chain fatty acids
in stools. The observed small increase in ethanol excretion is
unlikely to have any clinical significance (Stansbridge et al.
1993). Whether or not pre- and probiotics modulate gut
maturation and have relevant health benefits in infancy
remains to be elucidated.
6.7. Dietary regulation of xenobiotic metabolism
The nutritional status or specific nutrients may influence the
metabolic capacity of the liver, but also of other organs
(intestinal mucosa). The exact mechanism of drug–nutrient
interaction remains unknown. Moreover, the health effects
of such interactions have to be explored.
Diet could modulate the metabolism either through an
action on substrate availability or by modifying key
enzymes of metabolism. The influence of starvation on
conjugation and glucuronidation illustrates this point
(Mandl et al. 1995).
One important discovery of recent years is that some
components of food (antioxidants like butylated hydroxytoluene) are able to modulate the activity of key enzymes of
phase 1 or 2 metabolism, by modifying gene expression
(Kashfi et al. 1994). This is a promising area that requires
further work. In fact the modulation of gene expression by
nutrients is well described in the context of the influence of
food on carbohydrate or lipid metabolism but the effect of
macro- or micronutrients on xenobiotic-metabolizing
enzymes (XME) remains unelucidated.
Diet may also influence XME by inducing specific
pathology e.g. steatosis. The part played by nutrients and
morphological alteration in the modification of XME has to
be established (Leclercq et al. 1996).
Specific components of food (n-3 PUFA), even at very
low concentrations (piperine from black pepper, naringenine from grapefruit) may modify the activity of specific
isoforms of XME (e.g. glucuronosyltransferases) (Speck et
al. 1991). This could lead to interesting developments in
several fields. (1) Fundamental research: it will help in
studying this metabolic reaction in detail by using those
nutrients as activators or specific inhibitors. (2) Those
nutrients which can be used at low dose could, thus, be
considered as �toxico-modulators’. (3) Such compounds
have been proposed as therapeutic adjuvants, allowing
reduction of the dose of expensive drugs, or drugs with a
low safety-therapeutic index. The discovery of new nutrients with new targets could constitute a very promising area.
Finally, the validation of experimental models (and particularly in vitro models) allowing the study of drug–nutrient
interactions would give a new input in this area.
7. Development of the immune system
7.1. Introduction
Positive effects of particular foods or food ingredients on
the human immune system (e.g. inhibition of CHD and
cancer development etc.) could conceivably be related to
early nutritional events or may only be seen after decades of
intake or lifestyle changes. An important unresolved issue is
whether there are critical time periods during which a
provision may be especially beneficial to the immune
system. In view of these difficulties, in vitro surrogate
markers for study end-points are frequently used. A surrogate end-point can be defined as a laboratory measurement
or a physical sign used as a substitute for a meaningful endpoint that measures directly how a patient feels, functions or
survives. Changes to a surrogate end-point induced by a
dietary intervention are expected to reflect changes in a
B. Koletzko et al.
biologically meaningful end-point. However, surrogate endpoints do not always reflect the true clinical outcome and
can be misleading or even meaningless (Fleming & DeMets,
1996). Other important limitations of studies reporting
effects on the development of immunity are:
short duration;
lack of standardized tests;
lack of correlation of in vitro and/or in vivo findings with
immune protection or immune suppression;
lack of demonstration of health-enhancing effects in a
developing normal, presumably non-deficient population
of infants, but with changing nutritional requirements.
7.1.1. Which constituents of the immune system to
investigate? The immune system is a highly complex
regulatory cellular and humoral system of protection and
stimulation directed to avoid danger to the host. On this
basis a possible role of diet in cancer prevention could be
taken as summary evidence for beneficial effects of the diet
on the immune system. This extrapolation, however, seems
highly conjectural since it would argue that a number of
cancers are due to deficient immune surveillance mechanisms. This may or may not be the case and a number of other
protective mechanisms could be equally plausible.
The present report focuses separately on published evidence mostly in infants and children and, where appropriate,
in normal individuals and animals. Parenteral micronutrient
supplementation in disease states or after surgery, low level
toxicity and multiple chemical sensitivities will not be
7.1.2. Special considerations for the immune system of the
developing child. There is a dearth of reliable information of
the effects of vitamins, saturated and unsaturated fatty acids,
trace minerals and other normal food constituents on the
infant’s developing immune system. The majority of reports
examine the effects of corrections of severe or moderate
deficiencies on the immune responses in infants and
children (or animals). Very little is known of the effects
of supplementations, either in line with recommendations or
above, in a non-deficient population. As indicated earlier,
most measurements have been related to surrogate endpoints and the relationships between administration of test
substances and their effects on the immune system are not
strongly causal.
7.2. Antioxidants and vitamins
7.2.1. In general. Antioxidant vitamins generally enhance
different aspects of cellular and non-cellular immunity. The
antioxidant function of these micronutrients could, at least
in part, enhance immunity by maintaining the functional and
structural integrity of important immune cells. Multiple
effects attributed to antioxidants include risk reduction of a
variety of chronic diseases (Messina & Messina, 1996),
anti-(retro)-viral activity (Formica & Regelson, 1995),
immune enhancement in animals (Forni et al. 1986;
Gebhard et al. 1990, ), in man (Chavance et al. 1989; Penn
et al. 1991; Rall & Meydani, 1993), and evidence that certain
vitamins alone or in combination and other micronutrients
given in levels above the current recommendations have
critical, beneficial effects on human immune responses
(Bendich, 1995).
7.2.2. Vitamin A. The effects of vitamin A supplementation on measles in deficient and non-deficient children
have been the subject of several recent reports (Coutsoudis
et al. 1992, 1995; Rosales & Kjolhede, 1994; Semba et al.
1995; Stabell et al. 1995; Keusch, 1996). In general, in the
last decade epidemiological, immunological, and molecular
studies have yielded substantial evidence for a central role.
The recent discovery of retinoic acid and retinoid X
receptors has provided a molecular basis for the action of
vitamin A and its metabolites at the level of gene activation.
b-Carotene supplementation enhances the expression of
functionally associated molecules on human monocytes
(Hughes et al. 1996) and also enhances immune responses
to poor immunogens, which may be relevant to infants
receiving vaccines which are characterized by low seroconversion rates (Semba, 1996), however under certain
conditions seroconversion rates may be negatively affected
too (Semba et al. 1995). No difference was found (using
whole-blood culture techniques) in the in vitro proliferative
responsiveness of T-cells to concanavalin-A and tetanus
toxoid of children with normal or low–normal concentrations of vitamin A or Zn (Kramer, 1996).
7.2.3. Vitamin C. Vitamin C has gained great scientific
and media attention through the promotions of its effects on
the common cold. Early reports of systemic conditioning of
infants following an increased intake of vitamin C during
development seem to have been unfounded (Gerster &
Moser, 1988). Studies which failed to identify a positive
effect on the symptoms of the common cold have recently
been critically reviewed and it now seems likely that there is
a reduction in clinical symptoms associated with intake of
2–3 g ascorbic acid/d at the onset of the cold (Hemila,
7.2.4. Vitamin B complex. Few studies have addressed
the effects of supplementation with individual B vitamins
and/or the coenzyme CQ10. When studied, positive
enhancing effects on cell-mediated immunity, CD4 : CD8
ratios, delayed type hypersensitivity and antibody production have been reported (Gebhard et al. 1990; Miller &
Kerkvliet, 1990; Folkers et al. 1993). Tumour inhibition by
high dietary pyridoxine may be mediated by immunological
mechanisms that are lacking in the genetically immunodeficient (athymic) mice in which these studies have been
carried out (Gebhard et al. 1990).
7.2.5. Vitamin E. Vitamin E, in its role as a potent
antioxidant and immunostimulant, has received a great deal
of attention (Tengerdy, 1990; Shor Posner et al. 1995; Liang
et al. 1995; Finch & Turner, 1996; Liang & Watson, 1996).
Vitamin E supplementation enhances humoral and cellmediated immunity, and augments the efficiency of
phagocytosis in laboratory animals and human subjects.
Vitamin E deficiency has been suggested to contribute to
alterations of the neonatal neutrophil function, and supplementation with 120 mg/kg over the first 14 d of life of
healthy premature infants has been shown to increase
phagocytosis (Chirico et al. 1983).
7.2.6. Vitamin D. There is now increasing evidence that
the hormonal form of vitamin D, 1,25-dihydroxycholecalciferol (1,25(OH) 2D 3), is involved in the regulation of the
Growth, development and differentiation
immune system. 1,25(OH) 2D 3 exerts most of its actions
after it has bound to its specific receptors which are present
in monocytes and activated lymphocytes. The hormone
inhibits lymphocyte proliferation and immunoglobulin
production in a dose-dependent fashion. It interferes with
T-helper cell function, reducing T-helper cell-induction of
immunoglobulin production by B-cells and inhibits the
passive transfer of cellular immunity by T-helper cells in
vivo. Expression of Class II antigen by lymphocytes and
monocytes is also affected. In experimental in vivo studies
1,25(OH) 2D 3 is particularly effective in preventing autoimmune diseases (Schwartz, 1992; Mathieu et al. 1994;
Thomasset, 1994). There is no information on the effects of
vitamin D or its metabolites on the developing human
immune system.
7.3. Multiple micronutrient supplementation studies
Trace elements perform important functions in growth and
development. However, little information exists about dietary requirements of them during the demanding period of
infancy. Although several factors influence the dietary
needs of these essential elements, the basis for establishing
dietary needs in infants is hindered by the dearth of studies
that have assessed their bioavailability and effects on the
immunity in this age group. Thus, until it has been conclusively shown otherwise, the physiological response to
human milk is used as the standard for infant feeding
practices (Milner, 1990).
Key questions such as the risks to human health of altered
environmental distribution of Zn, assessment of Zn status in
man, effects of Zn status in relation to other essential metals
on immune function, reproduction, neurological function
and others remain (Sherman, 1992; Walsh et al. 1994;
Prasad, 1995; Ripa & Ripa, 1995; Sazawal et al. 1996).
Zn supplementation has recently been shown to reduce
persistent diarrhoea in children (Sazawal et al. 1996).
In vitro and in vivo studies show that antioxidants generally enhance different aspects of cellular and non-cellular
immunity. The antioxidant function of these micronutrients
could, at least in part, enhance immunity by maintaining the
functional and structural integrity of important immune
cells (Chew, 1995).
Effects of trace minerals on the outcome of pregnancy
have been reviewed. About 30 % of pregnant women suffer
from Fe deficiency worldwide, and while its effects on
neonatal Fe status are not severe, adverse sequelae include
impaired neonatal immune status (Allen, 1986; for review,
see Bryan & Stone, 1993). Low maternal intakes of Cu, Mn,
and Se have not been associated with adverse outcomes of
pregnancy. Se deficiency, however, appears to result in
immunosuppression affecting neutrophil function, antibody
production, cytotoxicity and lymphocyte proliferation
(Kiremidjian Schumacher & Stotzky, 1987).
7.4. Fatty acids
Several lines of evidence support the role of dietary lipids as
regulators of the immune system. This is demonstrated by
studies examining lipid alteration of the immune response to
allergens, malignancy, autoimmune disease, sepsis, trauma,
and transplantation. Both the quantity and quality of lipid
are important in immunoregulation. Both cell-mediated and
humoral immunity are affected by dietary lipids. Multiple
mechanisms probably contribute to the overall effects of
lipids, including alteration of arachidonic acid metabolism,
changes in cell membranes, production of inflammatory
cytokines, and impairment of the reticuloendothelial
system (Perez & Alexander, 1988; Yetiv, 1988; Fernandes
et al. 1990; Melnik et al. 1991; Watanabe et al. 1994;
Endres, 1996; Hellerstein et al. 1996).
7.5. Arginine
Many of the known roles of arginine (e.g. in immune
function, wound healing, and protection against NH 3
intoxication) are mediated by a metabolic pathway synthesizing NO in the liver. Both stimulatory and suppressive
functions have been identified with a prominent role of the
macrophage (Barbul, 1990; Rodeberg et al. 1995; Suzuki et
al. 1995; Krenger et al. 1996; Marcinkiewicz et al. 1996).
Particular effects on the developing human immune system
are uncertain. Oral administration may be less effective than
parenteral administration (Torre et al. 1993). A number of
reports investigate the potential benefits of arginine supplementation during surgical and other periods of stress in
human subjects and in experimental models. Beneficial
effects on wound healing, reduction of postoperative septicaemias etc. cannot always be attributed entirely to arginine,
since it was often given with other dietary modulations
(Daly et al. 1990; Cerra et al. 1991; Seidman et al. 1991;
Kemen et al. 1995; Senkal et al. 1995; Braga et al. 1996;
Kudsk et al. 1996; Marcinkiewicz et al. 1996).
7.6. Nucleotides
Dietary sources of preformed purines and pyrimidines seem
to be important for optimal function of the cellular immune
response. It was previously assumed that nucleotides were
not needed for normal growth and development, but the
results described in the present review demonstrate a need
for nucleotides in the response to immunological challenges. The need for sources of preformed nucleotides in
defined formulas such as parenteral and enteral formulas
and infant formulas is suggested in some studies (Carver,
1994; Kulkarni et al. 1994; Rudolph, 1994). An exogenous
source of nucleotides from the diet may optimize the
function of rapidly dividing tissues when growth is rapid
and the diet is low in nucleotides. Studies performed in
human infants are, at most, inconclusive (Carver, 1994;
Kulkarni et al. 1994) and further studies are required to
assess in infants the interesting findings about dietary
nucleotides reported in experimental models (Gil & Uauy,
1989; Sanchez Pozo et al. 1994, 1995; Ortega et al. 1995;
Lopez-Navarro et al. 1996; Navarro et al. 1996).
7.7. Maturation of the immune system in formula-fed v.
breast-fed infants
In a small study of systemic and secretory immunity of
breast-milk-fed v. formula-fed infants it was shown that
B. Koletzko et al.
there is a general stimulation of responsiveness by cytokines in milk and a reduction of specific responses by
antigen exclusion. Bottle-fed infants reached a similar level
of immunological maturity by 3 months of age, exhibiting
raised levels of serum antibodies against gut organisms and
milk proteins and demonstrating increased non-specific
activation of lymphoid cells (Stephens, 1986; Stephens et
al. 1986a,b)
7.7.1. Effects of antigen transfer via breast milk on the
infant’s immunity. Dietary antigen excretion into breast
milk seems to be a general phenomenon and has been
reported for milk, egg, wheat proteins and parasite antigens
(Kilshaw & Cant, 1984; Troncone et al. 1987; Petralanda et
al. 1988). Excreted amounts are in the range of вђ®g/l. The
immunological significance of transfer of dietary antigens
during breast-feeding is still unclear. It is generally accepted
that breast-feeding reduces the risk of food allergic reactions
and also of atopy in a population at risk (uni- or biparental
history of atopy) but sensitizing effects in infants have also
been described (Warner, 1980; Gerrard & Shenassa, 1983;
Savilahti et al. 1987; Lindfors & Enocksson, 1988). Studies
by Chandra et al. (1986, 1989a), Zeiger et al. (1992) and
others (Halken et al. 1993a; Vandenplas et al. 1995) suggest
that elimination of (significant) dietary antigen transfer via
breast milk for 6 months (amongst other preventive
measures) in a population at risk reduces the probability
of a food-specific sensitization (and possibly atopic
symptoms) for up to 48 months, an effect which persists
even after the diet of the infant has been liberalized. The
following points merit special consideration.
7.7.2. Maternal diet during pregnancy and effects on the
infant’s immunity. There are no reports which demonstrate
any preventive (or sensitization) effects in infants with a
parental history of atopy (FaВЁlth-Magnusson et al. 1987; Lilja et
al. 1989; FaВЁlth-Magnusson & Kjellman, 1992).
7.7.3. Maternal diet during pregnancy and lactation. In
view of the complexity of the studies and the number of
confounding variables, it is not entirely surprising that a
number of studies have come to different conclusions.
Maternal diet, while continuing breast-feeding, has been
shown to be of moderate benefit in the reduction of atopic
manifestations (mainly eczema) and milk allergy in infants
or children (Chandra et al. 1989a,b; Sigurs et al. 1992;
Zeiger et al. 1992; Zeiger & Heller, 1995). Other studies
have failed to show this effect (Lilja et al. 1989).
7.8. Role of the gut flora and probiotic bacteria in the
infant’s immunity and gut defence
Little is known about the immunomodulating capacity of
the first bacteria colonizing the gut. Breast-fed infants
develop a typical intestinal flora and this has been linked
to a certain resistance to enteric infections. However, breast
milk contains a host of other immunomodulating factors and
it is difficult to claim any causality in these studies. Infants
given breast-milk substitutes with various strains of lactic
acid-producing bacteria may also exhibit some resistance to
infections. In a small clinical study of thirty-nine children,
protective effects of the supplementation of an infant formula with oligosaccharides, fermented milk or lactic acidproducing bacteria on the reduction of the incidence of acute
diarrhoea were reported. A randomized controlled feeding
trial with Bifidobacterium breve in ninety-one very-lowbirth-weight infants demonstrated effective colonization,
fewer abdominal signs and better weight gain (Kitajima et
al. 1997). An enhancement of the circulating antibodysecreting cell response was observed in infants with rotavirus diarrhoea supplemented with a strain of Lactobacillus
casei, compared with a placebo group (Kaila et al. 1992).
The duration of this response and other protective or longerterm effects are unknown. Other small studies reported an
enhancement in the phagocytic activity of granulocyte
populations in the blood of human volunteers after consumption of fermented milk with Lactobacillus acidophilus
and B. bifidum (Schiffrin et al. 1995). It is unresolved
whether studies in a larger number of unselected infants
under different conditions in different countries would yield
similar encouraging results.
The gut microflora is an important constituent in the
intestine’s defence barrier. Probiotic bacteria have been
suggested to affect different aspects of gut defence:
immune exclusion, immune elimination and immune
7.8.1. Immune exclusion and elimination. Although
many clinical benefits have been ascribed to consumption of
candidate probiotic strains in gastrointestinal disease
(Isolauri et al. 1991; Saavedra et al. 1994), only a few
human studies have assessed the effects of these bacteria on
gut defence mechanisms. Early reports associated clinical
observations with the effects on the intestinal microflora
(Niv et al. 1963). Oral bacteriotherapy affected microbial
imbalances shown during rotavirus infections in infants
(Isolauri et al. 1994). Oral introduction of probiotic microorganisms has been associated specifically with reduction of
intestinal inflammation (Majamaa & Isolauri, 1997) and an
increase in circulating antibody-secreting cells in the serum
as an indicator of the intestine’s immunological barrier
function (Kaila et al. 1992). In children with rotavirus
diarrhoea, probiotic bacteria administered during the
diarrhoeal phase of the infection promoted clinical recovery
and enhanced intestinal immunoglobulin A (IgA) responses
(Kaila et al. 1992).
7.9. Effects of formulas with protein hydrolysates on the
infant’s immune responses
Extensively hydrolysed casein formula has been used in the
treatment of children with cow’s milk protein allergy and/or
intolerance. Recently, ultrafiltrated whey hydrolysates have
been also been used therapeutically (Halken et al. 1993b). In
an attempt to prevent and/or modulate the risk of developing
food-allergic and atopic manifestations in infants and children, less extensive (partial whey hydrolysates) (Chandra,
1991; Vandenplas et al. 1992, 1995) and extensively hydrolysed formulas (Chandra et al. 1989a; Zeiger et al. 1989;
Halken et al. 1993a; Zeiger & Heller, 1995; Oldaeus et al.
1997) have been used. Although these studies have been
performed in infants of different (atopic) family and ethnic
background, with different nutritional habits and different
measures of control for confounding variables and a lack
of standardized diagnostic protocols, they could be summarized as follows. (1) Infants with a high-risk family
Growth, development and differentiation
background of atopic disease are likely to benefit from
exclusive breast-feeding for 4–6 months with some added
benefit if the mother avoids certain foods such as milk, eggs,
fish and possibly nuts (including peanuts) during lactation.
The benefits include a reduction in the incidence of cow’s
milk and food allergies and atopic eczema for up to 4 years.
(2) If exclusive breast-feeding for 4–6 months cannot be
sustained, the use of a hydrolysed infant formula may help
reduce the overall incidence of atopic manifestations in the
child at risk. (3) Preventive effects of hydrolysed formulas
in infants with a normal risk of developing atopic manifestations and the respective benefits of extensively v. less
extensively hydrolysed infant formulas need to be further
7.10. Insulin-dependent type 1 diabetes mellitus and cow’s
milk exposure in infancy
Insulin-dependent diabetes mellitus (IDDM) is considered
to be a chronic autoimmune disease characterized by
gradual b-cell destruction mediated by autoreactive T-lymphocytes during an asymptomatic prediabetic phase of
varying duration (Knip, 1992). In a Finnish study, associations of infant feeding patterns and milk consumption with
cow’s milk protein antibody titres were studied in newlydiagnosed diabetic children, sibling-control children and
birth-date- and sex-matched population-based control
children. Inverse correlations were observed between the
duration of breast-feeding, or age at introduction of dairy
products, and antibody titres. High IgA antibody titres to
cow’s-milk formula were associated with a greater risk of
IDDM both among diabetic-population-control and diabetic-sibling-control pairs. The results suggested that
young age at introduction of dairy products and high milk
consumption during childhood increase the levels of cow’s
milk antibodies and that high IgA antibodies to cow’s milk
formula are independently associated with increased risk of
IDDM (Vaarala et al. 1996). Similar associations between
bovine serum albumin antibodies and onset of IDDM have
also been found in a low-incidence French population (Levy
Marchal et al. 1995). Human T-lymphocyte cultures of cells
taken from affected children allowed the detection of bovine
serum albumin-specific T-cells which were mapped to the
ABBOS peptide (pre-bovine serum albumin position
152–169) previously identified as a possible immunological
mimicry epitope which could explain the cross-reactivity
with pancreatic islet cell antigens (Cheung et al. 1994). A
currently ongoing prospective dietary intervention trial in
children genetically at risk will be able to address the
causality of this highly intriguing association and, it is
hoped, open the way for a primary nutritional prevention
8. Bone growth and mineralization
8.1. Cell biology of bone growth
Bone growth and mineralization is an ongoing process
during human fetal and postnatal development, stabilizing
at about 21 years of age. Skeletal Ca content increases from
30 g in the neonate to 1200 g in the adult, and skeletal P
from 17 to 700 g. Bone tissue possesses a series of enzymic
mechanisms that permit mineralization of its extracellular
matrix. This matrix is composed of collagen, proteoglycans
and other non-collagen proteins in which insoluble mineral
salts of hydroxyapatite and small amounts of other salts of
Mg, sodium carbonate and citrate are deposited, converting
it into a structure capable of supporting the organism. The
two most important bone cell types are osteoblasts and
osteoclasts. Osteoblasts are responsible for the formation
and organization of the extracellular matrix and its subsequent mineralization. Osteoclasts are large motile multinucleated cells, located on bone surfaces, responsible for the
resorption of bone matrix. Bone growth or bone modelling
is the result of two processes: first formation of new bone
and then resorption to maintain the same structural form
with, as a net result, acquisition of bone mass. At the age of
about 18 years, both male and female adolescents have
reached 95–99 % of their individual peak bone mass. After
adolescence the processes of bone resorption and formation
become quantitatively in balance and this situation is
referred to as bone remodelling. After 35–40 years of age
the processes of bone resorption and formation become
uncoupled and net bone loss will occur, eventually leading
to osteoporosis (Price et al. 1994; Anderson, 1996a,b). The
obvious strategies to prevent, or at least delay, the onset of
osteoporosis include: (a) optimizing the attainment of peak
bone mass in adolescents, and (b) preventing bone loss in
later life.
8.2. Methodological aspects in bone-mass-related studies
In the interpretation of the bone-mass-related results of the
various studies one needs to be aware of the actual technique used. The first non-invasive methods for measuring
bone mass were based on quantitative evaluation of standard
radiographs. During the last decade gamma- or X-ray
techniques were developed based on the variable effect of
matter on the passage of radiation. Most studies were
performed using either single photon absorptiometry
(SPA) or dual-energy X-ray absorptiometry (DEXA). SPA
is a relatively simple technique using 125I as the radioactive
source. Its application is limited to the peripheral skeleton,
particularly the radius. DEXA is becoming more and more
the preferred technique to assess bone density at various
sites of the skeleton with only minimal radiation exposure.
Although calibration of DEXA instruments seems to be a
rather trivial exercise, it relies strongly on highly specific
software, with the results that measurements on one patient
made with instruments of different brands do not necessarily
result in similar bone mineral density data (Slosman et al.
1995). An additional feature of DEXA is its ability to
measure whole-body mineral content as well as body
composition. Appropriate DEXA software for infants has
been developed recently and reference values from the first
studies are now beginning to become available in the
literature (Rigo et al. 1996). The fact that SPA and DEXA
data cannot be compared with each other is illustrated by the
fact that SPA produces a measurement of bone mineral
content (g/cm) and assumes that the site of measurement is a
small cylinder of constant width, whereas DEXA produces a
measurement of bone mineral density (g/cm2) by correcting
B. Koletzko et al.
Table 1. Additional increment in bone mineral density (BMD) (as a percentage) following supplementation of the diet with calcium or dairy
products, in children and adolescents from five different studies (From Kerstetter, 1995)
Johnston et al. (1992)*
Subject no., total
Entry age (years)
Intervention duration (months)
Baseline Ca intake (mg/d)
Total Ca intake (mg/d)
(diet Гѕ supplement)
Supplemental Ca source
BMD determination
Change in BMD‡
Midshaft radius
Distal radius
Lumbar spine
Femoral neck
Ward’s triangle
Greater trochanter
Total body
22 twin pairs
Boys and
6.9 (SD 1.4)
Lloyd et al. (1993) Lee et al. (1994)
11.9 (SD 0.5)
Boys and
7.2 (SD 0.2)
11.4 (SD 0.8)
11.1 (SD 1.0)
1315 1618
Ca citrate malate
Ca citrate malate
CaCO 3
Ca citrate malate
Dairy foods
23 twin pairs
Boys and
10.6 (SD 2.0)
Andon et al. 1994)†Chan et al. (1995)
DEXA, dual-energy X-ray absorptiometry; SPA, single photon absorptiometry.
‫ ء‬Two age groups were studied: prepubescent (6.9 (SD1.4) years) and pubescent (10.6 (SD2.0) years).
†Two levels of dietary Ca (1315 and 1618 mg/d) were studied.
‡ Change in BMD = percentage increase in supplemented group minus increase in unsupplemented group.
the bone mineral content for the projected area of bone
(Slosman et al. 1995). A shortcoming of the usual expression of bone mineral density obtained by DEXA (g/cm2) is
that this areal bone mineral density does not take the agerelated increase in bone thickness into account. Therefore
Cowell et al. (1995) have developed a measure of true bone
mineral density, volumetric bone mineral density (g/cm3), and
demonstrated its usefulness in assesssing patients with phenylketonurea (PKU), chronic renal failure and chronic asthma.
8.3. Peak bone mass and relative risk of osteoporosis
The relative importance of peak bone mass on the subsequent risk of osteoporosis has recently been reviewed by
Ribot et al. (1995). They started with the available in vitro
evidence on the relationship between low bone mass and the
risk of osteoporosis relating to the mechanical properties of
bone. The relevant in vivo studies include both crosssectional surveys and at least eleven prospective studies.
From these studies it can be deduced that the relative risk of
osteoporosis for each 1 SD decrease in bone mineral density
is increased by a factor of between 1.7 and 2.7. On
comparing this value with the relative risk of CHD for a 1
SD rise in serum cholesterol or that of stroke for 1 SD
increase in blood pressure being 2.1 and 1.3 respectively,
it is clear that low peak bone mass is a very strong risk factor
for later osteoporosis. Another appealing value relating to
the relevance of optimizing or increasing peak bone mass
can be deduced from the study by Gilsanz et al. (1991) on
comparing the development in peak bone mass in white and
black girls. They found a 10–20 % higher bone density in
black girls relative to white girls which is likely to correspond to an additional 10–20 years of protection against the
decline in skeletal mass, and might explain the relatively
low prevalence of osteoporosis in black women. About 80 %
of the variance in bone mineral density is accounted for by
genetic factors (Pocock et al. 1987), thus leaving only 20 %
of the variance to be influenced by environmental factors
such as diet. For this reason the investigation of twin pairs is
very attractive because the genetic bias may thus be minimized (Johnston et al. 1992). From a study with postmenopausal twins in Britain, Spencer et al. (1995) reported a
genetic linkage between the vitamin D receptor genotypes
and bone mineral density. The degree to which this might
explain the genetic factors is as yet unclear and a recent study
from Denmark failed to find any significant association
between common allelic variations at the vitamin D receptor
locus and bone mineral density (JГёrgensen et al. 1996).
8.4. Bone growth and mineralization in infants and young
The literature on the effects of different diets during infancy
on bone mineral content at 2 or 5 years of age is not yet
consistent. Exclusive feeding of breast milk during the first
6 months of life supports a bone growth considered adequate
even though breast milk contents of Ca and vitamin D are
relatively low. Infant formulas in general, and formulas for
premature infants in particular, contain higher levels of
these nutrients to meet the dietary requirements and to
provide a safety margin to correct for a likely lower
bioavailability. Surprisingly, Bishop et al. (1995) reported
a strong positive association between the amount of human
milk consumed and bone mineral content at the age of 5
years in their multi-centre cohort of prematurely born
infants. The authors raised two possible hypotheses to
interpret their finding. Bone mineral depletion in preterm
infants fed on unsupplemented human milk might
�programme’ these infants to be conservative with bone
mineral and to reduce the overall rate of growth so that
Growth, development and differentiation
�over-mineralization’ occurs at a later stage when the intake
of bone mineral substrates is normal. A second possibility is
that one or more of the human milk growth factors might
survive breast milk pasteurization and the immature digestion system and end up via the circulation at the target
organ. Until one of these hypothetical mechanisms is further
substantiated, the general goal in the nutrition of premature
infants remains to provide enough mineral supplementation
to allow attainment of bone mineral content comparable to
that accrued in utero and to support catch-up growth in the
first year of life. Several studies comparing bone mineralization in term-born breast-fed infants with that in infants
fed on formulas containing moderate or high Ca content
conclude that the bone mineral content of formula-fed
infants is higher at the ages of 2 and 5 years (Demirini &
Tsang, 1995). Whether this effect of cumulative Ca intake
during the first 2 years of life will be retained until
adolescence is still unclear. A negative impact on bone
growth and mineralization has been reported for a number
of chronic conditions during infancy and early childhood
like cystic fibrosis, IDDM, cerebral palsy, leukaemia, renal
disease, growth hormone deficiency and anorexia nervosa
(Shaw & Bishop, 1995). In a group of fifty-five children
with milk allergy showing a broad distribution of daily Ca
intake (quartiles: 409, 663, 950 and 1437 mg Ca/d), a clear
correlation was found between Ca intake and bone mineral
density, thus underlining the vulnerability of this group and
illustrating the efficacy of dietary measures or supplementation of Ca to achieve normal bone growth (Henderson
& Hayes, 1994).
8.5. Calcium supplementation in children and adolescents
and bone health
Despite the relatively poor contribution diet is supposed to
make to the variance of peak bone density, a substantial
number of studies have been published in recent years to
address the effects of nutrition, particularly of Ca, on bone
density in children and adolescents. These studies have
been reviewed by Kerstetter (1995) who concluded that
the cross-sectional and correlation studies have yielded
rather mixed results. Surprisingly, more consistent findings
were obtained from the five recent prospective Ca or dairy
supplementation studies in children and adolescents published between 1992 and 1995. In Table 1 the basic data
from these studies (Johnston et al. 1992; Lloyd et al. 1993;
Andon et al. 1994; Lee et al. 1994; Chan et al. 1995) are
compared. In all studies the baseline Ca intake was less than
1000 mg/d and the amount of supplemented Ca ranged from
300 to 700 mg/d. The percentage increase in bone mineral
density in all the supplemented groups amounted to 1–10 %
and was significant in all studies. Of those intervention
studies, the one by Chan et al. (1995) is most appealing
because in this study a near doubling of the Ca intake (from
728 to 1437 mg/d) was reached only by supplementation
with dairy products and the increase in total body bone
mineral density amounted to 7 %. However, the conclusion
which one might draw on superficial reading of the review
by Kerstetter (1995) that dairy products outperform other Ca
supplements cannot be substantiated yet. Nevertheless, it is
remarkable that in the study by Chan et al. (1995) the
supplemented dairy products also contained some vitamin D
and extra P, which can at least be interpreted as showing that
P has no negative effect on peak bone mass accretion in
adolescents under these conditions. In conclusion, the
results from the Ca and dairy-product supplementation
studies summarized have clearly demonstrate that it is
possible to increase peak bone mass at the end of adolescence simply by dietary means.
8.6. Nutrients other than calcium and environmental factors
involved in bone growth
The crystal salt of bone resembles hydroxyapatite
(Ca10(PO4)6(OH)2) which contains Ca and P in the proportion 2.15:1 (w/w); in addition approximately 60 % of the
body Mg and 30 % of the body Zn are present in the
skeleton. It is, therefore, obvious that P, Mg and Zn are
also important nutrients in the process of bone mineralization. Although there is little information about conditions in
developed countries where Mg or Zn would represent single
limiting factors causing impaired bone growth, it seems
reasonable and prudent to include these elements proportionally in supplements, possibly also taking their different
rates of absorption into account. The case for P is more
delicate and, except for premature infants where P intake
can be a limiting factor for bone growth, much more
concern has been shown about excessive dietary intakes of
P (Calvo & Park, 1996). High P intake results in an
increased serum P concentration which initiates several
hormonal respones, of which an increase in parathyroid
hormone level effects the balance between bone mineralization and bone resorption (Anderson, 1996a,b). It is for this
reason that in several supplementation studies no phosphate
was given in order to shift the Ca : P ratio as much as
possible in the direction of Ca. As already mentioned, the
results from the study by Chan et al. (1995) give support to a
concept where the absolute amount of Ca is more important
than the Ca : P ratio.
A nutrient which is most important in bone metabolism is
vitamin D. The major role of dietary vitamin D is to function
as precursor for 25-hydroxy- and 1,25-dihydroxycholecalciferol which maintain the plasma Ca concentration within
very narrow limits. This is accomplished by varying the
proportion of dietary Ca absorbed and excreted. As the body
becomes vitamin D depleted, the efficiency of Ca absorption
decreases from 30–50 % to no more than 15 %. In addition,
1,25-dihydroxycholecalciferol has a direct effect on osteoblast production and thus on bone formation and mineralization (Anderson, 1996a,b). The major source for vitamin
D in human subjects is exposure to sunlight which enables
cutaneous synthesis of vitamin D. However, several groups
ranging from premature infants to institutionalized elderly
people may not be able to receive sufficient exposure to
sunlight and thus require dietary vitamin D.
The involvement of vitamin K in bone metabolism is
through its action on maturation of osteoblastic bone proteins by carboxylation of their glutamate residues (Vermeer
et al. 1996). Vitamin K may be generated by the intestinal
microflora or obtained from dietary sources such as green
vegetables and meat. Except for the fact that it depends on
the quantity of fluoride ingested and the time of exposure,
B. Koletzko et al.
the role of F in bone health is still poorly understood. It has
been used as a therapeutic agent in bone pathology including osteoporosis, but also cases of skeletal fluorosis are
reported with radiologically demonstrable abnormal bone
densification (Boivin et al. 1993). Of the minerals for which
a role and/or essentiality in man is still unclear, B has been
connected with the mechanical properties of bone
(Mastrmatteo & Sullivan, 1994). Because the effects of B
supplementation are not striking, and plausible mechanistic
explanations still need to be presented, it is uncertain
whether B will become an important issue in bone health
research. In a cross-sectional study in about 500 children
and adolescents (aged 8–17 years) bone mineral density was
found to be related to diet, weight-bearing exercise and
daylight hours spent outdoors (Gunnes & Lehman, 1995).
Next to an effect in accordance with the literature with
respect to dietary Ca it is surprising that the authors found a
positive correlation between bone mineral density and
saturated fat, fibre and vitamin C. Any interpretation of
these correlations can only be speculative. The positive
association between fat intake and bone mineral density
might be attributed to an increased intake of vitamin D with
the fat or might possibly be mediated by a higher cholesterol
level. The positive association between bone mineral density and fibre intake is even more puzzling because one
might expect exactly the opposite based on impairment of
Ca absorption by dietary fibre. As a possible explanation for
the association between vitamin C and bone mineral density, the authors indicate the fact that vitamin C is a cofactor
in collagen synthesis. Moreover, they point to a possible
link between high intakes of Ca, fibre and vitamin C as
constituents of a more wholesome diet. Considering the
importance of a high Ca intake in achieving maximal bone
growth and peak bone mass, it seems logical to prevent any
negative dietary interactions which might interfere with
maximal Ca absorption. Of the dietary interactions affecting
Ca absorption reviewed by Licata (1993), the possible
negative effect of caffeine is mentioned because it increases
urinary Ca excretion. However, this effect is considered to be
fairly unimportant relative to other factors and is also not
consistently found in all the studies. A clear negative effect
on Ca absorption is represented by dietary oxalate-exemplified by the poor Ca absorption (only 5 %) from oxalaterich spinach. Although some authors report a stimulating
effect of lactose on Ca absorption in humans, others fail to
find any significant effect. Essentially the same holds true
for the effect of protein on Ca absorption: if there is any
stimulating effect, it will be rather small. In view of the
marginal gain on fractional Ca absorption which can possibly
be obtained by optimizing the nutritional matrix, selecting Ca
salts with a high bioavailability may be of particular relevance. In this respect an increase in fractional Ca absorption
of 0.26 to 0.36 which can be achieved by replacing CaCO 3
with calcium citrate–malate is illustrative (Peacock, 1991).
9. Nutrient effects on development of neural functions
and behaviour
9.1. Introduction
Pregnancy and the first postnatal months are critical time
periods for the growth, development and differentiation of
the human nervous system. There is good evidence that
availability of nutrients during these critical time periods
affects brain growth and development and can have longterm programming effects on an individual’s central nervous functions. In contrast to some other mammalian
species, in man the peak growth rate of the brain (�brain
growth spurt’) occurs both pre- and postnatally, with continued relatively rapid growth well into the second year of
life. Between the 24th week of gestation and the time of
term birth, brain weight increases more than fivefold. The
disproportionate speed of brain growth, compared with total
body growth, is apparent from the fact that at age 2 years the
weight of the human brain has already reached 80 % of adult
weight, while whole body weight at this age is less than
18 % of adult weight. An adequate substrate supply is
essential for a physiological brain composition and differentiation during this rapid perinatal growth.
9.2. Physiology of neural development
Development of neuronal tissues is characterized by the
sequential occurrence of mitosis, cell migration, differentiation, synaptogenesis, apoptosis and synaptic reorganization.
These consecutive steps begin during pregnancy at different
gestational ages and occur over a certain time period in
different brain areas; therefore, there is an overlap in time of
the various brain development steps in different areas of the
brain (Reisbick, 1996).
Mitosis of germinal cells located along the neural tube
and the brain ventricles, which form neuronal and glia cells,
begins in the sixth embryonic week, peaks in the second
trimester and is almost complete by the end of the second
trimester, even though a small number of cells may form
during the last trimester of gestation (Jacobson, 1991;
Rakic, 1995). Under the influence of hormones, cell adhesion molecules and other local factors, the created cells
migrate to the brain nuclei and cortex and the ganglia of the
peripheral nervous system (Jacobson, 1991; Kandel et al.
1991). Although largely occurring during pregnancy, nervous cell migration particularly in the superficial layers of
the cerebral and cerebellar cortices continues to occur after
term birth (Rakic, 1995). Cell differentiation into particular
neurons may begin during migration but generally is only
completed at its final destination (Kandel et al. 1995). The
differentiation of neuronal cells is modulated by induction
through surrounding cells, the kind of cell innervated,
growth factors and hormones such as glucocorticoids, and
nutrient intakes. For example, pre- and postnatal protein–
energy malnutrition has marked effects on brain cell
differentiation (Cravioto & Cravioto, 1996), and long-term
depletion of the n-3 PUFA DHA induced altered brain cell
levels of monoamines and monoamine receptors in rats.
When neurons have migrated to their destination and
reached their differentiation, they grow and form synaptic
connections (Kandel et al. 1995). Most cell growth in the
human cerebellar cortex occurs between 32 weeks of
gestation and 11 months after term birth (Rakic, 1995).
Synaptogenesis in primates peaks in the first 2–3 months of
life (Rakic, 1995), and the number of synapses in the human
visual cortex increases twofold between the ages of 2 and 8
months after birth, which is paralleled by a marked increase
Growth, development and differentiation
in the number of neurotransmitter receptors (Huttenlocher et
al. 1982). The number of synapses formed is far greater than
the number of synapses later found in adult brains. The early
rapid synaptogenesis depends on an extensive formation of
highly fluid cell membranes and, hence, on sufficient availability of substrates required for this membrane formation.
Brain structure and function is also greatly influenced by
apoptosis, since cell death affects between 20 and 80 % of
the neuronal cells formed during gestation before the time of
birth (Rosenzweig et al. 1996), which appears to be
regulated by genetically programmed apoptosis, protecting
neurotrophic factors, synaptogenesis, hormones and
eicosanoids. In the surviving cells, the synaptic connections
are reduced and rearranged at a rapid rate initially, but
continuing throughout the life of the organism in relation to
stimulation and learning. This synaptic rearrangement
appears to be regulated by many different modulators,
including Ca channels, second messengers such as phosphatidyl inositol, protein kinases, membrane lipids and
eicosanoid hormones (Reisbick, 1996; Wainwright, 1997).
9.3. Nutrition and neural development
In Europe and other developed countries, an associated
secular increase both of adult height and adult intelligence
quotients has been observed over the last few decades, and
the hypothesis has been raised that both these effects are
related to the early nutrient supply.
Several studies have documented that severe general
malnutrition in infancy or early childhood, which is
characterized by a combined deficiency of energy, protein
and many other substrates, is associated with a marked
reduction of cognitive ability (Cravioto & Cravioto, 1996;
Kretchmer et al. 1996). However, the adverse effects on
neural development cannot be attributed entirely to nutrient
depletion, but may also be modulated by other factors
typically associated with severe childhood malnutrition,
including infections, poverty, psychological depression
and lack of adequate stimulation. Evidence of organic
effects of malnutrition on brain growth and maturation
was provided by the demonstration of cerebral atrophy
and lasting organic brain damage (Ambrosius, 1966; Stoch
et al. 1982; Houscham & Devilliers, 1987) and by electrophysiological evidence of impaired information processing,
such as altered auditory evoked brain stem potentials
(Barnett et al. 1978; Bartel et al. 1986).
9.3.1. Protein. Randomized studies in premature
infants demonstrated that a low protein intake during the
early postnatal period resulted in poorer results of orientation, habituation and stability clusters when tested with the
neonatal behaviour assessment scale (Bhatia et al. 1991),
and in a markedly reduced mental and psychomotor
development when assessed at an age of 18 months postterm with the Bayley scales of infant development (Morley
& Lucas, 1993).
9.3.2. Iodine. Today I deficiency is considered the
most common cause of nongenetic inborn neurological
damage on a worldwide basis, and causes cretinism with
severe mental retardation (Stanbury, 1994). In I-deficient
populations, an early I supply beginning before or at the
time of conception can prevent neural damage of the infant,
whereas a later start of I supply in the second or third
trimester of pregnancy or after birth is associated with a
smaller preventive effect (Xue-Yi et al. 1994).
9.3.3. Iron. Fe uptake into the brain is mediated by
transferrin receptors on the endothelial surface of brain
microvasculature, reaches its maximum during the period of
rapid brain growth and peak myelinogenesis (Taylor &
Morgan, 1990) and continues throughout life. Analysis of
Fe distribution in the human brain during childhood with
magnetic resonance imaging showed the highest concentrations in the globus pallidus, caudate nucleus, putamen and
substancia nigra, while the cortex and cerebellum had
substantially lower contents. Fe serves as an essential
cofactor in a variety of cellular and metabolic functions,
including the synthesis of dopamine, serotonin, catecholamines and possibly g-aminobutyric acid as well as myelin
formation (Kretchmer et al. 1996), while Fe overload has
toxic effects. In young rats deprived of Fe in early postnatal
life, total brain Fe content was severely depleted to 27 % of
that of controls and was resistant to later restoration despite
aggressive treatment (Dallman & Spirito, 1977). Sustained
early Fe deficiency in rats causes persistent behavioural and
learning deficits, leading to the hypothesis that Fe
sufficiency throughout the critical phases of early brain
development is crucial to the achievement of normal brain
Fe content, function and behaviour. Several studies in
children have clearly documented that severe Fe depletion
resulting in Fe-deficiency anaemia results in a poor attention
span, poor performance in the Bayley mental development
index, low intelligence scores, some degree of perceptual
disturbance and altered affective behaviour (Lozoff &
Brittenham, 1986; Beard et al. 1993; Pollitt, 1993; Sheard,
1994; Kretchmer et al. 1996). Although children with Fe
depletion without anaemia also showed behavioural
abnormalities in some studies, these abnormalities appear
to have been influenced by poor environmental conditions
that were associated with the occurrence of Fe depletion
(Lozoff et al. 1996). At this time there is no conclusive
evidence that poor Fe status without anaemia has adverse
effects on neural development in children.
9.3.4. Zinc. In animal experiments, Zn deprivation
was shown to adversely affect brain growth, learning ability,
memory and activity (Smart, 1974; Halas & Sanstead, 1975,
1980; Peters, 1979). Low-birth-weight infants showed an
improvement of motor development when supplemented
with Zn (Friel et al. 1993). In a recent study in Indian
children aged 6–35 months who were not malnourished,
daily supplementation with 10 mg elemental Zn (as Zn
gluconate) for a period of 6 months resulted in a significant
increase of observed activity (Sazawal et al. 1996).
9.3.5. Polyunsaturated fatty acids. Some 50–60 % of
the structural matter in the central nervous system is
composed of lipids, which almost entirely serve structural
functions in cell membranes and myelin. Much of the rapid
lipid accretion during brain growth comprises lipids that can
be synthesized de novo in the fetus and infant, e.g.
cholesterol. In addition, the rapid brain growth occurring
perinatally and the extensive synaptogenesis occurring
during the first months of life require the incorporation of
relatively large amounts of essential PUFA, primarily the
highly unsaturated long-chain PUFA DHA and arachidonic
B. Koletzko et al.
acid (Koletzko, 1992). In experimental studies, the addition
of DHA and arachidonic acid to fetal mouse brain cultures
increased the number, diversity and complexity of synaptic
contacts (Tixier-Vidal et al. 1986). In addition to cell
growth and synaptogenesis, long-chain PUFA as well as
eicosanoids formed from long-chain PUFA may influence
neural cell apoptosis (Finstad et al. 1994; Wainwright, 1997).
Experimental studies in rodents and in non-human primates
indicated that the degree of long-chain PUFA incorporation
into the developing brain influenced reflex development,
memory, discrimination learning, retinal function and visual
acuity (Neuringer, 1993; Wainwright, 1993), i.e. functions
related to the efficacy of information processing.
The human fetal supply with preformed long-chain PUFA
by a materno-fetal placental transfer (Koletzko & MuВЁller,
1990) may be influenced by the maternal dietary long-chain
PUFA intake. Also, the postnatal long-chain PUFA supply
in human milk is affected by maternal diet and can be
altered by dietary supplements provided to lactating women
(Harris et al. 1984; Koletzko et al. 1992). In contrast to
human milk, most infant formulas do not contain preformed
DHA and arachidonic acid. Although newborn infants have
the ability to synthesize long-chain PUFA from EFA
precursors, the rate of synthesis appears to be low
(Demmelmair et al. 1995; Sauerwald et al. 1997) and
long-chain PUFA levels in blood lipids (Decsi & Koletzko,
1995) and brain (Farquharson et al. 1992; Makrides et al.
1994) of formula-fed infants are significantly lower than
those found in breast-fed infants.
Some randomized intervention studies comparing diets
without and with a supply of preformed long-chain PUFA
both in premature infants (Uauy et al. 1990; Carlson &
Werkman, 1996) and in healthy term infants (Makrides et
al. 1993, 1995; Agostoni et al. 1995) found indications of
improved retinal and visual function and of cognitive
development in infants receiving long-chain PUFA, while
other studies found no appreciable advantage (Innis et al.
1996). Effects of supplementing additional long-chain
PUFA during pregnancy or lactation on functional development of the infant have not been reported.
9.4. Early nutrition and development of taste preferences
Preferences of taste and smell are critical factors in selecting
foods and drinks throughout life. There are some indications
that sensory perception of tastes and flavours may be
modulated by early exposure.
Observations of fetal swallowing following the ingestion
of sweet- and bitter-tasting substances into the amniotic
fluid suggest that the fetus is sensitive to sweet- and bittertasting substances (de Snoo, 1937; Liley, 1972). It has been
concluded that both the olfactory apparatus and taste perception are fully developed in utero (Beauchamp et al.
1991). Molecules carrying flavours and aromas may cross
the placenta, and it has been proposed that early sensory
exposure may modulate the acquisition of later flavour
preferences. In animal studies, such effects of intrauterine
exposure to flavour-rich foods on postnatal food choices
have been documented.
Premature infants tested between 33 and 40 weeks postconception and newborns during the first hours after birth
show a clear and reproducible preference for sweet tastes
(Beauchamp & Mennella, 1980). In blinded controlled
studies, the sucking behaviour of breast-fed infants is
altered by supplementing their mothers with encapsulated
garlic compared with placebo, and repeated consumption of
garlic modifies the infantile response to this taste (Menella
& Beauchamp, 1993). Also the volatile flavours of vanilla
and alcohol modulate infantile sucking behaviour
(Beauchamp & Mennella, 1980; Mennella & Beauchamp,
1991). The question of to what extent food choices during
childhood and adult life are modulated by pre- and postnatal
experience is of major importance and needs to be further
9.5. Methodological aspects
An optimization of the quality of nutrient intakes during
pregnancy, lactation and infancy may well have the potential of improving developmental outcomes in the recipient
infants. Since even small improvements of the population
means by such potential effects are of major public health
significance, they need to be carefully examined. If plausible hypotheses can be raised and supported by data from
experimental models, epidemiological studies or pilot intervention trials, they should be subjected to rigorous testing
with adequate scientific methodology in double-blind
placebo-controlled randomized trials. While some electrophysiological measures of human neural function can be
assessed with sufficient precision to justify interventions in
small groups, behavioural methods tend to have a greater
degree of variation and, therefore, effects on such outcome
variables require large studies, particularly if one considers
that dietary factors must be expected to have relatively small
effects compared with genetic and other environmental
influences. The results of such studies can be greatly
influenced by the study design and factors such as the
time points chosen for testing effects as well as the adequacy of the method chosen for testing the targeted effect.
Large sample sizes may be required, for example, to show
an effect of a dietary supplement, resulting in a comparably
small mean difference of developmental scores due to the
many other variables that influence such end-points. Therefore, the realization of adequate trials to test for long-term
developmental effects of early food choices will usually
require a large budget. In view of the major public health
importance of the questions addressed by such trials, it
appears justified that well-designed trials addressing relevant and pertinent questions are supported by public funds.
10. Production of bioactive factors for inclusion into food
Interest in the production of human milk proteins, peptides,
growth factors and other bioactive substances with the use
of biotechnology and recombinant techniques is growing.
The inclusion of such substances into dietary products may
have beneficial physiological effects particularly in infancy
and early childhood, for example defence against infectious
agents, the optimization of nutrient uptake from the diet as
well as the differentiation and growth of cells and tissues.
Micro-organisms and transgenic animals can now be used
Growth, development and differentiation
for the production of bioactive proteins (LoВЁnnerdal, 1996).
However, the benefits and safety of each substance must be
evaluated in adequate studies in cells, animal models and
clinical studies before routinely adding them to products for
infants to improve their nutrition, health or development.
Proper manufacturing conditions must be developed for
introducing such substances into foods. The importance of
post-translational modifications must also be taken into
account. Some proteins may require proper glycosylation
or phosphorylation for physiological activity.
Several human milk proteins have been cloned and
sequenced. The majority were cloned at the level of
complementary DNA (cDNA). In a few cases the entire
gene has been characterized. One of the first milk proteins
which was cloned from a mammary gland library was alactalbumin (Hall et al. 1987). Other human milk proteins
that have been cloned include lysozyme, lactoferrin,
human b-casein and k-casein. For the production of
human milk proteins and bioactive factors, several expression systems can be used, such as bacterial expression of
recombinant proteins. The expression vector can be constructed so that the protein is made available in the supernatant fraction or in the bacteria. Bacterial expression will
lead to the production of proteins which are not phosphorylated or glycosylated. If this property is needed for
biological functions, yeast or fungi can be used as expression systems. Saccharomyces cerevisiae was used to produce human lactoferrin (Liang & Richardson, 1993) and
human b-caseins. Aspergillus nidulans and Aspergillus
oryzae were also used to produce human lactoferrin
(Ward et al. 1992a,b). Alternatively, living cells such as
baby hamster kidney cell can be used to produce human
lactoferrin (Ward et al. 1992a). As such expression systems will still provide proteins with different phosphorylation and glycosylation, physiological function
may be altered. Tissue-specific expression of human
milk proteins in transgenic animals should result in
recombinant protein glycosylations and phosphorylation
similar to the native human milk proteins.
The architecture of the transgene DNA that is introduced
into the germline of animals by microinjection plays an
important role in the level of expression of the transgene.
DNA that is introduced by microinjection into the pronucleus is usually inserted randomly into the genome as
head-to-tail concatemers. Because the eukaryotic genome is
organized into topologically constrained domains, random
integration can lead to position effects in which the transgene expression is influenced by the surrounding chromosomal sequences. Thus in many cases the level of transgene
expression will vary over several logarithms, depending on
the site of integration, and expression may be observed in
<50 % of the positive transgenic mice. Because of the
expense and time required to generate transgenic livestock,
this presents a major problem (Stowell et al. 1991). To date,
human lactoferrin (Rosen et al. 1996) and human lysozyme
(Kim et al. 1994) have been expressed in transgenic mice
with reasonable expression in the milk. Human lactoferrin
was recently produced in transgenic cows (Maga et al.
1994). Whether the glycosylation pattern of human lactoferrin within the cow’s mammary cells will alter the
biological function remains to be elucidated.
Similar to the gene farming of human milk proteins and
other important functional proteins in transgenic animals,
such bioreactors are used to produce human hormones and
growth factors. In transgenic rabbits, hGH was produced in
milk in suitable amounts without being affected by the
transgene expression (Limonta et al. 1995). Bovine
growth hormone was also placed by a recombinant technique into the milk of transgenic mice (TheВґpot et al. 1995). In
addition, human IGF1 was expressed in the milk of transgenic mice (Hadsell et al. 1996) and rabbits (Brem et al.
1994). Some oligosaccharides which may play an important
role in promoting growth and differentiation of intestinal
epithelial cells have successfully been expressed in the milk
of transgenic mice (Prieto et al. 1995). The enormous
potential of these methodologies for improving food products needs to be further explored.
11. Commentary on biomarkers
Drawing on experience from child health and development
it is possible to use experience of inborn errors of metabolism to demonstrate the evolution and use of biomarkers to
detect and predict a disease arising from an imbalance
between systemic homeostasis and essential and non-essential dietary components. A good example is hyperphenylalaninaemia (HPA) which arises from an inborn error of
metabolism of the essential amino acid phenylalanine
(Scriver & Clow, 1980; GuВЁttler, 1984). First of all, however,
it is helpful to consider some general aspects of the strategy
of biomarkers.
Biomarkers can be defined as indicators of actual or
possible changes of systemic, organ, tissue, cellular and
sub-cellular structural and functional integrity which can be
used, either singly or in batteries, to monitor health and
exposure to compounds in populations and individuals: as
such they are the essence of chemical pathology or clinical
biochemistry. Thus although the term �biomarker’ might
itself be relatively novel, the concept is far from being so:
the use of biochemical biomarkers dates at least from the
discriminatory use of the sweet taste of glycosuria to
diagnose and name diabetes mellitus. This intolerance of
glucose illustrates a disturbance of the usually tightly
controlled internal milieu as conceived by Claude Bernard,
and of the processes involved in maintaining this state, i.e.
homeostasis. These mechanisms lie at the core of the
regulatory adaptations which maintain a steady state in the
face of changes in environment, including our interaction
with desirable and non-desirable components of the diet,
which, of course, is one of our most intimate interactions
with the environment. Homeostasis is regulated by cellular
and systemic mechanisms which, in turn, are dependent on
gene-mediated responses. Genetic heterogeneity amongst
people underlies the intrapopulation variability of the interaction between nurture and nature. These generalizations
apply equally to exposure to abnormal compounds and to
excessive or inadequate exposure to normal components in
the diet or environment.
Homeostasis comprises several processes, some of which
act synchronously, but many of which act in a specific
sequence, each being triggered by the relative efficiency of
the preceding process. Some of these mechanisms are
B. Koletzko et al.
specific for individual compounds. This applies to essential
nutrients or generic groups of nutrients, but for non-nutrients and xenobiotics there appears to be a limited number of
common protective mechanisms. In the chain arising from
threatened or actual toxic exposure there would first be
compensatory adaptations such as metabolic biotransformations (phase I: oxidation, hydrolysis, reduction; phase II:
conjugations), excretion, and sequestration of compounds
(for example in adipose tissue, bone, hair, skin); when these
are inadequate biochemical and histopathological features
follow with structural cellular damage and functional
derangement. These lead to tissue damage with clinical
manifestations (e.g. neurological and muscular toxicity,
retinal toxicity, immunotoxicity, hepatotoxicity, nephrotoxicity, bone marrow damage, teratogenicity, cytotoxicity,
cancer, methaemoglobinaemia, haemolysis) and overt disease states many of which are major health issues (e.g.
cancers, cardiovascular disease, obesity, osteoporosis,
adverse reactions to foods and food additives and contaminants, atopic disease, behavioural abnormalities, fetotoxic
effects). This chain shows the series of genotypic (genetic),
and phenotypic (biochemical and clinical) biomarkers.
The underlying assumption here is that all diet-related
disease arises from an inappropriate interaction between
diet and systemic homeostasis and that the fundamental
basis of this imbalance lies in the relative imbalance of
dietary exposure and the genetic controlled response. At this
level the genome and its product would serve as an ideal
biomarker: unfortunately the processes involved might not
have been identified or might not be accessible to practical
and ethical sampling techniques. Alternative and less
immediate biomarkers have to be used. However, the
more remote the biomarker is from the primary event the
more it is attenuated and subject to confounding factors. It
becomes less specific. On the other hand the more immediate
it is to the basic interaction the more quantitatively related
and predictive it becomes: biomarkers represent Garrod’s
concept that genetic factors determine the nature of chemical
metabolism and human biochemical heterogeneity.
More immediate biomarkers are not only potentially
more predictive, they might also provide more immediate
outcomes which could be used to assess interventions in a
reasonable timescale and, in turn, replace the temporally
and aetiologically remote outcomes which are so often the
foci of epidemiological studies of diet and health: for
example the diseases mentioned earlier are probably both
metabolically and temporally remote from their aetiology.
Insight into the metabolic processes involved in the particular issue being investigated can inform the choice of tissue
or fluid to be sampled and the phenotypic marker to be
measured. This applies also to genotypic biomarkers designed
to assess adaptive phenomena but it is possible to measure
some predictive genotypic markers in tissues other than those
in which the gene product is expressed. Whatever the situation
the selection of biomarker(s) should be dictated by the
problem being considered rather than by the accessibility of
tissue or fluid to be measured or the ease of an assay.
These general points are represented by the disease PKU
which epitomizes the interaction between nature and nurture
and the benefits which can be attained by an appropriate
manipulation of the diet. PKU is a manifestation of HPA
which results from the altered activity of the principally
hepatic enzyme phenylalanine hydroxylase (EC;
PAH), functional defects of which arise from intrinsic
defects in the apoenzyme or from defective synthesis of
its cofactor (tetrahydrobiopterin) (Scriver & Clow, 1980;
GuВЁttler, 1984; Scriver, 1991; Scriver et al. 1996; GuВЁttler &
Guldberg, 1996). The condition was recognized in 1933
when a mother with a mentally retarded child sought help on
the peculiar smell of her child’s urine. She saw several
doctors, one of whom even referred her to a psychiatrist for
help with her delusion. In the end one physician, Dr Asbjorn
Folling confirmed the peculiar smell. He attributed it to the
presence of phenylpyruvic acid in the urine and he called the
condition �imbecillitas phenylpyruvica’ thereby incorporating both the initial clinical and biochemical biomarkers into
the condition’s nomenclature (Gu¨ttler, 1984; Scriver, 1991).
He developed the �FeCl 3’ colorimetric test of urine to detect
the excess metabolite and used it to screen mentally retarded
children and adults for the condition (Scriver & Clow,
1980). In 1950 Horst Bickel appreciated the implication of
raised blood phenylalanine concentrations in PKU patients
and explored the use of a phenylalanine-free mixture of
amino acids to feed affected infants. It was found that blood
phenylalanine levels fell and that the urine abnormality
decreased. There was also some clinical and behavioural
improvement, but the intervention was too late to affect the
established developmental delay and mental damage.
Nonetheless this progress in the phenotypic biomarkers
demonstrated the potential clinical and economic benefits
of screening and early diagnosis in the hope that early
dietary intervention could prevent the neurological damage.
Early dietary management was effective, but many cases
went undetected because the FeCl 3 biomarker was relatively
insensitive and non-specific (Scriver & Clow, 1980).
Phenylpyruvic acid is one of a number of normal metabolites of phenylalanine which are found in excessive amounts
in the urine in PKU as a consequence of the increased
activity of alternative pathways for the metabolism of
phenylalanine. Other variables affected the production of
these metabolites and it was appreciated that blood phenylalanine concentrations would be a better biomarker, but the
analytical techniques (paper chromatography) available at
that time were costly and time consuming. In the late 1950s
the abnormal gene product, i.e. defective PAH, was identified, but since this activity was in the liver this did not
provide an effective biomarker. The finding of this functional defect reinforced the opinion that screening should be
targeted at the HPA.
Guthrie solved the problem in 1961 by establishing a
semiquantitative bioassay based on the inhibition of bacterial growth by high concentrations of phenylalanine. The test
was done on blood drops collected on filter card from infants
at 4–7 d of age by which time the babies would have fed
and, in contrast to in utero, would have had the opportunity
to challenge the activity of their endogenous PAH with a
phenylalanine load. The biomarker for definitive diagnosis
and monitoring of management was direct quantitative
measurement of blood phenylalanine concentrations. This
enabled an appreciation that these concentrations could
correlate with the efficiency of management and with
behavioural and other clinical outcomes. However, not all
Growth, development and differentiation
patients responded similarly or predictably. Amongst this
heterogeneity was an atypical group with HPA which was,
in the mid 1970s, found to be secondary to a defect in the
synthesis of the tetrahydrobiopterin cofactor. Thus, in the
midst of the phenotypic and molecular heterogeneity which
was becoming increasingly obvious, it was realized that
PKU or HPA was not the result of a single gene defect. The
allele for PAH has since been found on chromosome 12q,
and that for the affected stage in tetrahydrobiopterin synthesis is on chromosome 4 (GuВЁttler & Guldberg, 1996; Scriver
et al. 1996).
HPA is, thus, evident as a complex entity with many
clinical and metabolic phenotypes. Consistent with this
heterogeneity and with the variation in dietary tolerance for
phenylalanine, almost 300 mutations in the PAH gene have
been identified. Some of these genetic mutations have been
specifically correlated with the activity of their enzyme
products and with the severity of the clinical disease (Scriver,
1991; GuВЁttler & Guldberg, 1996; Scriver et al. 1996).
In the context of biomarkers, HPA represents a continuum of genotypic, biochemical (PAH, tetrahydrobiopterin
defect), metabolic (HPA), and clinical (the PKU syndrome)
phenotypic biomarkers of an abnormal interaction between a
dietary component and an individual’s ability to achieve
effective homeostasis at customary dietary exposure.
The genotypic biomarker may be used for definitive
diagnosis and family screening and possibly for
prognostication, but the metabolic biomarker is most
useful for monitoring and tailoring the dietary reduction
of phenylalanine. The urinary biomarker (PKU) is no longer
of much use, and the clinical phenotypic biomarker is one of
a medical tragedy.
PKU and HPA demonstrate the movement of biomarkers
from remote and non-specific outcomes (many other conditions including other inborn errors of metabolism cause
epilepsy and developmental delay) to more specific and
informative outcomes, and ultimately to the basic genomic
mutation offering the opportunity of appropriately designed
diets. It is feasible that in due course a similar heterogeneity
will be found in the metabolism of other nutrients, and that
this will explain many of the conflicting phenomena found in
epidemiological studies of the interaction between diet and
health and in apparent heterogeneity of nutritional requirements. Such biomarkers should also provide more definitive
indicators of response to interventions, thereby enabling
shorter and more definitive epidemiological studies. Whether
or not people would accept the corollary of this, namely
specific diets tailored to their genotypic or surrogate metabolic characteristics, depends probably on the nature of the
diets involved and on the people’s motivation in the context
of the much less dramatic and immediate effects of an
inappropriate diet compared with that experienced with
PKU. The integration of markers of susceptibility with
appropriate follow up of long-term outcomes by epidemiological studies of characterized populations will demonstrate
the relevance or otherwise of formal interventions.
12. Conclusions
Food supply and the metabolism of food ingredients in
women during pregnancy and lactation and in their children
have implications for long-term health and child development. Epidemiological evidence and studies performed in
infants have highlighted the fact that maternal and intrauterine influences are of special importance during the
development of the infant and child. Early nutrition
modulates growth and functional development of the organism and appears to exert life-long programming effects that
modulate health, disease and mortality risks in adulthood,
neural function and behaviour, and quality of life.
The field of nutrient–gene interaction is in a phase of
rapid expansion and development. There are several areas
where dietary modulation of gene expression could exert
beneficial effects, for example with respect to lipid metabolism and risk of cardiovascular disease. Applied research
should further elucidate the interaction of nutrients and gene
expression. The genes affected by specific nutrients, e.g.
amino acids, must be characterized in animal models as well
as the underlying cellular and molecular mechanisms. Since
the vast majority of studies have been performed on animal
models or animal cell lines, strategies must be defined to
approach these questions in human subjects, for example by
using human cell lines responding to nutrients. Once the
mechanisms and relevance to man have been clarified, then
development and testing of existing or new �functional
foods’ could be performed both in animals and human
The relation between nutrients and differentiation needs
to be further explored by in vivo models, studying food
effects on cell differentiation and later performance. Concomitantly, the effects of nutrients on cell differentiation in
in vitro studies should be strongly encouraged.
The course of pregnancy, childbirth and lactation as well
as human milk composition and the short- and long-term
outcome of the child may be influenced by the intake of
foods and particularly micronutrients, e.g. PUFA, Fe, Zn
and I. Folic acid supplementation from before conception
through the first weeks of pregnancy was reported to
markedly reduce the occurrence rates of severe embryonic
malformations, including anencephaly and spina bifida. The
potential of exerting beneficial effects for mother and child
by modulating maternal nutrient supply should be further
The evaluation of dietary effects on child growth requires
epidemiological and field studies as well as evaluation of
specific cell and tissue growth. Novel substrates, growth
factors and conditionally essential nutrients (e.g. growth
factors, amino acids, unsaturated fatty acids) may be potentially useful as ingredients in functional foods and need to
be assessed carefully with respect to their potential effects
on growth, maturation and development of specific cell
types, tissues and organs under different physiological
conditions. In particular, the potential modulation of later
obesity by early food choices needs to be further explored.
Intestinal growth, maturation, intestinal adaptation and
regulation and long-term function may be influenced by
food ingredients. The roles of compounds such as dietary
oligosaccharides, gangliosides, high-molecular-mass glycoproteins, bile salt-activated lipase, pre- and probiotics as to
their physiological functions in the developing organism
need to be further explored. The interaction of the appropriate genes of intestinal substrate transporters and their
B. Koletzko et al.
substrates in early childhood is not well understood and
needs to be clarified.
There are indications for some beneficial effects of
functional foods on the developing immune response in
vitro and in vivo, for example induced by antioxidant
vitamins, trace elements, fatty acids, arginine, nucleotides,
and altered antigen content in infant foods. A general
unresolved issue is related to the lack of reliable surrogate
outcome markers when investigating the effects of nutrition
on the developing immune response and possible long-term
Peak bone mass at the end of adolescence can be
increased by dietary means, which is expected to be of
long-term importance for the prevention of osteoporosis at
older ages. Future studies should be directed to the combined effects of Ca and other constituents of growing bone,
such as P, Mg and Zn, as well as vitamins D and K, and trace
elements F and B. In addition to observational and intervention studies, in vitro and animal model studies might
provide a basis for new concepts on the interaction and
optimal relative proportions of the several macro- and
micronutrients involved in bone growth and mineralization.
Pregnancy and the first postnatal months are critical time
periods for the growth and development of the human
nervous system, processes for which adequate substrate
supplies are essential. Early diet may have long-term effects
on the structure and function of the nervous system, sensory
and cognitive abilities as well as behaviour. The potential
beneficial effects of a balanced supply of nutrients such as I,
Fe, Zn and PUFA need to be explored in further detail.
The question of a possible relationship between early
exposure to tastes and flavours and later food choice preferences may have a major impact on public health and needs
to be further elucidated.
Bioactive factors such as human milk proteins, peptides,
growth factors and other substances may be produced for
use in food products with the use of biotechnology and
recombinant techniques. The inclusion of such substances
into dietary products may have beneficial physiological
effects particularly in infancy and early childhood, for
example defence against infectious agents, the optimization
of nutrient uptake from the diet as well as the differentiation
and growth of cells and tissues. The enormous potential of
these methodologies for improving food products needs to
be further explored.
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б­§ Nutrition Society 1998
British Journal of Nutrition (1998), 80, Suppl. 1, S47–S75
Functional food science and substrate metabolism
W. H. M. Saris1В¬ , N. G. L. Asp2 , I. BjoВЁrck2 , E. Blaak1 , F. Bornet3 , F. Brouns4 , K. N. Frayn5 ,
P. FuВЁrst6 , G. Riccardi7 , M. Roberfroid8 and M. Vogel9
Nutrition Research Centre, Department of Human Biology, Maastricht University, PO Box 616,
NL-6200 MD Maastricht, The Netherlands
Chemical Centre, Applied Nutrition and Food Chemistry, University of Lund, PO Box 124, S-221 00, Lund, Sweden
Vilvoorde Research and Development Centre, Nutrition and Health Service,
Havenstraat 84, B-1800 Vilvoorde, Belgium
Novartis Nutrition Research Unit, PO Box 1350, NL-6201 BJ Maastricht, The Netherlands
Oxford Lipid Metabolism Group, Sheikh Rashid Laboratory, Radcliffe Infirmary, Oxford OX2 6HE, UK
Institute of Biological Chemistry and Nutrition, University of Hohenheim, PO Box 700 562,
Garbenstrasse 30, D-70593 Stuttgart, Germany
Dipartimento di Medicina Clinica e Sperimentale, Universita´ degli Studi di Napoli – Federico II,
Via Sergio Pansini 5, I-80131 Napoli, Italy
UCL, Ecole de Pharmacie, Tour Van Helmont, Avenue E. Mounier, B-1200 Brussels, Belgium
Dipl. Biochemiker–Biochemisches Lab, Su¨dzucker AG Mannheim/Ochsenfurt, Zafes, Wormser Strasse 11,
D-67190 Obrigheim, Pfalz, Germany
1. Introduction
2. Chronic diseases related to energy balance and
substrate regulation
2.1. Obesity
2.1.1. Genetic contribution to obesity
2.2.2. Regulation of energy balance
2.2.3. Costs of obesity
2.2. Insulin resistance syndrome
2.2.1. Features predisposing to the insulin
resistance syndrome
2.3. Diabetes
2.3.1. Aetiology
2.4. Undernutrition
2.4.1. Definition
2.4.2. Present position
2.4.3. Pathophysiology and adaptive responses
2.4.4. Body composition
2.4.5. Geriatric undernutrition
2.5. Conclusions and further research
3. Metabolic conditions related to these chronic
3.1. Body-weight control
3.1.1. Energy balance, macronutrient balance
and body-weight regulation
3.1.2. Type of carbohydrates
3.1.3. Type of fat
3.1.4. Alcohol
3.1.5. Macronutrient replacement
3.1.6. Dietary components stimulating
3.1.7. Physiological and metabolic consequences
of undernutrition
3.1.8. Conclusions and further research
Insulin resistance/sensitivity
3.2.1. Introduction
3.2.2. Dietary carbohydrates
3.2.3. Dietary fat
3.2.4. Niacin and insulin sensitivity
3.2.5. Minerals
3.2.6. Conclusions and further research
Blood glucose control
3.3.1. Introduction
3.3.2. Nutritional influence on fasting and
postprandial blood glucose levels
3.3.3. Food properties determining the glycaemic
3.3.4. Indigestible carbohydrates and glucose
metabolism: possible mechanisms of action
3.3.5. Conclusions and further research
Plasma triacylglycerols
3.4.1. Introduction
3.4.2. Dietary carbohydrates
3.4.3. Dietary fat
3.4.4. Conclusions and further research
Abbreviations: ALP, atherogenic lipoprotein phenotype; GI, glycaemic index; IDDM, insulin-dependent diabetes mellitus; IRS, insulin resistance
sydnrome; MCT, medium-chain triacylglycerol; NEFA, non-esterified fatty acids; NIDDM, non-insulin-dependent diabetes mellitus; P : S ratio,
polyunsaturated : saturated fatty acid ratio; PUFA, polyunsaturated fatty acids; SCFA, short-chain fatty acids.
*Corresponding author: Dr W. H. M. Saris, fax Гѕ31 43 367 0976, email [email protected]
W. H. M. Saris et al.
4. Nutrition, substrate metabolism and physical
4.1. Introduction
4.2. Carbohydrates
4.3. Fat
4.4. Protein
Fluid and electrolytes
Trace elements
Ergogenic supplements
Conclusions and further research
The present review addresses the role of food constituents in the aetiology of metabolic
conditions and chronic diseases, mostly related to energy metabolism and substrate regulation,
such as obesity and non-insulin-dependent diabetes mellitus. Second, attention is paid to
malnutrition, a major cause of mortality and morbidity in developing countries, which may be
a cause of concern in Europe because of the increasing number of elderly people in the
population. Finally, the role of diet during exercise, a condition of enormous substrate demands,
is evaluated. Based on a critical evaluation of the existing knowledge in the literature,
implications for future research in relation to functional foods are discussed.
Functional foods: Substrate metabolism: Energy balance: Physical activity
1. Introduction
It is clear that with modern food and physical activity
habits, the human metabolic regulatory mechanisms are
challenged. There is a growing imbalance between foodrelated energy intake and physical activity-related energy
expenditure. Also, the balance in macronutrient intake has
changed dramatically over the past few decades. Therefore,
a number of specific body functions, mostly related to
energy metabolism and substrate regulation, are at risk.
A number of chronic diseases such as obesity, non-insulindependent diabetes mellitus (NIDDM) and osteoporosis can
be identified as being directly related to changes in food intake
and physical activity-related behaviour. At the same time,
however, malnutrition thought of as a major cause of mortality
and morbidity only in the developing countries, is a cause for
concern in Europe, mainly because of the increasing number
of elderly people in the population. There are also specific
groups in whom undernutrition poses a particular problem,
especially those with concurrent chronic or severe illness.
This chapter will briefly evaluate the metabolic changes,
and the underlying specific metabolic conditions as well as
the role of specific dietary components in the aetiology of
the previously mentioned chronic diseases.
Of special interest is the relation between nutrition and
physical performance. During physical stress such as
exercise, the substrate demands are enormous. Nutrition
plays a crucial role in this process. Therefore, a balanced
diet with a carefully planned mix of food ingredients can
play an important role in the level of performance. In this
way well-designed and effective sports foods have been
proven to be clear examples of functional foods.
2. Chronic diseases related to energy balance and
substrate regulation
2.1. Obesity
Obesity is defined as an excessive accumulation of body fat.
Its prevalence may vary in different populations between 5
and 50 %, also depending on the definition of overweight
(mild v. moderate v. severe obesity). A BMI (body weight
(kg)/height (m)2 ) between 25 and 30 kg/m2 is defined as mild
obesity (in USA: BMI > 27 kg/m2 ), a BMI in the range of
>30–35 kg/m2 is moderate obesity, and a BMI > 35 kg/m2
is severe obesity (BjoВЁrntorp & Brodoff, 1992). The high
incidence of obesity in affluent societies is recognized as a
major health problem (VanItallie, 1992). Obesity is associated
with an increased risk of developing hypertension, insulin
resistance, diabetes and cardiovascular disease. Apart from
these major health hazards, obesity is thought to cause a great
variety of health problems, some of which are listed in Table 1.
2.1.1. Genetic contribution to obesity. It has been
recognized that there is an important genetic contribution
to obesity. The fact that a sedentary lifestyle and high-fat
Table 1. Health disorders and other problems thought to be caused or
exacerbated by obesity (Based on VanItallie, 1992 and Frayn, 1996)
Cardiovascular disease
Non-insulin-dependent diabetes
Impaired reproductive and
sexual function
Reduced fertility (males)
Polycystic ovary syndrome
Increased risk of breast and
endometrial cancer
Osteoarthritis in weight-bearing
Impaired self-image,
depression, suicides
Possible cause
Elevated LDL-cholesterol,
decreased HDL-cholesterol,
May result indirectly from insulin
Insulin resistance
Increased cholesterol flux into
bile (possibly related to insulin
resistance and high insulin
Decreased androgens,
increased oestrogen
production in adipose tissue
Increased oestrogen production
in adipose tissue*
Excess weight bearing
Psychological aspects of poor
body image
Loss of mobility
* Increased oestrogen production occurs because adipose tissue contains
the enzyme aromatase, which converts androgens (e.g. testosterone) into
Functional foods and substrate metabolism
diet may predispose towards obesity, independent of
genetics, has been equally well recognized and appreciated
(Prentice & Jebb, 1995). The interaction of genetic
predisposition and environmental factors is the most
commonly accepted model for the genetics of human
obesity (Bouchard & Perusse, 1993). The relative importance of these two factors in different populations may vary
across studies. The heritability of obesity on the basis of
studies of monozygotic twins raised together or apart has
ranged from 20 to 60 %. The best current estimate of
heritability of body fat is 35 % (Bouchard & Perusse,
1993). If another 15 % is allocated for subjects who are
predisposed to obesity when a �Western high-fat’ diet is
consumed, then genetic inheritance can account for about
50 % of the variation in percentage body fat (Campfield et
al. 1997).
2.2.2. Regulation of energy balance. Obesity develops
under conditions where energy intake exceeds energy
expenditure. Much of the research in the obesity field
has focused on the possible deviations in the different
components of the energy balance equation. There is some
evidence that a low resting energy expenditure may
contribute to the development of obesity and that the
metabolic rate during rest is, to some extent, genetically
determined (Ravussin et al. 1988). Also, a blunted dietinduced thermogenesis response has been reported in
obesity (Segal et al. 1990), but this has not been found
consistently (D’Allesio et al. 1992). Thus, the relative
importance of a low rate of energy expenditure in the
development of obesity and the extent of individual
differences in susceptibility to obesity remain controversial.
A major difference in energy balance between lean and
obese subjects is the increased energy expenditure in obese
subjects, proportional to their increased fat-free mass, and as
a consequence the proportionally increased energy intake
(Prentice et al. 1986). Within the past decade, attention
has been shifted to the investigation of the balances of the
individual macronutrients, proteins, carbohydrates and fat,
in the development of obesity. There are indications that
subjects predisposed to obesity, or obese subjects, may have
a diminished ability to oxidize fat, which would make them
more susceptible to a positive fat balance on a high-fat diet
(Zurlo et al. 1990; Blaak et al. 1994). These topics will be
dealt with further in section 3.1. Extensive attention to the
control of food intake is given in another paper in the
present supplement (Bellisle et al. 1998).
2.2.3. Costs of obesity. The direct economic costs of
obesity, defined as the costs related to diversion of
resources to diagnosis and treatment of obesity as well as
the treatment of obesity itself, have been estimated to vary
between 2 and 5 % of total health-care costs of various
countries. In this calculation, only the costs of moderate and
severe obesity (BMI > 30 kg/m2 ) are taken into account.
The costs associated with mild obesity (BMI between 25
and 30 kg/m2 ) may also be substantial, since a large
proportion of the adult population is involved. These costs
comprise costs of health services (visits to general
practitioners, consultation with medical specialists, medication; Seidell, 1997). In addition societal costs (loss of
productivity, disability pensions, premature death), and
personal costs (job discrimination, higher premiums to life
insurance companies) contribute substantially to health-care
costs, but many issues of these costs are too fragmentary to
allow calculation (Seidell, 1997). Thus, there is much direct
information to show that obesity contributes significantly to
health-care costs.
2.2. Insulin resistance syndrome
It has been recognized for many years that a number of
adverse metabolic changes tend to cluster within individuals
(Table 2). The underlying change appears to be a decrease in
insulin sensitivity (insulin resistance) although cause and
effect have never been properly disentangled. Nevertheless,
plausible mechanisms have been described whereby development of insulin resistance can lead to the other aspects of
the syndrome. Each of these changes has been shown
independently to relate to risk of cardiovascular disease,
and insulin resistance is a strong marker of risk of development of NIDDM (Reaven, 1995). The terms insulin
resistance syndrome (IRS) or metabolic syndrome are now
preferred to the original term syndrome X. This syndrome
may be very prevalent. Some studies suggest that about
25 % of non-diabetic adults manifest this syndrome
(Reaven, 1995).
The concept of the IRS has been enormously helpful in
understanding current patterns of chronic disease, especially
CHD and NIDDM. As a risk factor for CHD it is probably
greater in absolute terms than elevated cholesterol concentrations (DespreВґs, 1993). The characteristic dyslipidaemia
of insulin resistance includes elevated fasting plasma triacylglycerol and depressed HDL-cholesterol concentrations. These changes are associated with a preponderance
of small, dense LDL particles. This dyslipidaemia has been
called the atherogenic lipoprotein phenotype (ALP) (Griffin
& Zampelas, 1995). Postprandial triacylglycerol concentrations may be even more important than fasting plasma
triacylglycerol concentrations. It has been suggested that
the ALP results from exaggerated postprandial lipaemia
associated with insulin resistance (Frayn, 1993).
Insulin sensitivity is usually measured as the ability of
insulin to stimulate glucose disposal, usually by means of
the hyperinsulinaemic, euglycaemic clamp procedure.
Table 2. Metabolic changes associated with insulin resistance
Glucose metabolism
Glucose intolerance
Lipid metabolism
Exaggerated postprandial lipaemia
Decreased HDL-cholesterol concentrations
Preponderance of small, dense LDL particles
Increased coagulation
Clinical correlates
Cardiovascular disease
Breast cancer
NIDDM, non-insulin-dependent diabetes mellitus.
W. H. M. Saris et al.
Insulin is infused at a rate chosen to elevate the plasma
insulin concentration by a predetermined amount, and
glucose is infused at a variable rate, as necessary to
maintain a constant plasma glucose concentration (hence
the glucose concentration is �clamped’). Under these conditions of constant glycaemia, glucose disposal must equal
the rate of glucose entry into the plasma, which is known
since it is being infused. (A correction can be made for
endogenous glucose production by infusion of a glucose
tracer.) Thus, the rate of glucose disposal from plasma at a
predetermined insulin concentration is known, and may be
used to compare different subjects. An alternative procedure involves mathematical modelling of the disappearance
of glucose following its intravenous injection (Ader &
Bergman, 1987).
However, these specialized procedures are unsuitable for
large-scale epidemiological studies. A simple �proxy’ may
be measurement of the fasting plasma insulin and glucose
concentrations. These may be referred to a simple mathematical model which interprets them in terms of insulin
secretion and insulin sensitivity (Matthews et al. 1985).
Even the fasting plasma insulin concentration alone provides useful information, and elevated fasting plasma
insulin concentrations have been shown to be associated
(for instance) with development of CHD independently of
other lipid markers (DespreВґs et al. 1996).
Although insulin sensitivity is measured in terms of
glucose disposal, insulin resistance appears to affect many
metabolic processes other than glucose disposal. Insulin
resistance of multiple aspects of lipid metabolism may be
the explanation for the characteristic dyslipidaemia of
insulin resistance (Frayn, 1993). Thus, there must be
some common change leading to multiple aspects of
insulin resistance. The effects of insulin on metabolism
are mediated through binding to specific receptors on cell
surfaces. The intracellular domain of the insulin receptor
possesses an intrinsic tyrosine kinase (EC activity which is activated upon insulin binding, and this initiates
the intracellular signalling chains leading ultimately to
changes in enzyme expression and activity. In principle,
insulin sensitivity might affect any one of the steps in these
signal chains, which are themselves divergent for different
aspects of insulin action. However, because insulin resistance, as commonly observed, affects so many diverse
metabolic functions, it is likely that an early and common
step is mainly affected. Attention has focused on events
around the insulin receptor. One possible mechanism for
widespread modulation of sensitivity to insulin might be a
change in membrane fluidity caused by the incorporation
into the membrane of different amounts of cholesterol or of
phospholipids containing different fatty acids.
2.2.1. Features predisposing to the insulin resistance
syndrome. The IRS may exist in apparently healthy
individuals of normal body weight. There is evidence for
a genetic component (Austin et al. 1990; Mitchell et al.
1996). However, it is most commonly associated with
obesity, particularly obesity involving upper body fat
distribution (often called visceral obesity).
Since obesity and fat distribution are themselves in part
genetically determined (Bouchard, 1992), there may be a
considerable genetic component to the IRS. Nevertheless, it
must also be strongly influenced by environmental factors
including diet and activity level, because it is considered to
have markedly increased in prevalence in recent years in
Western countries. Development of the IRS may be the
reason for the high incidence of CHD and NIDDM in
immigrant people from the Indian sub-continent (McKeigue
et al. 1991). This highlights both the possible genetic
predisposition of some groups, and the effect of a change
in lifestyle imposed on a susceptible genetic make-up.
Many of the features of the IRS shown in Table 2 could
also be described as the effects of a sedentary lifestyle.
Therefore, physical inactivity, development of obesity and
genetic make-up probably interact with dietary factors to
explain the high prevalence of the IRS in Western societies.
A number of studies have indicated that about 50 % of
variation in insulin sensitivity amongst individuals is
accounted for by variations in body fat content together
with some measure of aerobic capacity (Bogardus et al.
2.3. Diabetes
Diabetes mellitus is a disease characterized by increased
plasma glucose concentrations due to a reduced insulin
action at its target tissues (insulin resistance) and/or an
impaired insulin secretion. High glucose levels sustained
for several years induce structural abnormalities in the walls
of small arteries at the level of the retina and kidney and in
the peripheral nerves; moreover, they contribute (directly
and/or indirectly) to acceleration of the atherosclerotic
process in the large arteries at the level of heart, brain,
and lower limbs (Table 3).
Diabetes mellitus is classified into two large groups in
relation to its clinical manifestations and aetiology
(American Diabetes Association, 1997a). Type 1 or insulindependent diabetes mellitus (IDDM) usually develops in
young lean individuals; it has an abrupt onset and requires
insulin treatment to prevent the occurrence of a severe
metabolic derangement that leads to death within a few
days. This type of diabetes is due to an almost complete
destruction of pancreatic b cells which represents a consequence of an autoimmune process. Therefore, IDDM is
characterized by plasma insulin levels that are very low or
even unmeasurable. Type II diabetes (NIDDM) usually
develops in elderly individuals who are very often overweight; it has a very slow onset (it may be asymptomatic
for several years) and does not necessarily require insulin
treatment. It develops as a consequence of two metabolic
derangements occurring together: (1) insulin resistance at
the level of liver, muscle, adipose tissue; (2) impaired
insulin secretion (which nevertheless is never completely
Table 3. Long-term diabetic complications and their possible health
Health implications
Foot ulcers, impotence
Stroke, myocardial infarction, foot gangrene
Functional foods and substrate metabolism
suppressed). In relation to the balance between these two
metabolic derangements, plasma insulin concentrations can
be increased, normal or slightly decreased in comparison
with non-diabetic individuals.
Nutrition has very important implications for diabetes
in relation to aetiology, management and prevention of
complications (Riccardi, 1994).
2.3.1. Aetiology. Different nutritional factors have been
implicated in the aetiology of IDDM and NIDDM (WHO
Study Group, 1994). For IDDM, two nutritional factors have
been described in association with increased susceptibility
to the disease. The first one is represented by a short
duration of breast-feeding. It is not clear, so far, whether it is
breast-feeding per se which has a protective effect on
IDDM development or if an early introduction of dairy
products and/or solid foods in the infant diet might have a
predisposing effect. However, since duration of breastfeeding and age at introduction of dairy products and/or
solid foods are highly correlated, it is difficult to dissect
the independent effect of each of them on the risk of
IDDM. The second nutritional factor linked with IDDM is
represented by consumption of N-nitroso compounds
(chemically related to streptozotocin, a well-known b-cell
toxin). Studies in laboratory animals and ecological surveys
in man have shown an increased risk of developing IDDM
when N-nitroso compounds are consumed both by the
parents at the time of conception and by the progeny during
infancy. Further studies are needed, particularly intervention trials in human subjects, before the link between
these dietary factors and IDDM is fully established (Virtanen
& Aro, 1994).
In relation to the aetiology of NIDDM a large body of
evidence indicates obesity, particularly the visceral type, as
an important cause of NIDDM (WHO Study Group, 1994).
It is now generally accepted that both degree and duration of
obesity are associated with an increased risk of NIDDM.
Moreover, visceral adiposity represents a risk factor for the
development of NIDDM independently of the degree of
overweight. As a matter of fact, obesity, particularly visceral obesity, induces resistance to the action of insulin at its
target tissues (probably due to high non-esterified fatty
acid (NEFA) concentrations) thus deteriorating glucose
tolerance in the presence of an impaired insulin secretion
(this might also be secondary to high NEFA concentrations).
Weight reduction can lower the risk of NIDDM and, in
persons already affected by the disease, it can ameliorate
insulin sensitivity and glucose tolerance.
Dietary factors for treating diabetes should be able to
limit glycaemic excursions, which are difficult to keep
under control, even with the use of the available glucose
lowering medications, and which negatively affect the
overall blood glucose control. In addition, the diabetes
diet should be able to improve the cardiovascular risk profile
by reducing plasma lipid levels and blood pressure values,
which are often elevated in diabetic patients. This aim can
be achieved by dietary measures that are in principle
identical to those recommended for the non-diabetic
population (Riccardi & Rivellese, 1991; Diabetes and
Nutrition Study Group EASD, 1995). Nutritional factors
that might possibly play a role in the aetiology of NIDDM
are: (1) a low consumption of fibre-rich foods; (2) a low
consumption of fish; (3) a high consumption of fat,
particularly the saturated type. Two recent epidemiological
studies indicate that low-glycaemic-index (GI) diets (low
glycaemic load) might protect against the development of
NIDDM (SalmeroВґn et al. 1997a, b). However, so far none of
these nutritional factors has been unequivocally proven as
being implicated in the aetiology of NIDDM (Virtanen &
Aro, 1994; Storlien et al. 1996).
2.4. Undernutrition
2.4.1. Definition. A condition of undernutrition or
malnutrition occurs due to consumption of a diet deficient
in one or more food constituents (specific undernutrition), or
insufficient consumption of an otherwise adequate diet
(general undernutrition). This statement is not a tautology; it
makes the point that the adequacy of a diet, the extent to
which it meets the requirements, can only be defined in
terms of functions of the consumer: growth, health, activity
etc. (Waterlow, 1992).
Nutritional deficiency results from an imbalance between
the body’s requirements for nutrients and energy and the
supply of these substrates of metabolism. Specific undernutrition is usually associated with biochemical changes
preceding the clinical sign, thus allowing the early diagnosis
of subclinical or impending deficiency. A general undernutrition is reflected by failure to grow or by loss of weight.
It results usually from a quantitatively inadequate intake.
The two conditions may or may not coexist.
2.4.2. Present position. Despite the extensive understanding of human nutritional requirements, malnutrition
(undernutrition) is one of the main causes of morbidity and
mortality in developing regions of the world, especially in
young children. In technologically advanced societies
undernutrition no longer constitutes a major hazard to
health, but occurs in especially vulnerable groups in various
ways. Some of these are related to the introduction and
widespread use in recent years of techniques such as
parenteral feeding and renal dialysis, or other treatments in
the frame of artificial nutrition. Ageing, chronic alcoholism,
drug abuse or even the medically supervised use of drugs,
and food faddism may lead to deficiency disease states.
2.4.3. Pathophysiology and adaptive responses. Undernutrition may develop gradually (days or months). This
process allows a series of metabolic and behavioural
adjustments that result in decreased nutrient demand. If
the supply of nutrients becomes persistently lower than that
to which the body can adapt, a critical situation supervenes.
Metabolic equilibrium can also be disrupted in aged
individuals during the progression of a disease or as a
result of inadequate therapeutic measures (ToruВґn & Viteri,
2.4.4. Body composition. An undernourished individual
differs in two ways from a normal subject: in the relative
proportions of the various organs and tissues, and in the
chemical composition of the body.
In malnutrition there is a preferential loss of muscle tissue
which in the resting state has a low metabolic activity, while
organs with high rates of activity are relatively well preserved. It is generally accepted that in undernutrition the
proportion of extracellular water is increased. This is in
W. H. M. Saris et al.
contrast to the condition of severe malnutrition in which
excess extracellular fluid is only measurable in about 50 % of
the cases. A decreased share of intracellular water is consistently found in malnutrition and undernutrition. This is
important considering the recent hypothesis concerning the
regulatory role of the cellular hydration state (HaВЁussinger et al.
1993). Therefore, the measurement of the distribution of body
water is fundamental to understanding the changes in body
composition that occur in undernutrition.
It is not inconceivable that undernutrition is associated
with intracellular K or Mg depletion, while moderate
decreases of these cations might be observed in serum. Ca
and P have received very little attention. It seems reasonable
to assume that Ca deficiency may well occur in undernutrition. The intracellular Ca concentration is about 1000 times
lower than the extracellular; the constancy of the cytoplasmic
Ca concentration is of great importance, since it plays a
fundamental role in the integrated control of membrane
permeability, the cellular response to stimulation and intracellular signalling. There seems to be a clear case for
studies on the possibility of P deficiency in undernutrition.
Nevertheless, in the case of muscle wasting, total body K
might be decreased. The low insulin action and diminished
intracellular energy substrates reduce the availability of
ATP and phosphocreatine. This process probably alters
the cellular exchange of Na and K, leading to K loss and
increased intracellular Na. Water accompanies the Na
influx, and although total body intracellular water, as mentioned, is decreased because of losses in lean body mass,
there may occasionally be intracellular overhydration.
These alterations in cell electrolytes may explain, at least
in part, the increased fatiguability and reduced strength of
skeletal muscle.
2.4.5. Geriatric undernutrition. At present, about
12.5 % of the population are over 65 years of age; and in
the next 25 years the number of 80-year-old individuals
may grow to over 20 %. Nutrition and ageing research
yields several unique challenges. Undernutrition may be
common among older persons (morbid consequences) due
Table 4. Prevalence of risk factors for protein–(energy) undernutrition in the population of 65 years and older (Adapted from
Miller et al. 1995)
Risk factor
Social factors
Psychological factors
Physical factors
impaired mobility
needs help with shopping, meal preparation, feeding
visual deficit
poor dentition
chewing difficulty
Functional impairment
impaired instrumental ADL
impaired basic ADL
ADL, activities of daily living.
to under-consumption of macro- and micronutrients, protein
malnutrition being frequently observed, and deficiencies in
the amino acids, glutamine and arginine, should be
Changes in nutrient digestion, absorption or metabolism
may contribute to undernutrition though the nutrients are
within the recommended limits for the general public. Ageing
is associated with impaired immune responses and
increased infection-related morbidity. Protein undernutrition impairs, especially, cell-mediated immunity. Previous
studies substantiate the hypothesis that an optimum intake of
nutrients (protein) leads to a striking reduction of proteindeficiency-induced cardiac failure and decreases the risk of
infection. These findings are of considerable clinical and
public health importance (Chandra, 1992; Miller et al. 1995).
Several factors common among older adults have been
shown to increase the risk of undernutrition (Table 4). The
prevalence of risk factors for protein (energy) undernutrition
in the elderly population ranges between 2 and 69 %.
Despite the common occurrence of protein–energy
undernutrition in older persons, its presence is rarely
recognized and even when it is observed, it is often not
treated (Move & Bohmer, 1991). In Table 5 the major
conditions that appear to be secondary to the development
of undernutrition are listed.
2.5. Conclusions and further research
Obesity. Obesity is a major health hazard, associated
with a great variety of health problems. The research with
respect to body-weight regulation has shifted from the study
of possible abnormalities in the energy balance equation
towards the study of the individual macronutrients. These
concepts, conclusions and recommendations for further
research will be further discussed in section 3.
Insulin resistance syndrome. The IRS may be an
important conceptual link between obesity, NIDDM and
cardiovascular disease. Insulin resistance of severity
comparable with that of NIDDM is thought to be present
in 25 % of non-diabetic individuals. The IRS is associated
with a characteristic dyslipidaemia, the ALP. Impaired lipid
metabolism in the postprandial period (exaggerated postprandial lipaemia) may be the link between insulin
resistance and the ALP.
The present knowledge base is lacking in several
important areas.
What is the basis for the IRS in people of normal body
weight? (This will require epidemiological and genetic
Table 5. Effects of protein undernutrition in ageing (Adapted from
Morley, 1995)
Decubitus ulcers
Immune dysfunction (decreased CD4Гѕ, CD8Гѕ)
Euthyroid sick syndrome
Anaemia (decreased maximum breathing capacity)
Decreased bone mass
Decreased glomerular filtration rate
Weight loss
Functional foods and substrate metabolism
How prevalent is the ALP in non-obese, non-diabetic
The ALP has been described as a genetic condition, and yet
it also appears to be a secondary consequence of the
IRS. Can the genetic and environmental components be
Diabetes. Diabetes can be classified on the basis of its
clinical manifestations and aetiology into type 1 or IDDM,
due to almost complete destruction of the pancreatic b-cells
as a consequence of an auto-immune process, and type 2 or
NIDDM, associated with insulin resistance and impaired
insulin secretion. Two nutritional factors linked with IDDM
are the duration of breast-feeding and the consumption of
N-nitroso-compounds during infancy and by the parents at
the time of conception. Further studies are required, in
particular intervention trials in human subjects, before the
link between these dietary factors and IDDM is fully
established. Type 2 diabetes is associated with ageing and
obesity, particularly visceral obesity. Further research is
necessary to elucidate the mechanisms behind the impaired
insulin resistance, in particular in relation to obesity and
intermediary carbohydrate and fat metabolism.
Undernutrition. Undernutrition is an important risk
factor, being the main cause of morbidity and mortality in
developing regions, especially in children. In the Western
world, undernutrition occurs in some vulnerable groups
especially in the elderly as outlined in section 2.4. The
physiology of undernutrition related to various organ
functions and its influence on immunity as well as
recommendations for further research are outlined in section
Protein undernutrition, in particular, impairs immunity.
Furthermore, with the development of undernutrition,
changes occur in the extra- and/or intracellular concentrations of K, Mg and Ca, leading to a shift in extra- and/or
intracellular fluid and control of membrane permeability
and intracellular signalling. Muscle wasting is frequently
observed and the observed shift in muscle electrolytes may
explain the reported fatiguability and reduced strength.
3. Metabolic conditions related to these chronic diseases
3.1. Body-weight control
3.1.1. Energy balance, macronutrient balance and bodyweight regulation. According to the classical energy balance
equation, obesity develops when the equilibrium between
energy intake and energy expenditure shifts towards a positive
balance. The excess energy is stored in the form of
More recently, evidence has accumulated that energy
balance can only be achieved in the case of macronutrient
balance and interest has shifted to an investigation of the
balances of the different macronutrients, carbohydrates,
fats and proteins, in the aetiology of obesity (Blaak &
Saris, 1995; Frayn, 1995; Hill & Prentice, 1995; Flatt,
1996). Achievement of macronutrient balance requires
that the net oxidation of each nutrient equals the average
amount of the same macronutrient in the diet. Oxidation
of the different macronutrients appears to take place in a
hierarchical manner, some substrates being more readily
oxidized than others. Oxidation of carbohydrates and
proteins tends to vary in response to the recent intake of
each fuel in an autoregulatory manner. Adjustments in
carbohydrate oxidation are capable of efficiently maintaining carbohydrate balance in the face of large changes
in carbohydrate intake. Many experiments confirm this
carbohydrate-driven autoregulation (Hill & Prentice, 1995;
Flatt, 1996). In contrast, in the shorter term fat intake does
not promote its own oxidation when carbohydrate content is
constant. It has been demonstrated that over a 9 h period the
same amounts of fat, carbohydrate and protein are oxidized
whether or not the test meal is supplemented with extra
fat (Schutz & JeВґquier, 1989). The underlying mechanisms
between the differences in macronutrients may be related to
the characteristics of the macronutrient stores. This theory
fits in the framework described by Flatt (1996) and is based
on the fact that the carbohydrate stores of the body are
limited (illustrated in Table 6) and are only capable of
covering oxidation for a few days, which requires a tight
metabolic control of carbohydrate balance. In contrast, the
capacity for fat storage is enormous (Table 6), which
implies that rapid adjustment of fat oxidation to intake is
not necessary. When a diet high in fat is consumed over
longer periods of time, the inability to acutely adjust fat
oxidation will result in a positive energy balance and
expanding fat stores and obesity. This will in turn result in
an increased NEFA release from the expanded fat stores and
an increased fat oxidation, until a situation of fat balance is
again reached. Recent studies indicate that subjects predisposed to obesity, or obese subjects, may have a diminished
ability to oxidize fatty acids (Zurlo et al. 1990; Blaak et al.
1994), which would make them even more susceptible to
positive fat balance on a high dietary fat intake. However,
further long-term well-controlled studies have to be
performed in different types of subjects to obtain more
information on the long-term effect of carbohydrate–fat
exchange on energy expenditure and substrate utilization.
The size of the protein pool, in relation to daily intake,
may lead one to predict that the regulation of protein
balance would resemble more the regulation of fat than
carbohydrate balance, whereas the opposite is true. In this
respect, it may be more realistic to think in terms of the
body’s free amino acid pool since most of the body’s protein
pool is in a relatively inert form (Frayn, 1995).
Also, in several studies it has been shown that the
carbohydrate : fat ratio of the diet may affect food intake.
It has been shown that over a 2-week period compensation is
less accurate when the diet has been diluted by removal of
fat than by removal of carbohydrates (Lissner et al. 1987).
There is now substantial evidence which shows that high-fat
diets undermine the body’s ability to regulate energy intake
Table 6. Macronutrient reserves and daily and annual macronutrient
intake (Data from Flatt, 1996)
Intake (kg/year)
Body content (kg)
Intake (kg/d)
(% body content)
W. H. M. Saris et al.
in line with requirements and that they induce high-fat
overfeeding. Several reports on the relationship between
diet and the prevalence of obesity show that a higher fat
intake is associated with a higher BMI (Gibney et al. 1987;
Miller et al. 1990; Lovejoy & DiGirolamo, 1992). There
remains a debate as to whether high-fat overfeeding is a
passive overconsumption simply due to an energy density
effect or whether carbohydrates play a specific role in
inhibiting food intake (macronutrient effect; Flatt, 1996).
As mentioned earlier, Flatt’s glycogenostatic hypothesis
argues that the body’s different storage capacities for
carbohydrates and fats result in mechanisms which give
priority to the maintenance of stable glycogen levels, and
that changes in these glycogen stores provide the primary
feedback signal to regulate appetite (Flatt, 1996). In contrast, studies by Prentice and colleagues (Prentice & Poppit,
1996) showed that high-fat hyperphagia was eliminated
when diets were manipulated to have the same energy
density. Further studies are necessary to elucidate this issue.
In the whole debate on the interaction of the carbohydrate : fat ratio of the diet and energy metabolism, less
attention has been paid to possible differences of various
types of carbohydrates and fats in the regulation of energy
and macronutrient balance.
3.1.2. Type of carbohydrates. Monosaccharides, disaccharides and starch are defined as �digestible’ carbohydrates
because they are digested and absorbed in the human small
intestine. A second category of carbohydrates, defined as
�non-digestible’, (i.e. dietary fibre) cannot be digested by
intestinal enzymes. This latter category of carbohydrates,
including a fraction of starch called resistant starch, may
still have an important nutritional role because of their
inhibitory effects on food intake (Blundell & Burley, 1987)
and their possible role in weight management (Blundell &
Burley, 1987; Hamilton & Anderson, 1992). This paragraph
will further focus on the metabolic effects of digestible
carbohydrates, also called glycaemic carbohydrates (Food
and Agriculture Organization/World Health Organization,
The ingestion of different types of digestible carbohydrates may lead to varying metabolic postprandial
responses, which implies the possibility that different
types of carbohydrates have varying effects on thermogenesis and substrate utilization. Indeed, differences in
postprandial thermogenesis among various types of digestible carbohydrate have been reported, with sucrose and
fructose being more thermogenic than glucose and readilydigestible starch (Tappy et al. 1986; Blaak & Saris, 1996).
Additionally, carbohydrate oxidation, glycogen formation
and the decrement in lipid oxidation have been reported
to be higher after fructose than glucose ingestion. The
higher thermogenic response with sucrose and fructose
ingestion may be ascribed to differences in post-ingestive
substrate utilization and is probably related to particularities
of fructose metabolism in the liver. The higher increase in
post-ingestive carbohydrate oxidation with fructose and
sucrose compared with glucose may be explained by the
finding that fructokinase (EC, responsible for
fructose phosphorylation, is about ten times as active as the
combined activities of glucokinase (EC and hexokinase (EC, required for glucose phosphorylation
(Blaak & Saris, 1996). Despite an increased carbohydrate
oxidation with fructose, leaving less fructose available for
storage, it has previously been reported that fructose is also
a better substrate for hepatic glycogen synthesis (Nilsson &
Hultman, 1974). Moreover, glycogen formation from fructose requires more energy than glycogen formation from
glucose and starch, and may therefore be of importance in
the higher thermogenic response with fructose and sucrose
as compared with glucose and starch (Blaak & Saris, 1996).
Furthermore, acute thermogenesis studies showed no
differences in thermogenesis after ingestion of an easilydigestible maize starch and glucose (Blaak & Saris, 1996)
or cooked manioc starch and glucose (Ritz et al. 1991),
indicating that there are no differences in the thermogenic
efficiency with which the body handles available carbohydrates with varying chain lengths. All the previously
mentioned studies are acute experimental studies. Evidence
for differences in the effects of digestible carbohydrates on
long-term energy expenditure and substrate utilization is
As indicated earlier the carbohydrate : fat ratio of the diet
has a distinct effect on appetite regulation. In the current
literature there are no indications that various types of
digestible carbohydrates may differ in their effects on
food intake or energy intake. However, it is a popular
belief that starches and mono- and disaccharides may have
varying effects on energy intake and that the attractiveness
of �simple’-sugar-rich foods may promote overeating and
may thereby contribute to the development of obesity
(Yudkin, 1988). In man, taste preferences for both fat and
sugar have been investigated. Studies examining sweetness
have not revealed any difference in sensory functioning
between normal-weight and obese individuals (Grinker,
1978). In addition, several studies indicated a negative
relationship between preference for sweet taste and degree
of body fatness and a strong positive relationship between
body fatness and preferences for fatty foods (Drewnowski et
al. 1985, 1992). This seems consistent with reports on the
relationship between diet and the prevalence of obesity,
showing that a higher fat intake is associated with a lower
carbohydrate and sugar intake, which is in turn associated
with a higher BMI (Gibney et al. 1987; Miller et al. 1990;
Lovejoy & DiGirolamo, 1992; Bolton-Smith & Woodward,
1994). In summary, it can be said that the available literature
indicates a closer regulation of carbohydrate compared
with fat balance and that there is no conclusive evidence
indicating differences in the effects of different types of
digestible carbohydrates on long-term energy and substrate
3.1.3. Type of fat. As for the type of carbohydrate, the
impact of type of fat on substrate metabolism and
balance is often ignored. Modifications in dietary fat
profile may affect body weight and adiposity through
changes in partitioning between oxidation and storage
and/or alterations in membrane structure (Pan et al. 1994).
Significant differences were observed in oxidation of
stearate, oleate and linoleate with the oxidation rates for
oleate sixteen times as high as for stearate (Jones et al.
1985). Furthermore, a low polyunsaturated : saturated fatty
acid (P : S) ratio of the diet was associated with an increased
basal fat oxidation, and a lower contribution of fat to the
Functional foods and substrate metabolism
thermic effect of feeding than a diet with a high P : S ratio in
man, suggesting that the long-chain fatty acid composition
of dietary fat modulates the oxidation of fat and carbohydrate after chronic feeding and after meal feeding (Jones
& Schoeller, 1988). Another study showed that obese
subjects consuming low P : S ratio diets exhibited a reduced
contribution of fat oxidation to the thermogenic response,
compared with lean individuals consuming low or high P : S
ratio diets (Jones et al. 1992). Postprandial thermogenesis
has been reported to be higher after a medium-chaintriacylglycerol meal than after a long-chain-triacylglycerol
meal, indicating that besides the long-chain fatty acid
composition of the diet the chain length of the fatty acid
may be important in determining its metabolic effect
(Seaton et al. 1986). Although these results indicate that
the type of fatty acid may affect energy expenditure and
substrate utilization, more (long-term) research is needed
for these effects to result in nutritional implications.
3.1.4. Alcohol. The role of alcohol in human energy
metabolism and human obesity is still a matter of debate.
The consumption of excessive amounts of alcohol is usually
discouraged because this may be a causal factor in the
development and maintenance of obesity. The issues that
receive most attention in the literature are whether alcohol is
as efficiently used as an energy source as other macronutrients, i.e. carbohydrates and fats, and whether alcohol is
added to, or substituted for, non-alcoholic energy in the
diet. Several studies suggest that alcohol consumption may
increase the risk of a positive energy balance and overweight (MacDonald et al. 1993; Tremblay et al. 1995).
However, other epidemiological surveys show a negative
association between alcohol consumption and adiposity and
body weight (Colditz et al. 1991; MacDonald et al. 1993).
Numerous studies have shown that the efficiency of use of
alcohol for the maintenance of metabolizable energy is
the same as for carbohydrates (Westrate et al. 1990;
MacDonald et al. 1993; Sonko et al. 1994), whereas others
have shown that ingested ethanol is less efficiently used as
an energy source (Suter et al. 1992; MacDonald et al.
1993; Klesges et al. 1994). Other studies have shown that
alcohol may have a fat-sparing effect (Suter et al. 1992),
indicating that when consumed in excess this may
promote fat gain, especially upper body fat. Although
results remain controversial, alcohol does not seem to be a
major determinant of body weight when consumed in
moderate amounts, suggesting that replacing a moderate
amount of energy from carbohydrates and fats with alcohol
in non-alcoholics is not expected to decrease energy
retention significantly or to be useful as an adjuvant in
weight-reducing regimens.
3.1.5. Macronutrient replacement. Macronutrient substitutes are ingredients which are added to foods to replace
macronutrients such as various digestible carbohydrates and
fats in volume and in their technological functions (Finley &
Leveille, 1996). The replacement may help the consumer to
achieve dietary objectives, e.g.:
reduced energy intake for reduction of body weight or
maintenance of a normal body weight;
reduced and modified fat intake (reduced cholesterol and
saturated fat intake);
reduction of the risk of developing dental caries by
replacing sugars with polyols.
Carbohydrate replacement. Carbohydrates such as
sucrose and glucose are bulking agents in foods such as
chocolate, candies, cookies and cakes. Their replacement
can be achieved by using a carbohydrate of similar or lower
sweetness and similar taste but different physiological
properties (e.g. reduced absorption and digestibility in the
small intestine). In this respect polyols (sugar alcohols) have
to be mentioned first (BaВЁr et al. 1994). Complex polymeric
carbohydrates e.g. polydextrose or inulin in combination
with intensive sweeteners are the second group of bulking
agents which can replace sugars in special cases (Finley &
Leveille, 1996).
Metabolic studies of polyols have been reviewed elsewhere (Schiweck & Ziesenitz, 1996) and can be summarized as follows: monosaccharide-derived polyols are
more slowly absorbed from the small intestine than glucose.
The absorption is thought to take place by means of passive
diffusion along a concentration gradient. The rate of
absorption differs among the polyols. Erythritol is well
but not totally absorbed, whereas mannitol, sorbitol and
xylitol are absorbed only slowly and incompletely. The
absorbed part of the polyol is either excreted unchanged,
mainly in the urine as in the case of erythritol and mannitol,
or is converted to fructose by specific dehydrogenases, as
found for sorbitol and, partly, mannitol. Xylitol is oxidized
to xylulose by a polyol dehydrogenase and then enters
the normal pentose pathway after phosphorylation by
The disaccharide alcohols isomalt, lactitol and maltitol
are hydrolysed at varying rates from slow and partial
hydrolysis to almost none by the various glycosidases
located in the small intestine. Hydrogenated starch hydrolysates also contain di- and oligomeric polyols which are
hydrolysed to glucose, sorbitol and maltitol.
Most polyols have practically no impact on blood glucose
concentrations and only a moderate influence, if any, on
postprandial serum insulin profile. In the case of maltitol
and hydrogenated starch hydrolysates substantially higher
glycaemic responses were found (Felber et al. 1987).
Significant amounts of ingested polyols reach the large
intestine and the colon, where they are readily fermented by
micro-organisms to H2 , CO2 , CH4 and short-chain fatty
acids (SCFA). The latter lower the pH of the gut content
and may, thereby, influence the composition of the colonic
microbial flora.
The most recent assessment of the energy value of polyols
was that of the Expert Scientific Panel of the Life Sciences
Research Office of the Federation of American Societies for
Experimental Biology (1994). According to that evaluation
the following energy values were given: mannitol 6В·7 kJ/g,
lactitol 6·7–9·2 kJ/g, isomalt approximately 8·4 kJ/g, sorbitol
7·5–13·8 kJ/g, xylitol approximately 10 kJ/g, maltitol and
hydrogenated starch hydrolysates 11·7–13·4 kJ/g.
Fructose as a replacer for glucose and sucrose should be
mentioned in this context as a sweetener for diabetics, due to
its low GI and low insulin response. Nutritional aspects
are reviewed elsewhere (Bowman & Forbes, 1993). Whilst
attention has been paid mainly to the use of polyols as sugar
W. H. M. Saris et al.
Table 7. Polysaccharides as fat replacers (mimic fat)
Further types
resistant starch
gums (xanthan, guar etc.)
replacers, a number of carbohydrates (including polyols) may
also act to partly or totally mimic fat in food (as stabilizers
and thickeners), i.e. as fat replacers. Carbohydrates relevant
in this aspect are summarized in Table 7.
The degree of digestion and absorption of these carbohydrates in the small intestine varies according to the type
of carbohydrate. All poorly digestible carbohydrates have
in common the fact that the part which is not absorbed in
the small intestine reaches the large intestine and is there
fermented, to a greater or lesser extent, by the colonic
microflora to SCFA and gases (H2 , CO2 , CH4 ). Due to the
increase in substrate reaching the large intestine and the
production of SCFA there is also an increase in the osmotic
load in the large intestine leading to an increase of water
within the gut content. These circumstances might be
noticeable for the host by e.g. an increase in flatulence
(gas) or soft-to-watery stools. Certain advantages with
respect to bacterial composition (e.g. bifidogenic effects)
or the properties of some SCFA (e.g. butyric acid and its
possible preventive effects with respect to colon cancer)
should be taken into account as well (Scheppach, 1994), but
to clarify this, or other positive effects on health, further
research work is required.
Fat replacement. Generally, fat replacers are ingredients that are designed to replace all or part of the fat which is
normally in a food without influencing the taste and texture
quality of the food products (Jones, 1996). There are three
categories of such fat replacers as indicated in Table 8.
As fat mimics are carbohydrate- or protein-based ingredients, at least 20 kJ/g less energy is provided compared
with fat. Some of the carbohydrate replacers mentioned
provide even less than 16 kJ/g, making them an important
tool in recipes for energy-reduced foods. Their metabolism
has already been described.
Fat substitutes are physically similar to fats and oils and
therefore heat-stable. In the case of sucrose polyesters there
are up to eight fatty acids (8–22 C atoms, saturated or
Table 8. Categories of fat replacers
Fat mimics
Fat substitutes
Low-energy fats
microparticulated proteins П® carbohydrates e.g.
modified starches
sucrose polyesters
unsaturated) per sucrose molecule (Food and Drug Administration, 1996). The resulting molecules are too big to be
split by lipases and are therefore neither digested nor
appreciably absorbed. The small amount of material that
is absorbed is metabolized to sucrose and fatty acids that are
further metabolized normally in the body. These sucrose
polyesters pass intact through the colon and are not used as a
substrate for the bacterial microflora. The hydrophobic
properties of these sucrose polyesters have influence on
other hydrophobic substances in the gastrointestinal tract.
For example fat-soluble vitamins are not well absorbed and
cholesterol is removed in the faeces in the same way. Due
to the fact that these sucrose polyesters pass through the
small intestine undigested, gastrointestinal effects such as
flatulence and soft stools occur in some people.
Low-energy fats are true triacylglycerols with a new lipid
structure. Triacylglycerols may be composed of mixtures of
long-chain saturated fatty acids and SCFA esterified on a
glycerol backbone. It is stated that their available energy is
approximately 20 kJ/g (Finley et al. 1994). This reduced
energy content is based on the poor absorption of stearic
acid and the lower energy value of SCFA compared with
long-chain fatty acids which normally occur in fats. SCFA
are rapidly absorbed and converted to CO2 . Stearic acid in
the 1- and 3-positions of the triacylglycerol would be
hydrolysed by lipases. Free stearic acid would be poorly
absorbed and stearic acid in the 2-position would be likely
to remain on the glycerol and absorbed as monoacylglycerol, and further be converted to oleic acid (50 % of
the absorbed stearic acid).
Another example of a low-energy fat is a triacylglycerol
prepared by the esterification of glycerol with capric,
caprylic, and behenic acids resulting in caproacrylobehenin
(Life Sciences Research Office/Federation of American
Societies for Experimental Biology, 1991). Due to the
limited intestinal absorption of behenic acid and the lower
energy yield from capric and caprylic acids, an energy value
of 20 kJ/g has been stated. This triacylglycerol is digested,
absorbed, and metabolized by the usual pathways of triacylglycerol metabolism. Its medium-chain fatty acid component is readily absorbed. The long-chain component,
behenic acid, is absorbed more slowly and less completely.
An alternative to fat replacement is fat-binding. Dietary
fat might be sequestered by binding to an appropriate nonabsorbed material. Derivatives of chitin such as chitosan
have been shown to reduce plasma cholesterol concentrations in animals (Sugano et al. 1988), probably by binding
of bile acids and interruption of the entero-hepatic circulation, but not to affect body weight. These derivatives of
chitin and similar products are being widely marketed for
human weight reduction, but we are not aware of scientific
evidence of their efficacy. Their effectiveness might be
estimated by comparison with the pharmacological agent
tetrahydrolipstatin which is an inhibitor of pancreatic lipase
(EC This agent can lead to useful weight loss
(typically a few kg greater than placebo) in controlled
studies, although its usefulness is limited by unwanted
side-effects of fat malabsorption and by reduction in absorption of fat-soluble vitamins (Drent & Van der Veen, 1993).
The idea of preventing absorption of dietary fat or fat
replacement is appealing because it could, in principle,
Functional foods and substrate metabolism
mitigate many adverse features of metabolic diseases. First,
energy intake would be reduced, helping with body-weight
control. The limited number of controlled experiments
that have been conducted with low-fat foods indicate that
they may possibly help to reduce fat intake (Jones, 1996),
although it also has been suggested that the �fat-free foods’
may actually cause consumers to increase consumption
(Rolls & Shide, 1992). Thus, more information on population-based consumption is needed to assess the impact of
such foods in reaching the goal of fat reduction. Second,
there would be a direct reduction of postprandial lipaemia,
and given the evidence reviewed earlier (section 2.2) linking
postprandial lipaemia with the ALP, this is likely to be
beneficial in terms of cardiovascular risk.
3.1.6. Dietary components stimulating thermogenesis.
The most important mechanism controlling thermogenesis
is the activity of the sympathetic nervous system. The
pharmacological approach to enhance metabolic rate has
centred on the development of novel Гџ3 adreno-receptor
agonists (Stock, 1989). However, �natural’ ingredients of
food can also interact with the adrenergic system for
thermogenic stimulation. Minor food constituents such as
caffeine and associated methylxanthines in coffee and tea
have a profound effect on metabolic rate.
Also other minor constituents, such as spices, have
thermogenic properties. Inclusion of these types of �natural’
food ingredients into food products could be a viable
approach in stimulating energy expenditure to keep energy
balance and thus body weight within acceptable limits.
Methylxanthines: caffeine, theophylline and theobromine. Caffeine and other methylxanthines are alkaloids
derived from at least sixty-three species of plants, including
the familiar coffee bean, the tea leaf and the cocoa bean.
Most human societies use caffeine regularly, most often
in beverages, for its stimulant effect and flavour. Caffeine
contents of beverages vary, depending on the plants they
were made from and the food technological methods applied.
The caffeine in cola soft drinks is added, using the
purified compound that is obtained from the decaffeination
of coffee beans. The Food and Drug Administration lists
caffeine as a multipurpose generally recognized as safe
(GRAS) substance that may be added to foods and beverages.
It has been known since 1915 that ingestion of caffeine
provokes an increase in the metabolic rate and subsequent
investigations have confirmed this original observation
(Acheson et al. 1980). Current theories attempting to explain
the diverse pharmacological actions of dietary methylxanthines, favour their actions as antagonists of adenosineinhibitory effects on noradrenaline-induced cyclic AMP
formation. The net result is an elevated cellular level of
cyclic AMP, a critical intracellular mediator for the actions
of catecholamines on thermogenesis. In a study by Dulloo
et al. (1989), the effect of normal caffeine consumption on
thermogenesis was studied. Single-dose oral administration of
100 mg caffeine (equal to a small cup of coffee) increased
the metabolic rate by 3–4 % over 150 min. Measurements
of energy expenditure in a room respiration chamber
indicated that repeated caffeine administration (100 mg)
at 2 h intervals over a 12 h daytime period, increased
energy expenditure by about 10 %. Comparable results
have been found by other groups. Acheson et al. (1980)
also observed an increase in fat oxidation, mediated by an
increased lipolysis leading to higher blood NEFA levels. In a
double-blind placebo-controlled study in moderate habitual
coffee drinkers, Astrup et al. (1989) found increases in energy
expenditure of 38.5, 30.1 and 136 kJ/h with 100, 200, 400 mg
caffeine ingestion respectively. These effects were positively
correlated to plasma caffeine response. In contrast, no significant correlations were found for plasma responses of
theophylline and theobromine.
It is suggested that most people develop caffeine tolerance due to a decrease in the inhibitory effect on adenosine.
However, in the study of Dulloo et al. (1989) habitual
caffeine intake of the subjects was 250–500 mg/d. In the
study of Astrup et al. (1989) habitual intake of caffeine was
100–200 mg/d. Although a certain degree of tolerance to the
thermogenic effect of caffeine may have been developed,
these results suggest that a substantial effect remains during
moderate daily caffeine consumption.
Pungent ingredients of ginger and spices. Ginger is
extensively used as a flavouring additive in foods, beverages
and confectionery. Ginger is known for its apparent ability
to subjectively warm the body.
The pungent principles of ginger are present as two
phenylalanine-derived homologous series: the gingerols
and shogaols (Eldershaw et al. 1992). In a number of
spices such as hot chillies, the compounds capsaicin and
dihydrocapsaicin have been isolated and found to have
thermogenic effects in isolated perfused rat hindlimb
(Cameron-Smith et al. 1990).
From the ginger components it turned out that gingerol
especially induces thermogenesis. Interestingly, this effect
was not inhibited by a- or b-adrenergic antagonists, suggesting that neither adrenergic receptors nor secondary
catecholamine release was responsible for the observed
Gingerols, shogaols and capsaicinoids have some similarities in terms of both structure and function. All contain
the 4-methoxy, 3-hydroxy phenylvanillyl moiety as well as
a carbonyl-containing allyl side-chain. Each group of homologues is responsible for the pungent taste of the parent
The only human study on the thermogenic effects of
spices was performed by Henry & Emery (1980) with chilli
(component: capsaicin) and mustard (component: allyl isothiocyanate). Subjects were given test meals with or
without 3 g mustard sauce and 3 g chilli sauce. Diet-induced
thermogenesis over a 3 h period was 25 % higher after the
spiced meal. This is a substantial increase in thermogenesis
compared with other thermogenic substances. Further
research is needed to investigate the role of spicy ingredients in human nutrition and the metabolic origins of their
effect on diet-induced thermogenesis. The thermogenic
properties are substantial. However, detailed human
research on tolerance and identification of active compounds and metabolic interactions is lacking. Recently,
also the catechin teoline, purified from tea leaves, showed
thermogenic effects that are synergistic with caffeine
(Dulloo et al. 1996).
3.1.7. Physiological and metabolic consequences of
undernutrition. In undernutrition certain functions are
affected and some nutrient reserves decrease, making
W. H. M. Saris et al.
the undernourished individual more susceptible to injuries
that a well-nourished individual can withstand with little
Cardiovascular and renal functions. In severe undernutrition cardiac work decreases, as does functional reserve,
and central circulation takes precedence over peripheral
circulation. Cardiovascular reflexes are altered, leading to
postural hypotension and diminished venous return. Haemodynamic compensation occurs primarily from tachycardia
rather than from increased stroke volume. Renal plasma
flow and glomerular filtration rates may be reduced as a
consequence of the decreased cardiac output, but water
clearance and the ability to concentrate and acidify urine
appear to be unimpaired.
Gastrointestinal functions. Impaired intestinal absorption of lipids and disaccharides and a decreased rate of
glucose absorption occur only in severe protein deficiency.
The greater the protein deficit, the greater the functional
Although the average protein requirement may not differ
with advancing age, at least certain categories of elderly
people have difficulties maintaining N balance when consuming the recommended daily amount (0.8 g/kg per d)
(Young, 1992). To assess more accurately the needs of the
elderly, they are usually evaluated in two age groups (65–75
years and 76 years and older). Distinctions are also made
between healthy elderly people and those with chronic
disease (Durnin, 1992; Morley, 1995). Due to the diminished efficiency of protein utilization in the elderly, the
prudent dietary recommendations should ensure a minimum
intake of 0.9 g/kg.
A decrease in gastric, pancreatic, and bile production is
also observed, with normal to low enzyme and conjugated
bile acid concentrations. These alterations further impair
the absorptive functions. Nevertheless, the ingestion of
nutrients in high, therapeutic amounts usually allows for
their uptake in sufficient quantity to permit nutritional
recovery. Undernourished elderly people are prone to
have diarrhoea probably due to irregular intestinal motility
and gastrointestinal bacterial overgrowth.
The immune system. The major defects are seen only in
severe undernutrition. This seems to involve T-lymphocytes
and the complement system. A marked depletion of
lymphocytes from the thymus and atrophy of the gland
occur. In addition, cells from the T-lymphocyte regions of
the spleen and lymph nodes are depleted, probably owing to
decreases in thymic factors. The production of several
complement components, the functional activity of the
complement system assessed by both the classic and
alternative pathways, and the opsonic activity of serum
are depressed. These deficiencies may explain the high
susceptibility of severely undernourished patients to Gramnegative bacterial sepsis. Phagocytosis, chemotaxis, and
intracellular killing are also impaired, partly due to the
defects in opsonic and complement functional activities.
The B-lymphocyte areas of spleen and lymph nodes and the
circulating levels of B-cells and immunoglobulins are
relatively normal, but there may be defects in antibody
production, such as secretory immunoglobulin A.
The overall consequences of all these alterations in severe
undernutrition are a greater predisposition to infections and
complications of otherwise less important infectious diseases. The defects in immune functions disappear with
nutritional rehabilitation (Chandra, 1992).
3.1.8. Conclusions and further research. There seems
to be a consensus view that a high-fat diet, resulting in a
positive energy and fat balance, is an important risk factor in
the aetiology of obesity. However, more information is
necessary to elucidate whether (moderate) manipulation of
the macronutrient content of the diet may affect body
weight. In theory, epidemiological studies would be the best
way to study these relationships. However, in this type of
study there are too many variables that cannot be controlled.
For this reason, long-term controlled intervention studies
where the carbohydrate : fat ratio and types of carbohydrate
and fat are manipulated would be more suitable. These
studies have to cover at least 6 months, since changes in
body weight are likely to be small. In this type of study acute
experimental mechanistic studies can be included at several
time points since more mechanistic research is necessary to
elucidate the regulation of carbohydrate and fat balance
within the body.
In the latter studies, attention has to be focused on the
issue of whether the different storage capacities for carbohydrate and fat within the body give rise to a specific role of
carbohydrate (stores) in the regulation of appetite (Flatt’s
theory) or whether the obesity-promoting effect of a high-fat
diet is simply a passive overconsumption effect due to the
high energy density of the diet. In mechanistic studies
regarding the regulation of carbohydrate and fat balance,
the type of fat or carbohydrate also has to be taken into
account, since mechanisms behind the relationship of type
of fat or carbohydrate and energy metabolism or substrate
utilization are largely unknown. More specifically, mechanisms behind the increased sucrose-induced thermogenesis
and decrement in fat oxidation and mechanisms behind the
impact of the P : S ratio of the diet on fat oxidation require
further study, since these metabolic effects may have
important consequences for energy and macronutrient balance. Mechanistic studies have to include techniques for
studying intermediary and tissue metabolism in man (stableisotope techniques, tissue balance studies, tissue biopsies,
microdialysis), since only these types of studies will add
information to the existing knowledge of body-weight
regulation in man.
With respect to alcohol, more mechanistic research is
necessary to elucidate whether alcohol is handled in the
body according to the law of thermodynamics (Macdonald
et al. 1993). Additionally, long-term controlled experimental studies are necessary to study the relationship
between alcohol consumption and body weight per se
without confounding variables. However, it does not seem
realistic to regard addition or substitution of alcohol in the
diet in the concept of future functional foods.
With respect to their digestion, most of the macronutrient
replacers discussed herein do have in common that they are
not, or are only partially, hydrolysed and absorbed in the
small intestine. They are partially or completely fermented
in the large intestine by the colonic flora. As a result, these
macronutrients provide less energy to the body than
completely absorbed and metabolized substrates. This
makes them an important tool in the development of
Functional foods and substrate metabolism
energy-reduced foods and an important tool to achieve a
balanced energy and fat intake or a reduction in energy and
fat intake. More research has to be performed to investigate
the long-term effect of these macronutrient replacers, in
particular the fat replacers, on energy and fat balance and on
body-weight control. Attention has to be paid to the suggestion that consumption of fat- or energy-reduced foods
may be compensated by an increased total food intake,
resulting in similar energy intake.
Due to the very limited absorption of the carbohydrateand fat-replacers in the small intestine, the substances are
fermented mostly in the large intestine thereby increasing
the amount of SCFA produced. The energy content provided
by the different fermented carbohydrates as well as the
impact of SCFA like butyric and propionic acids on health
may be a field of further research.
A number of plant ingredients can be identified which
elevate the diet-induced thermogenesis after ingestion. This
elevation is mainly mediated by a prolonged activation of
the sympathetic nervous system leading to an increased
catecholamine release from the sympathetic nerve endings
or an inhibitory effect on the action of adenosine. Both
lead to an increase in cyclic AMP essential for the
cellular increase in metabolism. However, other unknown
mechanisms must also be involved since adrenergic
blockade does not counteract the thermogenic properties
of some ingredients.
From the group of methylxanthines caffeine seems to be
the most potent in thermogenic response after ingestion of a
normal dose of 100 mg (þ 3–4 %). Another interesting
group is the pungent ingredients from spices, such as
ginger, chilli and mustard. With the increased interest in
the non-nutrient compounds such as polyphenols in plants, it
is clear that more ingredients that can increase thermogenesis will be discovered. Some of the known ingredients
have already shown efficacy in the treatment of overweight
Undernutrition affects cardiovascular, renal and gastrointestinal functions. Impaired intestinal absorption of nutrients occurs only in severe conditions, yet in certain
categories of elderly people, maintenance of N balance is
difficult with the recommended daily amount. The immune
system is apparently depressed in (severe) undernutrition.
The defects in immune functions disappear with adequate
nutritional rehabilitation.
In the field of undernutrition, the following points
may need further research: (1) development of analytical
methods (bioassay) and identification of biomarkers may
help in making estimates of nutritional status and of nutrient
requirements; (2) suitable animal and in vitro cell (cell
culture) models would facilitate the investigation of
metabolic handling of essential substrates (absorption,
transport, receptor sites); (3) nutrient interactions are an
essential focus of future studies because of alterations
caused by the ageing process; (4) nutrient mechanisms
that have an impact on genetic expression or immunological function should be examined with modern
methods of molecular biology and immunology. Special
interest should be directed to appraise post-translational
modifications related to various proteins, diets or indispensible
3.2. Insulin resistance/sensitivity
3.2.1. Introduction. As reviewed earlier, the strongest
factor predisposing to insulin resistance is obesity, particularly of the upper body. Therefore nutritional influences on
body weight will also have a profound effect on insulin
sensitivity. Since abdominal obesity is an even stronger
predisposing factor, specific nutritional influences on body
fat distribution are important. However, they are not clearly
understood. Although the concept of the �beer belly’ is
widespread, studies of the effect of alcohol on fat distribution have been conflicting (reviewed by Macdonald et al.
1993). More research is needed on this point. There is some
evidence from animal studies that saturated fats may lead to
intra-abdominal fat accumulation and that n-3 polyunsaturated fatty acids (PUFA) may protect against this (Hill et al.
1993), but no data on this point in human subjects are
available. There is indirect evidence that a high dietary fat
intake is associated with visceral obesity in women (Nicklas
et al. 1995). On the whole, the most consistent explanation
for the accumulation of intra-abdominal fat is an interaction
between lifestyle, including stress factors, and excessive
energy intake leading to obesity (BjoВЁrntorp, 1991a). Thus,
again, relevant dietary factors are those predisposing to
3.2.2. Dietary carbohydrates. In rats, feeding a fructoseenriched diet induces insulin resistance (reviewed in Frayn
& Kingman, 1995). There is no clear evidence for this effect
in human subjects at realistic levels of fructose intake.
High-carbohydrate, low-fat diets are consistently found,
at least in the short term, to raise plasma triacylglycerol
concentrations (see p. S66) and also plasma insulin concentrations (Hollenbeck & Coulston, 1991). This is not
surprising in view of the potentiation of insulin secretion
by carbohydrates, but it has been interpreted as an adverse
change indicative of insulin resistance (Hollenbeck &
Coulston, 1991). This may be an over-interpretation: there
is no prospective evidence that such diets have adverse
consequences on insulin sensitivity or CHD. Their welldocumented beneficial effect on body-weight regulation
(reviewed earlier) is likely to outweigh any possible direct
adverse effect on insulin sensitivity.
A reduction in the GI of the diet may improve insulin
sensitivity. This has been shown indirectly by an improvement in glycaemic control observed in many studies of lowGI diets in NIDDM (Brand Miller, 1994), and directly in
patients with CHD (Frost et al. 1996).
3.2.3. Dietary fat. Clearly excessive intake of fat will
lead to obesity, and possibly specifically visceral obesity,
and thus to development of the IRS. Of more interest are
specific effects of the quality of dietary fat. There is
considerable evidence in experimental animals that saturated fat in the diet may lead to insulin resistance (Vessby,
1995). The effects of saturated fat may be reversed by the
addition of n-3 PUFA (Vessby, 1995; Storlien et al. 1996).
The mechanism is likely to relate to a change in membrane
fluidity affecting processes around the insulin receptor and
recruitment of glucose transporters to the membrane.
In man there is indirect evidence for the same effect. The
phospholipid-fatty acids of skeletal muscle biopsies show a
relationship to insulin sensitivity measured in vivo: a more
W. H. M. Saris et al.
saturated fatty acid pattern is associated with insulin resistance and a high prevalence of PUFA is associated with
increased insulin sensitivity (Storlien et al. 1996). Similar
findings have been made with respect to plasma cholesterylester fatty acids (Vessby et al. 1994). Since these fatty acid
patterns reflect long-term dietary intake, a link between
saturated fat in the diet and insulin resistance is reasonably
firmly established (Fig. 1). Prospective studies of dietary fat
change and insulin sensitivity in human subjects have not
been conclusive, perhaps because periods of study have not
been long enough (Storlien et al. 1996). Thus, it would be
premature at this stage to make any functional claim for an
effect of dietary PUFA on sensitivity to insulin.
3.2.4. Niacin and insulin sensitivity. A common metabolic mechanism in insulin resistance may be an elevated
concentration of NEFA in the plasma (Frayn et al. 1996).
Nicotinic acid (one form of niacin: the other is nicotinamide) exerts a powerful suppressive effect on adipocyte
lipolysis and thus NEFA release, and has been used as an
effective hypolipidaemic agent (Farmer & Gotto, 1995).
However, it has a number of side-effects (Farmer & Gotto,
1995). Over-the-counter niacin preparations are popular in
the USA. Their long-term safety and efficacy have not been
properly evaluated. It might be predicted that a short-term
reduction in NEFA concentrations could improve insulin
Fig. 1. Relationships between insulin sensitivity and the fatty acid
composition of skeletal muscle phospholipids in normal men. The
insulin sensitivity index was derived from a glucose clamp study; units
are mg/m2 per min. Redrawn with permission from Borkman et al.
sensitivity, but in the longer term consistent entrapment of
fatty acids in adipocytes might lead to obesity with adverse
effects on insulin sensitivity. This may not happen in
practice because a well-known aspect of nicotinic acid
action is a marked �rebound’ of plasma NEFA levels
between doses.
3.2.5. Minerals. Some minerals have been associated
with insulin sensitivity.
Chromium. Cr may form a complex with nicotinic acid
in plasma which has been called the glucose tolerance factor
and may be associated with improved glucose tolerance
(Mertz, 1993). It has been suggested that Cr deficiency
might underlie the IRS (Mertz, 1993).
Vanadium. Inorganic and organic compounds containing
V, such as vanadyl sulfate and vanadate, have an insulin-like
effect both in vitro and in vivo (Shechter, 1990). V is available
as an over-the-counter preparation in some Southern American countries. As in the case of Cr supplementation, there are
no prospective human data on safety or efficacy.
Magnesium. There is a large body of evidence showing
low plasma and intracellular Mg concentrations in diabetes.
It is not clear whether these low levels represent Mg
deficiency although it has been suggested that supplemental
dietary Mg may be beneficial both in improving glycaemic
control and in reducing the complications of diabetes
(White & Campbell, 1993). In non-diabetic subjects low
dietary Mg intake has been linked with a number of aspects
of cardiovascular disease including insulin resistance (Ma
et al. 1995).
3.2.6. Conclusions and further research. Insulin sensitivity is closely related to body fat content and body fat
distribution. Thus, factors leading to obesity will increase
insulin resistance, and those leading to upper-body or
visceral obesity will have a greater influence. There are
direct and indirect pieces of evidence that diets based on
low-GI foods may improve sensitivity to insulin. The
evidence linking saturated fat in the diet and insulin
resistance appears to be reasonably firm, based on crosssectional studies, although it has not been proven in longterm intervention studies. Critical areas for future research
are as follows. (1) Long-term prospective studies of the
effects of high-carbohydrate, low-fat diets on insulin
sensitivity and on plasma triacylglycerol concentrations
(see p. S20) are needed in view of suggestions that such
diets might be harmful in these respects. These studies need
to be conducted in subjects with a range of sensitivities to
insulin (e.g. relatives of those with diabetes) and from
different ethnic backgrounds, to illuminate potential gene–
nutrient interactions. The potential of low-GI foods to
improve insulin sensitivity needs further investigation. (2)
Long-term prospective studies of the effects of manipulation of the quality of dietary fatty acids on insulin sensitivity
are also required to investigate whether insulin resistance
can be ameliorated by dietary means. Properly controlled
studies of micronutrient and mineral supplementation
(niacin, Cr, V and Mg) in insulin resistance are required.
3.3. Blood glucose control
3.3.1. Introduction. One of the characteristics of
untreated diabetes is hyperglycaemia. This is not only the
Functional foods and substrate metabolism
cause of a large proportion of the symptoms that heavily
affect the quality of life of these patients but represents also
the primary cause of the long-term specific complications of
diabetes (retinopathy, nephropathy, neuropathy) and is an
important contributor to the excess risk of cardiovascular
disease (The Diabetes Control and Complications Trial
Research Group, 1993). High blood glucose concentrations
(but below the diagnostic level of diabetes) may also
represent a cardiovascular risk factor in the general
population. This has been particularly substantiated in
individuals with impaired glucose tolerance (characterized
by mildly elevated postprandial and normal fasting blood
glucose concentrations), but there are some indications that
even within the normal population blood glucose values in
the upper part of the normal range might represent a
cardiovascular risk factor (Gerstein & Yusuf, 1996). A
recent contribution has shown that near optimal blood
glucose control is able to prevent most cases of microvascular complications occurring in diabetes (Stamler et al.
1993), indicating that preventive strategies to control blood
glucose level may reduce the occurrence of chronic
complications in diabetes.
In order to evaluate the effects of diet on blood glucose
control, it is not sufficient to measure blood glucose concentrations in the fasting state since large fluctuations can
occur throughout the day, particularly in patients with
IDDM. Therefore, reliable information on blood glucose
control needs to be based not only on fasting but also on
postprandial evaluation of blood glucose concentrations;
pre- and postprandial measurements can also be repeated
at each meal. An additional useful marker of glucose control
is the measurement of glycated haemoglobin, as it represents an integrated measure of blood glucose control during
the preceding 2–3 months or, alternatively, fructosamine
which reflects blood glucose levels during the previous 2–3
weeks. Relevant indices of blood glucose control are listed
in Table 9, and any evaluation of functional foods in
facilitating normoglycaemia needs to focus on these
variables (American Diabetes Association, 1997b).
Measurements of plasma insulin concentrations and insulin sensitivity are also relevant to evaluate the effects of
foods on glucose metabolism but they have been considered
The most powerful measure to improve blood glucose
control in overweight diabetic patients is weight reduction.
This is particularly effective in NIDDM patients who are
very often (70–80 %) overweight, a condition which has a
major impact on insulin resistance and, consequently, on
Table 9. Relevant measures of blood glucose control
Fasting blood glucose
Postprandial blood glucose
Daily blood glucose profile*
Glycated haemoglobin†Fructosamine†Oral glucose tolerance test‡
* Several blood glucose measurements performed throughout the day either by
conventional laboratory methods or by strips read on a reflectometer.
†Only in diabetic patients.
‡ Only in non-diabetic individuals or in patients with a milder form of diabetes.
plasma glucose levels. However, in IDDM patients, also, the
presence of overweight (occurring in about 30 % of cases)
impairs the hypoglycaemic effect of exogenously administered insulin, thus hampering the achievement of optimal
blood glucose control (Diabetes and Nutrition Study Group
EASD, 1995; American Diabetes Association, 1996). However, dietary composition can also affect blood glucose
control, particularly in the postprandial period, as discussed
in the next paragraphs.
3.3.2. Nutritional influence on fasting and postprandial
blood glucose levels. Although closely related, fasting and
postprandial blood glucose levels are regulated by mechanisms that are, to some extent, different; in fact, while
postprandial blood glucose concentrations are largely
dependent on meal composition, fasting values are only
minimally influenced by the amount and/or rate of glucose
absorption during the previous meal, and reflect the rate of
glucose production in the liver (the two key processes being
glycogenolysis and gluconeogenesis).
Among the various dietary constituents, the one with the
strongest influence on blood glucose levels in the postprandial period is the amount of digestible carbohydrate in the
diet. Digestible carbohydrates include monosaccharides
(glucose, fructose), disaccharides (sucrose, lactose) and
polysaccharides (starch), which are digested and absorbed
in the human intestine, thus contributing to the glucose
inflow to the bloodstream (Table 10) (Asp, 1996). In
diabetic patients, postprandial blood glucose levels are
directly related to the amount of digestible carbohydrate
in the diet and although they may be regulated by appropriate pharmacological treatment this is not always feasible
or fully successful (Perrotti et al. 1984). Therefore, diets
with a very high content of digestible carbohydrate are not
without problems in the treatment of diabetic patients,
particularly those with IDDM who have a severely impaired
endogenous insulin secretion and are therefore more susceptible to exogenous influences on blood glucose metabolism. On the other hand, a drastic reduction in the intake of
digestible carbohydrates is not feasible since in a weightmaintaining diet, this reduction should be compensated by
an increase in protein or fat intake. Very high intakes of both
protein and fat are not recommended because of their
possible untoward effects on the development of chronic
diabetic complications (Diabetes and Nutrition Study Group
EASD, 1995). Therefore, it is important to identify food
characteristics able to reduce the impact of digestible
carbohydrates on postprandial blood glucose levels.
The glycaemic index. The GI is defined as the incremental blood glucose area after the test product has been
ingested, expressed as a percentage of the corresponding
area after a carbohydrate-equivalent amount of white bread
(Jenkins et al. 1981).
The ratio between mono-, di- and polysaccharides is no
longer regarded as important in relation to the effects on
postprandial blood glucose since amylase and disaccharidase activities in the human duodenum are sufficient to
hydrolyse starch and disaccharides within minutes. The
meal content of protein and fat, although able to influence
postprandial glucose values, has limited clinical significance because the magnitude of these effects is rather small.
More important are all dietary factors able to delay the
W. H. M. Saris et al.
Table 10. Main food carbohydrates and their digestibility in the small
intestine (From Asp, 1996)
glucose, fructose
glucose, galactose
glucose, fructose
fructose, glucose
e.g. raffinose, etc.
modified starches
algal polysaccharides
uronic acids
�New’ carbohydrate food ingredients
various sugar alcohols
* Limited in some individuals when ingested without glucose.
†Except in disaccharidase deficiency.
‡ Resistant starch is indigestible.
process of digestion and/or absorption of carbohydrates in
the intestine, thus reducing the glycaemic response to
carbohydrate-rich foods (Table 11).
Due to the variety of factors that influence the glycaemic
impact of a meal, it is not possible to predict the glycaemic
response of each food on the basis of its physical and
chemical characteristics (Parillo & Riccardi, 1985). Therefore, it is necessary to test carbohydrate-rich foods in vivo to
evaluate their glycaemic response, thus allowing the selection of low-GI foods. Although the procedure used for GI
determination varies in different laboratories and needs to
be standardized (Food and Agriculture Organization/World
Health Organization, 1998), it allows the classification of
carbohydrate-rich foods into broad categories (high,
medium and low glycaemic responses) (Riccardi & Rivellese, 1987). In vitro methods for the prediction of the
glycaemic response to starchy foods seem promising (Granfeldt et al. 1992); however, no methods are at hand to
predict the glycaemic impact of foods containing lowmolecular-mass carbohydrates.
It is now well documented that a diet preferentially
containing low-GI foods improves the metabolic control
in diabetic patients and has a number of other possible
metabolic benefits. In fact, such a diet has been reported to
lower the day-long blood glucose profile, reduce glycated
haemoglobin or fructosamine, and improve glucose tolerance. In addition, in some studies, fasting blood glucose
levels were decreased in diabetic subjects (Brand-Miller,
1994). Moreover, low-GI diets have also been shown to
have beneficial effects on blood lipid metabolism and other
cardiovascular risk factors (for review, see BjoВЁrck, 1996;
Food and Agriculture Organization/World Health Organization 1998). As to the mechanism of these beneficial
effects, the slow rate of digestion and absorption per se,
i.e. the GI features, may be important. In addition, low-GI
foods may be more efficient in suppressing NEFA concentrations between meals, leading to an improved tissue
uptake of glucose when the second meal is ingested 4 h
later (Jenkins et al. 1982).
Cumulative effects of low-GI foods, extending beyond
the improved insulin economy in the acute prandial phase,
may also stem from a more extensive colonic production of
SCFA, since such foods are frequently richer sources of
indigestible carbohydrates. Generation of SCFA through
fermentation might, thus, explain improvements seen in
fasting blood glucose and glucose tolerance at breakfast
when preceded by a low-GI evening meal (Thorburn &
Proietto, 1993).
Effect on fasting glucose. The knowledge of nutritional
factors influencing blood glucose metabolism in the fasting
state is scarce. Liver glucose production, the major
determinant of fasting glucose levels, is under the control
of insulin and therefore not adequately suppressed when
insulin resistance is present; thus nutritional factors
influencing fasting plasma glucose concentrations are
primarily those acting on insulin resistance. In this line it
is well known that a diet with a very high fat and a very low
carbohydrate content increases the production of ketone
bodies, therefore impairing insulin sensitivity and elevating
blood glucose levels, particularly in the fasting state.
However, the relevance of this phenomenon in everyday
life is questionable since such an extreme nutritional
condition rarely occurs.
In relation to different dietary fats there are some
indications that saturated fat could impair insulin sensitivity, thus increasing blood glucose levels. More controversial are the effects of polyunsaturated fats; in fact it
seems that while n-6 PUFA could decrease blood glucose
concentrations, n-3 PUFA (if consumed in large amounts)
could have a hyperglycaemic effect. However, the influence of dietary fat composition on blood glucose control
has limited clinical significance because it is small and has
still to be properly documented in randomized controlled
intervention trials of sufficiently large sample size
(Rivellese et al. 1996).
Non-digestible carbohydrates (resistant starch, NSP, and
oligosaccharides) escape digestion in the small intestine and
are fermented by colonic bacteria in the large bowel
generating SCFA (see p. S19). These metabolites could
influence liver glucose production and, thus, the fasting
glucose concentration.
Functional foods and substrate metabolism
Table 11. Properties of carbohydrate foods that can be utilized to modify the postprandial blood glucose
response (the glycaemic index)
Chemical structure of the digestible carbohydrate
Monomeric composition
Amylose : amylopectin ratio
Physical structure of carbohydrates
Degree of gelatinization
Starch–lipid and starch–protein interactions
Viscous properties of dietary fibre
Food form
Botanical integrity (cells and/or tissue)
Physical structure, e.g. pasta
Other food components/supplements
Viscous dietary fibre
Organic acids
Amylase inhibitors
Finally, alcohol intake also has significant, although
conflicting, clinical effects on plasma glucose levels. In
fact, alcohol intake acutely suppresses hepatic glucose
production thus lowering plasma glucose levels. Conversely, if habitually consumed in large amounts it impairs
insulin sensitivity, thus impairing glucose tolerance and
increasing plasma glucose levels.
3.3.3. Food properties determining the glycaemic
index. As already mentioned, dietary carbohydrates
represent the major dietary constituent influencing blood
glucose control. However, the impact of dietary carbohydrates on glucose metabolism depends not only on the
amount consumed, as believed in the past, but also on some
specific food properties which can profoundly influence the
metabolic effects (Parillo et al. 1985) (Table 12). These
properties are a consequence not only of the ratio
indigestible : digestible carbohydrates, but also of the food
structure and of some specific physico-chemical characteristics of carbohydrates present in the food. This section will
try to illustrate the importance of these properties in relation
to the metabolic effects of different dietary carbohydrates.
Table 12 lists some important properties of carbohydrate
foods that can be utilized for the production of functional
foods with a reduced postprandial blood glucose response,
i.e. a low GI. It should be noted in this context that major
sources of carbohydrates such as potatoes and bread are
characterized by high GI values.
Raw starch granules. Plant cells store starch in semicrystalline granules with variable size and structure. Raw
starch granules are slowly digested by amylases. Cereal
starches with an A pattern on X-ray diffraction analysis
display a rather high digestibility in the small intestine,
whereas B-type granules, e.g. from potatoes or highamylose maize starch, are mainly indigestible (Langkilde
& Andersson, 1994). Formulas with raw maize starch have
been utilized to provide extremely slow-release carbohydrates to children with glycogen storage disease, making it
possible to avoid repeated tube-feeding during the night.
The potential of raw starch in producing low-GI foods is
limited to products produced below the gelatinization or
melting temperature of starch granules.
Gelatinization and retrogradation. Heating of starch in
excess water results in swelling, leakage (especially of
amylose), and eventually disintegration of the granule
structure rendering the starch soluble or dispersible in
water. This process is called gelatinization and occurs at
different temperatures for different starches, usually
between 60 and 80РЉ. Dispersed or soluble starch is highly
susceptible to salivary and pancreatic amylase, which is
present in excess in relation to the final hydrolysis and
absorption at the brush-border level. When, for example,
rolled cereals are produced under mild enough conditions
the starch may be only partly gelatinized. A very low degree
of gelatinization, however, is required to lower the GI of
such products significantly (Granfeldt et al. 1995).
Retrogradation is the process of recrystallization of starch
from a solution. This occurs especially during slow cooling;
amylose forms dense crystals resistant to amylase action,
and staling of bread is related to retrogradation of amylopectin. Amylose retrogradation is a well-documented
mechanism for resistant starch formation, but its effect on
the GI is incompletely understood.
Amylose : amylopectin ratio. Amylose, the virtually
unbranched form of starch, forms double helixes with about
six glucose residues per turn. The interior of the helixes can
accommodate the hydrophobic end of polar lipids, forming
inclusion complexes with reduced availability of the amylose
for enzymic hydrolysis (Holm et al. 1986). This, and the
propensity of retrogradation, are factors behind the usefulness
of high-amylose starch varieties for production of foods with
low GI and/or high resistant starch content.
Cellular structure. Legumes such as beans have low GI
and comparatively high resistant starch content. A main
feature behind these properties is intact cell walls forming
physical barriers to starch digestion even after boiling
(Wursch et al. 1986). A rather high amylose content may
contribute as well. Preservation of the intact cell structure is
essential for keeping these properties in processed foods.
Table 12. Properties of food carbohydrates modifying their effects on
glucose metabolism
Rate of absorption
Type of absorbed monomers
Extent of absorption
Extent and rate of colonic fermentation
Site and metabolites of colonic fermentation
W. H. M. Saris et al.
Gross structure. The presence of intact grains in bread
and other cereal products has been demonstrated to be a
main determinant of the GI as well as a source of resistant
starch (Jenkins et al. 1986; BjoВЁrck et al. 1996). Pasta is
another well-documented group of low-GI foods in which
structural effects are important. In this case a protein
network is responsible for the slow enzymic hydrolysis of
the starch. At least in some studies, the maintenance of an
intact botanical structure also reduces GI features of fruit
products (Haber et al. 1977).
Organic acids. Another factor influencing the glycaemic and insulinaemic impact of foods is the presence of
organic acids produced on fermentation of foods e.g. sourdough baking. The acids appear to differ in gastrointestinal
effect, and whereas some may reduce the rate of starch
digestion, others reduce the gastric emptying rate (Liljeberg
& BjoВЁrck, 1996). Thus, fermentation processes represent
one way of reducing the GI of carbohydrate foods.
Amylase inhibitors. The presence of certain antinutrients such as phytate, polyphenols, and lectins has been
discussed in relation to the low-GI features of legumes
(Thompson et al. 1987). However, due to other effects such
factors can hardly be used for optimizing starch properties
in food products.
Glucose : fructose : galactose ratio. Fructose occurs in
free form in fruits, berries and honey, as part of sucrose and
in high-fructose syrups. Its use as a sweetener is based on its
high sweetness, at least under certain conditions. The low
glycaemic response (GI about 20 % of glucose) has made
fructose an alternative sweetener for use in diabetic diets
(see also section 3.1.6.). The low-GI features of fructose
probably explain the moderate GI range of certain fruits and
the fact that sucrose has a moderate GI.
The main concern about fructose as a sweetener has been
the possibility that it induces elevated triacylglycerol concentrations, relevant in diabetic patients and individuals
with the IRS (Truswell, 1994).
The absorption capacity for fructose is limited when
given alone (Truswell et al. 1988). Together with glucose,
however, which is the normal situation in foods or diets, the
absorption is improved and intolerance problems avoided.
Galactose is part of lactose, but significant amounts of
free galactose are found only in fermented milk products,
due to the preferential utilization of glucose by the microorganisms.
3.3.4. Indigestible carbohydrates and glucose metabolism: possible mechanisms of action. The main types of
indigestible carbohydrates are: dietary fibre (NSP), resistant
starch and oligosaccharides. Although not the determinant
of low-GI features per se, many low-GI foods are rich
sources of these carbohydrates. The possible effects of
indigestible carbohydrates on glucose metabolism may be
related to different upper gastrointestinal events e.g.
reduced motility and/or absorption of carbohydrates due to
viscous properties of dietary fibre components or a reduced
rate of carbohydrate digestion and/or absorption due to
entrapment of the substrate within a fibre matrix at cell or
tissue level. However, the SCFA produced on colonic
fermentation of indigestible carbohydrates are increasingly
being discussed in relation to systemic effects on glucose
and lipid metabolism (Cummings & Macfarlane, 1991). Thus,
carbohydrate fermentation has been reported to enhance
suppression of hepatic glucose production and NEFA levels
in man, leading to lowered fasting blood glucose and
improved glucose tolerance (Thorburn & Proietto, 1993).
Few studies are, however, available on this topic.
NSP. Among the nutritional factors able to influence
blood glucose levels, dietary fibre is certainly the one that
has been most extensively studied. NSP constitute the main
part of dietary fibre in most foods, the daily average amount
in European diets being about 20 g/d (Cummings, 1993).
Plant cell walls are the main source of naturally occurring
NSP, such as cellulose, hemicellulose and pectic substances,
although storage polysaccharides such as inulin and guar
gum, as well as exudate gums, are also indigestible
polysaccharides included in dietary fibre. A large body of
evidence clearly shows that a diet consisting of a high
consumption of fibre-rich foods of natural origin induces
lower blood glucose levels particularly in the postprandial
period in comparison with a diet containing the same
amount of digestible carbohydrate but not rich in fibre
(Riccardi & Rivellese, 1991). More controversial is the
issue of whether dietary fibre represents the marker or the
cause of a low glycaemic response (Nuttal, 1993). It is now
clear that more important than the amount of fibre is the
interaction between fibre and carbohydrates within foods.
The presence of cells with intact walls composed of fibre
polysaccharides is important as they are able to encompass
carbohydrates, slowing their accessibility and thus their
As already mentioned, soluble viscous types of NSP are
those affecting the postprandial glucose and insulin
response after a meal. This has been demonstrated repeatedly for a large number of such polysaccharides when added
to meals or incorporated into foods. The effect is related to
viscosity, which inhibits mixing and diffusion in the intestinal tract and possibly delays gastric emptying, and which
can be abolished by hydrolysis with loss of viscosity. A
main obstacle in utilizing these properties to design functional foods lies in the organoleptic limitations to include
enough viscous polysaccharides. The importance of viscosity in conditioning the GI of food naturally rich in soluble
fibre, however, has been questioned. Even in products with
high fibre content, e.g. flaked oats or bread with oat flour,
structural properties seem more important in obtaining lowGI products. However, even if this is the case, the importance of fibre in conditioning the structural properties of
foods (thus influencing the GI) cannot be neglected.
In addition to their effects on the postprandial blood
glucose response, NSP act as the fermentation substrate
for colonic bacteria with production of SCFA which might
influence liver glucose production.
Resistant starch. Resistant starch, defined as starch and
hydrolysis products thereof that are not absorbed in the
small intestine, has emerged as a main substrate for the
human intestinal microflora. Although the present intake in
Europe seems low, about 4 g/d only (Dysseler & Hoffem,
1994), there is considerable potential for increasing it by
providing food with elevated resistant starch content.
Three main forms of resistant starch have been identified:
(1) physically enclosed starch, (2) resistant B-type starch
granules and (3) retrograded amylose (Englyst et al. 1992).
Functional foods and substrate metabolism
Chemically modified food starches and pyrodextrin are
other forms that may contribute in processed foods. Methods for determination of resistant starch are designed to
estimate the starch residue after treatment of the sample
with enzymes simulating normal starch digestion in the
small intestine. A critical step, not yet fully solved with
any one of the suggested methods, is simulation of the
normal disintegration of foods by chewing. This is essential
to recover the physically enclosed fractions of resistant
The main interest in resistant starch stems from its
properties as a fermentation substrate. Studies with human
faecal flora in vitro have indicated a high yield of butyrate
from resistant starch, which has been supported by some
human in vivo data (Scheppach et al. 1988). The site, extent
and SCFA pattern of fermentation of resistant starch from
various sources need further study. There seems to be considerable potential for designing resistant-starch-containing
foods for specific effects on colonic health (for review, see
Asp & BjoВЁrck, 1992; Asp et al. 1996).
Oligosaccharides. The main types of indigestible
oligosaccharides in foods and food ingredients are:
(1) a-galactosides (raffinose, stacchyose, verbascose)
found mainly in legumes;
(2) fructans (fructo-oligosaccharides, inulin);
(3) galacto-oligosaccharides (derived from lactose); and
(4) pyrodextrins and cyclodextrins.
The present interest in oligosaccharides stems from their
properties as low-energy more-or-less sweet bulk substances (see section 3.1.6.) and their effects as fermentation
substrates producing metabolites with local and/or systemic
effects, as well as prebiotics promoting desirable intestinal
micro-organisms (Mitsuoka et al. 1986).
Fructans have been studied most extensively and shown
to promote bifidobacteria with a concomitant inhibition of
other species such as bacteroides and clostridia. In vitro
studies have indicated a potential to inhibit pathogens. Other
potentially beneficial effects include lower activities of
hydrolytic and reductive enzymes thought to be involved
in colonic carcinogenesis. A comparatively high yield of
butyrate has been reported, which is also of interest in this
Effects of fructans on lipid metabolism (decreased
plasma triacylglycerol and cholesterol concentrations)
have been demonstrated in rats. Human data are still
scarce, however, and the potential of oligosaccharides to
reduce plasma lipids in man needs further exploration. With
respect to glucose metabolism, a daily supplementation with
oligofructose at 8 g/d for 14 d significantly reduced fasting
blood glucose in type II diabetics (Yamashita et al. 1984).
Whereas fructo-oligosaccharides are being studied extensively, the potential of other indigestible oligosaccharides for
prebiotic or systemic effects needs further exploration.
Metabolic effects of short-chain fatty acids. An important mechanism by which food characteristics may influence
glucose metabolism is represented by intestinal fermentation of carbohydrate in the large bowel which may play a
role in modulating glucose metabolism through the
production of SCFA. All carbohydrates (particularly fibre,
oligosaccharides and resistant starch) escaping digestion
and absorption in the small intestine, pass to the large bowel
for subsequent bacterial fermentation. There is some
evidence, mainly from in vitro studies, that the proportion
of the various SCFA (acetate, propionate, butyrate) formed
during fermentation differs depending on the specific
carbohydrate acting as substrate, with concomitant differences in physiological effects (Macfarlane & Cummings,
Several studies show that dietary supplementation of
propionic, acetic and lactic acids may diminish the postprandial glucose and insulin response (Brighenti et al. 1995;
Liljeberg & BjoВЁrck, 1996). Such effects are probably
mediated by mechanisms in the upper gastrointestinal
tract, such as inhibition of gastric emptying or inhibition
of digestive enzymes. Furthermore, long-term propionate
administration has been shown to lower the fasting glucose
concentration (Venter et al. 1990), an effect possibly related
to inhibition of glucose release from the liver.
In spite of extensive fermentation, lactulose was demonstrated in one study (Jenkins et al. 1991) to increase plasma
cholesterol levels in healthy subjects. The high yield of
acetate obtained on fermentation of this substrate was
suggested as an explanation.
The gross energy values for SCFA range from 15 kJ/g for
acetate to 25 kJ/g for butyrate. Of this, 75–85 % is metabolizable. The true energy value of fermented carbohydrate
is dependent on the yield of SCFA as well as the fractions
used for biomass and combustible gas production. Resistant
starch, for example, has been estimated to provide about
8.4 kJ/g fermented substrate (Livesey, 1994).
3.3.5. Conclusions and further research. The recent
progress in understanding mechanisms determining both
rate and extent of carbohydrate absorption have provided
tools for designing foods with specific nutritional effects.
One such tool is represented by the GI concept, and
accumulating data have demonstrated facilitated control
of blood glucose concentrations in diabetes with diets
characterized by low-GI foods. Some recent epidemiological data also suggest the hypothesis that low-GI foods could
be protective against NIDDM. The importance of blood
glucose levels for protein glycosylation implies a potential
role in ageing. Further, low-GI foods may help to reduce
plasma cholesterol concentrations and insulin resistance,
and thus be of more general importance in defeating the
metabolic syndrome.
The emerging knowledge of properties important for
the rate and site of fermentation of indigestible carbohydrates, as well as properties of various fermentation
products, lays the ground for designing foods with optimal
prebiotic effects, with the potential of influencing
also metabolic variables through absorbed fermentation
However, proper clinical testing (also long term) is
required before the functional properties of foods active
on glucose metabolism can be fully assessed.
The European study group on diabetes (EASD) recommended in 1995 an increased use of low-GI foods in diabetes.
Recently, a report (Food and Agriculture Organization/World
Health Organization, 1998) recommended that the bulk of
carbohydrate-containing foods consumed should be those
rich in NSP and with low GI. The lack of variety of such
W. H. M. Saris et al.
foods on the market, not least regarding cereal foods, makes
the development of products with low GI a priority area for
functional food development.
3.4. Plasma triacylglycerols
3.4.1. Introduction. An association between fasting
plasma triacylglycerol concentrations and development of
CHD has been recognized for decades. However, there has
been some controversy over the nature of the link. In
multivariate statistical analyses fasting plasma triacylglycerol concentrations have often been non-significant
contributors compared with plasma HDL-cholesterol concentrations, with which they are strongly negatively related.
More recently, however, meta-analysis has shown that
elevation of the fasting plasma triacylglycerol concentration
is a risk factor for development of CHD even when adjusted
for plasma HDL-cholesterol (Hokanson & Austin, 1996). In
fact, the combination of elevated plasma triacylglycerol and
depressed HDL-cholesterol concentrations is a particularly
strong risk marker for CHD. This is the typical dyslipidaemia
associated with insulin resistance.
In the fasting state the plasma triacylglycerol originates
from the liver, which secretes VLDL particles containing
triacylglycerol. The triacylglycerol in these particles is
hydrolysed by the enzyme lipoprotein lipase (EC
in peripheral tissues which include adipose tissue, skeletal
muscle and myocardium. As the particles shrink they may
be taken up by receptors or they may remain in the circulation, becoming smaller and more cholesterol-enriched
as they lose further triacylglycerol until they are classed as
LDL particles.
After a meal containing fat, dietary triacylglycerol is
packaged within the enterocytes into chylomicron particles,
which are released into the plasma via the lymphatics. The
metabolism of chylomicron particles is similar to that of
VLDL but they are a better substrate for lipoprotein lipase,
and their triacylglycerol is rapidly removed before their
remnants are taken up by receptors. Since the 1940s it has
been realized that postprandial triacylglycerol concentrations may be related to CHD. It is now recognized that the
magnitude and duration of elevated postprandial triacylglycerol concentrations (postprandial lipaemia) is a strong
marker of CHD risk, far stronger, at least in some groups,
than the fasting triacylglycerol concentration (Griffin &
Zampelas, 1995). The inverse relationship between plasma
triacylglycerol and HDL-cholesterol concentrations may be
explained by events in the postprandial period (Frayn,
1993). Increased postprandial lipaemia, i.e. prolonged residence of triacylglycerol-rich particles in the circulation,
leads to the exchange of their triacylglycerol for the cholesterol esters of HDL and LDL particles, leading to loss of
HDL-cholesterol. Thus, low HDL-cholesterol concentrations may be a marker of impaired postprandial triacylglycerol metabolism. Loss of cholesterol esters from LDL
particles makes them smaller and denser, thus accounting
for the third component of the ALP (see section 2.2.).
Dietary components may affect fasting triacylglycerol
concentrations mainly through changes in the rate of hepatic
VLDL-triacylglycerol secretion. The fasting triacylglycerol
concentration is one determinant of the postprandial
lipaemic response, perhaps because VLDL particles containing endogenous triacylglycerol compete with the chylomicrons for clearance by lipoprotein lipase. Therefore
lowering of fasting triacylglycerol concentrations will
usually also reduce postprandial lipaemia. Other dietary
components may affect postprandial lipaemia more directly.
3.4.2. Dietary carbohydrates. When a low-fat, highcarbohydrate diet is introduced, a consistent change is an
elevation of fasting (and in some studies postprandial)
triacylglycerol concentrations (reviewed in Frayn & Kingman, 1995). This has led to the belief that such diets may be
adverse in terms of their effects on blood lipids and thus
CHD (Hollenbeck & Coulston, 1991). However, there is
no evidence that this is so: rather, their beneficial effects
on body weight are probably predominant. The effect on
plasma triacylglycerol concentrations may be specifically
to increase the proportion of large, buoyant VLDL
particles and this might be a beneficial change since such
particles are not major precursors of LDL. However, this is
controversial and this area needs further work.
Ingestion of fructose in large amounts (e.g. 50 g) also has
a marked effect in potentiation of postprandial lipaemia.
The mechanism is thought to be potentiation of hepatic
lipogenesis and thus VLDL secretion, although impairment
of plasma triacylglycerol clearance has also been suggested
(reviewed in Frayn & Kingman, 1995). Again, further work
is needed to elucidate the mechanism of this effect and its
3.4.3. Dietary fat. Diets enriched in n-3 PUFA lead
consistently to lower fasting plasma triacylglycerol concentrations (Nestel, 1990). Other than this the effects of
dietary fat quality are more on plasma cholesterol than
on fasting triacylglycerol concentrations (reviewed by
Hornstra et al. 1998).
Of more interest are dietary factors which may affect
postprandial lipaemic responses, since this is an important
determinant of the dyslipidaemia of the IRS. The greater the
amount of fat eaten, the greater will be the lipaemic
response. Again, different qualities of dietary fat affect
this response, and in particular n-3 PUFA have a strong
effect in diminishing postprandial lipaemia (Griffin &
Zampelas, 1995). PUFA of the n-6 series lead to lesser
postprandial lipaemia than saturated fatty acids, but the
effects of monounsaturated fatty acids are not clear (Griffin
& Zampelas, 1995): more research is needed in this area.
3.4.4. Conclusions and further research. Both fasting
and postprandial plasma triacylglycerol concentrations are
markers of risk of CHD. They are potentially modifiable by
nutritional means. The short-term influence of a low-fat,
high-carbohydrate diet is to increase plasma triacylglycerol
concentrations. Whilst this had led to speculation that such
diets may be disadvantageous in terms of CHD risk, it could
also be argued that if such diets lead to weight loss they will
be beneficial. The quality of dietary fat may be an important
influence. Supplementation with long-chain n-3 PUFA
reduces fasting and postprandial triacylglycerol concentrations, but the effects of other unsaturated fatty acids in this
respect are not well documented.
Critical areas for future research are as follows. It is
critically important that long-term (at least 6 months)
studies of the effects of low-fat, high-carbohydrate diets
Functional foods and substrate metabolism
on plasma lipid constituents are carried out. The confusion
which presently reigns over the effects of such diets is
preventing their widespread acceptance (or avoidance).
Important questions to be answered by such studies are:
(1) Is the elevation of plasma triacylglycerol concentration
on a high-carbohydrate diet transient?
(2) What is the nature of the elevation of plasma triacylglycerol concentrations (large, buoyant or small, denser
VLDL particles) and what is its impact on other lipid
constituents (e.g. the density distribution of LDL
(3) Is it reasonable to apply conclusions from epidemiological studies to the effects of dietary manipulation
within individuals (e.g. an elevated plasma triacylglycerol concentration may be a risk factor for CHD in
epidemiological terms, but does elevation of plasma
triacylglycerol by dietary means confer equivalent
risk?). (This will require prospective studies lasting at
least 5 years with hard end-points such as incidence of
(4) Do beneficial effects of such diets on body-weight
regulation outweigh direct effects on plasma lipid
Studies are needed of the effects of replacement of
saturated fatty acids with monounsaturated fatty acids and
n-6 PUFA, on plasma triacylglycerol concentrations, both
fasting and postprandial. Any such effects must be related to
changes in sensitivity to insulin and genetic factors (see
section 3.2.).
Research is needed into factors acutely affecting postprandial lipaemia, including dietary fructose or sucrose, and
the effects of different types of dietary fat.
4. Nutrition, substrate metabolism and physical
4.1. Introduction
The most important metabolic characteristic of physical
exercise is the increased need for energy. Training or
competition will increase the daily expenditure by 2–
> 4 MJ/h depending on duration and intensity.
Athletes must adapt their food consumption to meet the
energy needs. This increased food intake should be well
balanced, with respect to the macro- and micronutrients.
However, this is not always simple. Many specific athletic
events may be characterized by extremely high exercise
intensities. Running a marathon, for example, costs about
10–12 MJ. Depending on the time needed to finish, this may
induce an energy expenditure of approximately 3 MJ/h in a
recreational athlete and 6 MJ/h in the elite athlete who
finishes in approximately 2–2.5 h. A professional cycling
race, such as the Tour de France, costs about 28 MJ/d, a
value which will be increased to approximately 36 MJ/d
when cycling over several mountain passes (Saris et al.
Compensating for such high expenditures by ingesting
normal solid meals will pose a problem to any athlete
involved in such competitions, since digestion and absorption processes will be impaired during intensive physical
activity. These problems are not exclusively restricted to
competition days. During intensive training days the values
for energy expenditure are impressive as well. In such
circumstances athletes tend to ingest a large number of
�in-between meals’, often composed of energy-rich snacks,
which can be low in protein and micronutrients. As such,
their diet may become imbalanced. Especially adapted
foods and fluids which are easily digestible and rapidly
absorbable may solve this problem. During endurance
sports activity the body will use its own energy stores (fat
stored as adipose tissue-triacylglycerol and carbohydrate
stored as glycogen in liver and muscle). Additionally, small
amounts of functional proteins (in the liver, gastrointestinal
tract and muscle) will be broken down due to mechanical
and metabolic stresses. These losses have to be compensated by supply of the necessary nutrients. At the same time
heat will be produced, which to a large extent will be
eliminated by production and evaporation of sweat. As a
result, fluids and electrolytes will be lost (Brouns et al.
Large sweat losses may pose a risk to health by inducing
severe dehydration, impaired blood circulation and heat
transfer, leading to heat exhaustion and collapse (Sawka
& Pandolf, 1990; Maughan & Noakes, 1991). Insufficient
replacement of carbohydrate may lead to hypoglycaemia,
central fatigue and exhaustion (Wagenmakers et al. 1991).
Inadequate protein intake induces protein loss, especially
of muscle and consequently a negative N balance and a
reduced performance (Lemon, 1991).
These observations show that increased needs for specific
nutrients should be met according to the level of daily
physical activity and exercise. These requirements depend
on the type, intensity and duration of the physical effort.
Depending on these factors, specific nutritional measures
and dietary interventions can be taken, particularly in the
phases of preparation, competition and recuperation.
Some groups of athletes compete in sport events where a
low body weight forms a prerequisite to perform well or to
compete in a certain weight category. These athletes are on
the one hand training frequently and intensively, but on the
other hand they have to maintain a low body weight. The
low energy intakes may in these situations lead to a low
intake of essential nutrients such as protein, Fe, Ca and
vitamins; the required carbohydrate intake to balance the
carbohydrate used in training may also be marginal (Van
Erp-Baart et al. 1989a,b). This aspect should receive special
In the next paragraphs sports nutritional aspects, specifically those related to the macro- and micronutrients which
make up the daily nutrition of individuals involved in heavy
physical work or exercise, will be described (for review, see
Brouns, 1993).
4.2. Carbohydrates
Carbohydrate is the most important nutrient for highintensity performance. Energy release from carbohydrate is
up to three times as fast as from fat. However, carbohydrate
stores in the form of liver and muscle glycogen in the body
are small. This limits the duration of high-intensity exercise.
Apart from decreasing performance, carbohydrate depletion
W. H. M. Saris et al.
induces an increased utilization of protein for energy production (Wagenmakers et al. 1991). This results in the
production of NH3 , which may enhance fatigue. Carbohydrate ingestion during exercise induces sparing of the
body’s carbohydrate stores, reduction of protein utilization
and NH3 production, and a delay of fatigue or improvement
of performance (Costill, 1988; Coyle, 1991a,b; Wagenmakers et al. 1991). Adequate carbohydrate ingestion
between training sessions or intense performance is of
utmost importance to avoid progressive glycogen depletion
and resulting fatigue development or overtraining. Carbohydrate sources to be used during exercise should be rapidly
absorbable, i.e. have a high GI, and should be combined
with sufficient fluid intake.
Factors that determine whether foods are �fast or slow’
carbohydrate sources have been reviewed earlier. Food
properties such as particle size, integrity of cellular structure, dietary fibre content, presence of organic acids, etc.
determine the rate at which the carbohydrates are absorbed.
The differences between starch-containing products disappear when the starch is extracted from the original
source and is ingested as pure starch. Glucose, sucrose
and maltodextrins (glucose polymers) solubilized in water
are all absorbed at similar rates and lead to equal oxidation
rates (Coyle, 1991b; Hawley et al. 1992). Exceptions are
fructose and galactose which are absorbed relatively slowly
and also have lower oxidation rates than the aforementioned
carbohydrate sources. The effects of training and dietary
factors in the modulation of muscle glycogen as well as
substrate utilization have recently been reviewed (Hargreaves, 1995; Brouns, 1997a).
4.3. Fat
Fat is a �slow’ energy source (Newsholme & Start, 1973).
When using fat as a prime energy source, athletes can only
work at 40–60 % of their maximal capacity. Nevertheless,
increased fat utilization, as a result of training, reduces the
use of carbohydrate from the stores in the body, and thus
will influence carbohydrate availability and fatigue (BjoВЁrntorp, 1991b). The idea that high-fat diets lead to adaptations
which enhance fat oxidation during exercise in favour of
glycogen sparing and performance capacity has been shown
in animals. However, there is currently no evidence that
this is also the case in human subjects. This topic has
been reviewed by Brouns & van der Vusse (1998) and
Jeukendrup et al. (1998).
Recently a number of studies have focused on the effect
of medium-chain triacylglycerol (MCT) ingestion on substrate oxidation and performance. The results show that
MCT is rapidly absorbed and oxidized. However, MCT
oxidation does not lead to an increase in total fat oxidation,
nor to a sparing of muscle glycogen. Additionally it was
shown that the amount of MCT which can be consumed
without causing gastrointestinal upset is < 30 g. From these
data is was concluded that MCT is a rapidly available
energy source for athletes but that its consumption does
not lead to measurable performance benefits (Jeukendrup
et al. 1996).
Daily fat intake in athletes should be relatively low,
20–30 % energy, allowing for an increase in the proportion
of carbohydrate in the diet in favour of restoring tissue
glycogen levels after the daily training or competition
sessions (BjoВЁrntorp, 1991b; Coyle, 1991a,b). From a general health point of view, saturated fat sources should be
avoided and vegetable-, fish- and plant-oil-based foods
should be promoted.
4.4. Protein
Protein is needed for muscle growth, repair of tissues and
enzymic adaptations. The protein requirement of athletes is
increased and, according to present knowledge, amounts to
approximately 1.2–1.8 g/kg body weight (Lemon, 1991a,b).
The reason for this increase is an enhanced utilization of
amino acids in oxidative energy production during physical
exercise, a process which is known to be intensified at
higher work levels and in a state of carbohydrate store
depletion (Wagenmakers et al. 1991). Generally, however,
the increased protein requirement is covered by an increased
food intake to cover the daily energy needs.
Studies carried out during the Tour de France, for
example, have shown that the mean daily energy intake
amounted to < 24 MJ (6000 kcal) (Saris et al. 1989; Van
Erp-Baart et al. 1989a). Since the daily protein consumption
(% of energy intake) remained the same, the cyclists
ingested > 3 g protein/kg body weight per d, more than
enough to cover the increased requirement.
However, there are examples of athletes who may need to
be protein-supplemented or need to increase the protein
density of the diet. Athletes who ingest low-energy diets
will generally have low protein intakes, which may not
compensate for the net N loss from the body and will
influence synthetic processes and training adaptations. To
these categories belong, amongst others, body builders,
athletes who have to fit into certain weight categories,
gymnasts, dancers and female long-distance runners, and
under certain circumstances vegetarian athletes (Van ErpBaart et al. 1989a). Protein supplements such as milk and
vegetable-protein hydrolysates may be useful in these
cases. However, one should keep in mind that protein intake
and/or supplementation above levels normally required
(1.2–1.8 g/kg body weight) will not enhance muscle
growth or performance (Lemon, 1991a,b).
The use of single amino acids, such as arginine,
ornithine, tryptophan and branched-chain amino acids to
influence metabolic pathways involved in fatigue development and hormone production, needs further research to
make definite statements, especially because data on the
safety aspects of high intakes of single amino acids in
exercising athletes are generally lacking. Although amino
acids, when supplied intravenously in high dosages, have
been observed to enhance hormone release, there are no
indications that this is also the case after oral ingestion of
amounts which are normally present in supplements.
Recently the ingestion of glutamine has been proposed as
a means of supporting the immune competence of athletes
involved in intense daily training. In addition, positive
effects of branched-chain amino acid ingestion on the
aetiology of central fatigue as well as on immune variables
have recently been suggested. However, the currently
available scientific data do not allow us (yet) to make
Functional foods and substrate metabolism
any claim in these respects (van Hall et al. 1995; Klarlund
Pedersen & Rohde, 1997).
4.5. Fluid and electrolytes
During prolonged physical exercise an adequate supply of
fluid is of prime concern for the performance capacity as
well as the health status of the athlete, especially when
performing in the heat. Progressive fluid loss from the
body, by means of sweating and breathing and in endurance
events also by diarrhoea, is associated with a decreased
blood flow through the extremities, a reduced plasma
volume and central blood volume, a reduction in sweating
and heat dissipation, and under circumstances of high
intensity work in the heat, with heat stroke and collapse
(Sawka & Pandolf, 1990; Maughan, 1991; Maughan &
Noakes, 1991).
Dehydration of > 1.5 litres is known to reduce the O2
transport capacity of the body and to induce fatigue.
Appropriate rehydration is known to counter these effects
and to delay fatigue (Sawka & Pandolf, 1990; Maughan,
1991; Maughan & Noakes, 1991). In contrast to plain water,
the addition of Na and carbohydrate (up to 80 g/l) to
rehydration drinks is known to stimulate water absorption
(Maughan, 1991; Maughan & Noakes, 1991), as well as to
supply energy. Addition of other electrolytes should generally not exceed the levels of loss with whole-body sweat
(Brouns et al. 1992). Sport rehydration drinks should not be
hypertonic because this reduces the rate of net water
absorption (Maughan, 1991). Recently the importance of
inclusion of a high amount of Na in post-exercise rehydration beverages has been underlined by Maughan et al.
(1997). Aspects of dehydration and rehydration have
recently been reviewed by Brouns (1997b).
4.6. Minerals
Exercise is known to be associated with increased mineral
losses, through sweating during exercise and through urine
in the post-exercise phase (Costill, 1988). In general,
athletes may develop an impaired mineral status in cases
of poor selection of food items which may lead to an
inappropriate intake of some minerals, compared with the
daily losses. Impaired Fe, Zn and Mg status are known to
induce malperformance and muscle weakness and are often
associated with the occurrence of muscle cramp. The latter
needs further research to validate the direct influence of
mineral deficits. As with most nutrients, mineral intake
depends on the quality of the diet and the amount of
energy consumed (Van Erp-Baart et al. 1989b). Therefore,
athletes consuming low-energy diets may be at risk of
marginal mineral intake, especially of Zn, Fe and Mg
(Clarkson, 1991). Vegetarian athletes are especially prone
to Fe deficiency. In these cases the ingestion of a daily
supplement may be recommended.
4.7. Trace elements
The importance of an adequate trace element status for
athletic training, performance and recovery has only
received attention relatively recently (Kieffer, 1986). As
with minerals, trace elements are increasingly lost as a result
of intensive physical training. Trace-element losses with
sweat (Cu) and urine (Cr) may exceed the daily recommended intakes. The diet itself may strongly affect these
losses. High carbohydrate intakes, especially of high-GI
carbohydrates, are known to enhance losses of Cr, whereas
diets rich in dietary fibre, often consumed by endurance
athletes and vegetarians, are known to reduce trace-element
absorption. Since science has become aware of the fact that
exercise leads to enhanced tissue and cell damage, the
importance of Se, which acts within the free-radical scavenging processes, has received attention. Much research is
needed in this field, but it is considered that supplementation
with amounts not exceeding the recommended safe daily
intakes will contribute to adequate daily intakes in athletes.
4.8. Vitamins
Vitamins belong among the nutrients which have received
the most attention. Vitamins are essential co-factors in many
enzymic reactions involved in energy production and in
protein metabolism. Any shortage of a vitamin is therefore
linked to sub-optimal metabolism, which in the long term
will result in decreased performance or even illness (Van
der Beek, 1985). Additionally, some of the vitamins act as
antioxidant substances and therefore have a protective role
for tissue and cell integrity, which may be threatened in the
case of metabolic stress (Bendich, 1991).
Vitamin supplementation has, thus, been shown to restore
performance capacity in cases of a vitamin deficit and also
to reduce tissue damage due to free radicals (Bendich, 1991;
Brouns, 1993). Vitamin supplementation with quantities
exceeding those needed for optimal blood levels has never
been shown to improve performance (Van der Beek, 1985).
As with minerals and trace elements, athletes involved in
intensive training, but consuming low-energy diets, are the
most prone to marginal vitamin intakes. In general it can be
concluded that vitamin restoration of energy-dense processed foods for supplementation with preparations will
not enhance performance but may, in athletic populations,
contribute to adequate daily intakes.
Daily intake of a low-dose vitamin preparation or nutrient
preparations, supplying not more than the recommended
daily intake or safe intake, may be advisable in periods of
intensive training or in any situation where athletes abstain
from a normal diet, such as during periods of limited food
intake combined with intensive training (especially in
females and in athletes who have to fit into certain weight
4.9. Ergogenic supplements
Substances such as creatine, caffeine, L-carnitine, aspartate,
NaHCO3 , bee pollen, specific amino acids, etc. have
recently received scientific attention due to their possible
influence on performance, fatigue and recovery. In many
cases such substances need further research to come to
conclusive scientific evidence and recommendations
(Brouns, 1993).
In the case of L-carnitine, for example, it has long been
suggested that the oral intake of this compound leads to
W. H. M. Saris et al.
improved fat oxidation which may improve athletic performance and reduce body-fat levels (Wagenmakers, 1991).
These suggestions were based on in vitro studies. However,
more recent and well-controlled human studies showed no
change in muscle carnitine content after L-carnitine supplementation, nor effects on fat oxidation and performance.
The intake of bee-pollen preparations and of royal jelly
has been said to initiate allergic responses in susceptible
athletes, which may lead to anaphylactic shock and even
death. Since there is no evidence for any performance effects
of bee pollen its use by athletes should be discouraged.
Recently a number of publications have appeared with
respect to creatine. The use of creatine has so far been
shown to be safe and to enhance physical performance in
events lasting 30 s–3 min as well as in the case of repetitive
sprinting. Creatine supplementation (four doses of 5 g/d for
4–6 d) has been shown to increase the muscle total creatine
content in most but not all subjects. It is hypothesized that
this effect causes a better ATP transfer from mitochondria to
cytosol by the creatine shuttle. This results in a reduced
formation of lactic acid and NH3 at a given work load and
improved performance at a freely-chosen maximal workload (Balsom et al. 1994). Creatine is a clear example of a
new functional food ingredient.
Since there is a growing awareness of functional food
ingredients which may affect athletic performance and
health status positively, there will be a significant increase
in the number of scientific studies needed to obtain evidence
to support any functional claim. This development will be
stimulated with the increasing knowledge about nutritional
substances, which are involved in the different metabolic
pathways, including brain metabolism. Judgement of the
role of food-derived substances as being nutritional or
pharmacological, requires research and optimal interaction
between science and the food industry.
4.10. Conclusions and further research
Oral rehydration products for athletes were one of the first
categories in the segment of functional foods and drinks for
which scientific evidence was obtained on all levels of
functionality: rapid gastric emptying, fast intestinal absorption, improved water retention, improved thermal regulation
and improved physical performance and delayed fatigue.
Generally, research on nutrition for athletes has shown
that the exercise-induced stress on the human body depends
on the duration, intensity and type of exercise. Intense
endurance exercise is characterized by changes in the
functionality of the gastrointestinal system. This leads to
the fact that nutrient and fluid supply during endurance
exercise cannot be realized by ingestion of normal meals.
Liquid food formulas, established to deliver fluid and
energy-providing nutrients in a convenient way and easily
digestible form, have been shown to be of benefit to
athletes. Exercise-induced losses of N, minerals, vitamins
and trace elements should be replenished by ingesting
larger amounts of high-quality food with normal meals.
However, this may be problematic in all cases where lowenergy diets are combined with intense training or in the
case of multiple-day competitions such as cycling tours.
The use of special meals or food products and micronutrient
supplementation will help ensure adequate intakes under
these conditions.
The ever increasing amount of daily high-intensity training leads to a high stress on the metabolic machinery, the
musculo-skeletal system and the hormonal system. There is
a growing awareness that the supply of food ingredients or
food-derived substances may interact with the biochemical
and physiological systems involved with physical and
mental performance, as well as with recovery from intensive
training and hence with the physical well-being and health
of the athlete. Therefore it is emphasized that future studies
are directed to the following goals:
(1) to define the safety or toxicity of promising functional
food substances or formulas, to be taken shortly before,
during or after exercise. This should be realized with
respect to short-term, single dosage intake, as well as
long-term chronic intake;
(2) to define the functionality of these substances in terms
of their influence on factors limiting performance and
(3) to support any claim made with respect to such
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б­§ Nutrition Society 1998
British Journal of Nutrition (1998), 80, Suppl. 1, S77–S112
Functional food science and defence against reactive oxidative species
A. T. Diplock1 *, J.-L. Charleux2 , G. Crozier-Willi 3 , F. J. Kok4 , C. Rice-Evans1 , M. Roberfroid5 ,
W. Stahl6 and J. VinЛњa-Ribes7
International Antioxidant Research Centre, UMDS, Guy’s Hospital, St Thomas Street, London SE1 9RT, UK
F. Hoffmann-La Roche Ltd, Business Unit Carotenoids, Headoffice Kaiseraugst VM1 Building, CH04002 Basel, Switzerland
Nestec Ltd, NestleВґ Research Center, Vers-Chez-Les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland
Division of Human Nutrition and Epidemiology, Wageningen Agricultural University, PO Box 8129, NL 6700 EV,
Wageningen, The Netherlands
UCL, Ecole de Pharmacie, Tour Van Helmont, Avenue E. Mounier, B-1200 Brussels, Belgium
Heinrich-Heine-University DuВЁsseldorf, Medizinische Einrichtungen, Institut fuВЁr Physiologische Chemie I, Postfach 10 10 07,
D-40001 DuВЁsseldorf, Germany
Universidad de Valencia, Facultad de Medicina, Departamento de Fisiologia, Avenida Blasco Ibafiez 17, E-46010 Valencia, Spain
1. Introduction
2. Oxidative damage, antioxidant defence and the role of
prooxidants in disease
2.1. Antioxidant defence system of the human organism
2.1.1 Origins and nature of free radicals and
other oxidants
2.1.2. Enzymic and non-enzymic defence systems
in vivo
2.1.3. Dietary antioxidants; nutrient and nonnutrient
2.2. Oxidative damage to bodily functions and its
implications in disease
2.2.1. Coronary heart disease
2.2.2. Carcinogenesis
2.2.3. Cataract and age-related macular
2.2.4. Neuronal diseases
2.3. Conclusions
3. Available methodologies for evaluating and
quantifying ex vivo damage to DNA, lipids and
proteins by prooxidants in vivo
3.1. Oxidative damage to DNA
3.1.1. Measurement of guanine damage products
in DNA by HPLC and gas chromatography–
mass spectrometry (GC–MS)
3.2. Oxidative damage to lipids
3.2.1 Lipid peroxidation
3.2.2. Can some measure of �total’ peroxidation
be obtained?
3.2.3. LDL oxidation
3.3. Oxidative damage to proteins
3.4. Measurement of antioxidant nutrients, carotenoids
and flavonoids extracted from human plasma
3.5. Conclusions
4. Nutritional options modulating oxidative damage and
antioxidant defence systems
4.1. Introduction
4.2. Dietary antioxidants
4.2.1. Sources of dietary antioxidants
4.2.2. Antioxidant intake and status
4.2.3. Bioavailability of antioxidants
4.2.4. Fat intake and antioxidant status
4.3. Epidemiological studies on protective effects of
4.3.1. Cardiovascular disease
4.3.2. Cancer
4.3.3. Other age-related diseases
4.4. Human intervention studies of antioxidants
4.4.1. Cardiovascular disease
4.4.2. Cancer
4.5. Conclusions
5. Potential safety implications related to antioxidant
nutritional enhancement
5.1. Introduction
5.2. Vitamin C
5.3. Vitamin E
5.4. Carotenoids
5.5. Non-nutrient antioxidants (flavonoids and other
related compounds)
5.5.1. Absorption
5.5.2. Possible adverse effects
6. Role of food technology in nutritional and safety
aspects of antioxidants
6.1. Introduction
6.2. Physical processes
Abbreviations: AMD, age-related macular degeneration; ATBC study, a-tocopherol b-carotene study; FOX method, ferrous oxidation in xylenol orange
method; GC–MS, gas chromatography–mass spectrometry; 8-OHdG, 8-hydroxy-deoxyguanosine; 8-OHG, 8-hydroxyguanosine; 8-oxodG, 8-oxo-7,8dihydro-20 -deoxyguanine; PG, prostaglandin; PUFA, polyunsaturated fatty acids; RDA, recommended daily allowance; RNS, reactive nitrogen species;
ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, TBA-reactive substances; a-TE, a-tocopherol equivalents.
* Corresponding author: Professor A. T. Diplock, fax +44 (0) 171 403 7195, tel +44 (0) 171 955 4521.
A. T. Diplock et al.
6.2.1. Structural integrity
6.2.2. Moisture content
6.2.3. Temperature
6.2.4. Minimizing oxygen
6.2.5. Protection from light
6.2.6. Irradiation
6.3. Chemical processes
6.3.1. Enzymes
6.3.2. Supplementation
6.4. Conclusions
7. Critical assessment of the science base and
7.1. Identification of criteria
7.2. Critical evaluation of the present knowledge base S101
7.2.1. Conclusions from section 2
7.2.2. Conclusions from section 3
7.2.3. Conclusions from section 4
7.2.4. Conclusions from section 5
7.2.5. Conclusions from section 6
7.3. Evaluation of criteria
7.4. Final conclusions
8. Recommendations for future research
8.1. Introduction
8.2. Specific recommendations
8.2.1. Oxidative damage and antioxidant defence
systems of the human organism
8.2.2. Ex vivo methodologies for quantitating and
validating damage in vivo to biological
8.2.3. Nutritional options modulating oxidative
8.2.4. Safety implications of nutritional
enhancement of antioxidants
8.3. Priorities for the recommendations made
This paper assesses critically the science base that underpins the argument that oxidative damage
is a significant causative factor in the development of human diseases and that antioxidants are
capable of preventing or ameliorating these disease processes. The assessment has been carried
out under a number of headings, and some recommendations for future research are made based
on the present day knowledge base.
The knowledge database
(1) Consideration of the basic science that underlies understanding of the role of free radicals in
causing cellular pathologies, and the role of antioxidants in preventing this, shows that an
imbalance of reactive oxygen species and antioxidant defence systems may lead to chemical
modifications of biologically relevant macromolecules. This imbalance provides a logical
pathobiochemical mechanism for the initiation and development of several disease states.
Experimental data obtained in vivo provide evidence that antioxidants function in systems that
scavenge reactive oxygen species and that these are relevant to what occurs in vivo. The relevance
in vivo of these observations depends inter alia on knowledge of the uptake and distribution of the
antioxidant within the human body, and on what tissue levels of the antioxidant may be expected
in relation to dietary levels.
(2) There is some way to go until validated precise methods are available for measuring
biomarkers of oxidative damage in human subjects in vivo under minimally invasive conditions.
With respect to oxidative damage to DNA, HPLC and GC–mass spectrophotometry methods
have both merits and limitations. Lipid oxidation products in plasma are best measured as
isoprostanes or as lipid hydroperoxides using specific HPLC techniques. Development of
isoprostane measurement will advance specificity and precision. The measurement of oxidative
damage to proteins has some potential but such methods have not been effectively exploited.
(3) Epidemiological studies support the hypothesis that the major antioxidant nutrients vitamin E
and vitamin C, and b-carotene (which may or may not be acting as an antioxidant in vivo), may
play a beneficial role in prevention of several chronic disorders. More research is needed on the
impact of other non-nutrient compounds, such as other carotenoids and flavonoids, on human
health. In general, human intervention studies using hard end-points are the gold standard. Trials
are restricted mainly to the major antioxidants and do not allow firm conclusions because of
inconsistent findings, an insufficient number of studies and the use of varying doses. There is
evidence that large doses of b-carotene may be deleterious to the health of certain subgroups of
the population such as heavy habitual smokers.
(4) With respect to the safety of administration of supplementary vitamins, vitamin C is safe at
levels of supplementation up to 600 mg/d, and higher levels, up to 2000 mg/d, are without risk.
Vitamin E has a very low human toxicity and an intake of 1000 mg/d is without risk; 3200 mg/d
has been shown to be without any consistent risk. Large intakes of b-carotene must be viewed
with caution because they have been shown to confer detriment to a population at high risk of
lung cancer when administered after many years of high risk (smoking) behaviour. Until further
work clarifies the situation in heavy smokers with respect to taking supplements, larger doses
should be avoided by such individuals. There is little reliable information about the human
toxicology of flavonoids and related non-nutrient antioxidant constituents of the diet.
Defence against reactive oxidative species
(5) The food industry has long experience in the control of oxidative damage in foods and this
experience can be used to advantage for the protection of food antioxidants which are beneficial.
Some of these, such as vitamins C and E and b-carotene, are well known, and strategies for their
protection in foods are already exploited by food technologies. Food technology strategies for the
preservation of those antioxidants which have been shown to be beneficial to health can be
applied in a cost-effective manner.
Research needs
(1) The review of the available scientific database enables the identification of areas where further
research is required. Improvement in dietary antioxidant intake in human populations is expected
to result in lowering of the risk of a number of degenerative diseases. While desirable as an
ultimate objective per se, the impact on public health and the resultant decrease in health-care
costs make it imperative that substantial sums of money should be spent on research in this
important area.
(2) Direct measurement of prooxidants in vivo is difficult or impossible. It is imperative to
establish which are the critical free radical �hits’ that are the relevant ones in the aetiology of
diseases. Are the processes examined really relevant to the disease causation? There is a need to
identify which are the important antioxidants in terms of the maintenance of health, and what is
their relationship to one another. Clarification is needed as to whether it is the antioxidant role of
the substance that is important, or whether it is some other function, and the possible nonantioxidant effects of antioxidants, in particular with respect to modulation of gene expression,
also need further research. The primary aim is to identify the active components in the overall
system that promote health.
(3) Ex vivo methodologies for quantitating and validating damage in vivo to biological
macromolecules urgently need further attention before meaningful work can proceed on
providing evidence of the level of antioxidants needed to maintain health and well-being. It is
necessary to refine and validate methods that are already available for measurement of oxidative
damage in human subjects in a non-invasive manner. Validation in many centres by measurement
of oxidative damage in the same biological material needs to be undertaken using the same
methodology. There is a somewhat longer term need for development of techniques to be used ex
vivo as measures of protein oxidation in vivo.
(4) Nutritional options modulating oxidative damage. For proper epidemiological research, as
well as for human intervention studies, it would be desirable to put special emphasis on the
following. (a) Chemical analysis of the antioxidant content of foods. (b) Studies of bioavailability
of antioxidants from the diet, and the factors that influence the absorption, distribution and tissue
uptake of the compounds and the likely impact of the antioxidants on metabolic processes. (c)
Development and validation of biomarkers of intermediate end-points, both biological response
markers and early disease markers, and emphasis on the relevance of the biomarker to the disease
end-point as well as the disease process. (d) Application of the validated biomarkers of
intermediate end-points in randomized controlled trials testing the efficacy of antioxidants in
functional foods for the maintenance of health and well-being.
(5) Safety implications of nutritional enhancement of antioxidants. The detailed evidence that is
already available which demonstrates that vitamin C and vitamin E are safe at quite high levels of
inclusion in the human diet, means that is unnecessary to recommend further work in this area.
The safety of b-carotene was not questioned before the results of the Finnish and American
intervention studies, which showed an apparent exacerbation in the incidence of lung cancer in
heavy smokers who were given supplements of b-carotene. This observation needs urgent
clarification. With respect to the flavonoids and other polyphenols, it is likely that their
bioactivity will be explored, and the key question of their bioavailability clarified, in the near
future. It will be necessary to examine the safety of such bioflavonoid compounds.
(6) With regard to the role of food technology, there is no particular direction that research needs
to take at the present time. Developments in food technology will be based on, and adapt to,
nutritional recommendations resulting from biologically driven work.
Prooxidants: Antioxidants: Oxidative damage: Diet and health
�Good health is more than the mere absence of disease’.
Mark Twain
1. Introduction
This paper presents a comprehensive assessment of the
literature to the end of December 1997 relevant to the role
and importance of dietary antioxidants in human health.
Section 2 reviews the basic science concerning free-radical
damage and the ameliorating role at the cellular level of
antioxidants, and introduces the concept of maintenance of
health, and the prevention of some major human degenerative diseases. In order to assess the extent of free-radical
damage in human subjects in vivo, and the modifying effects
of antioxidants, it is necessary to have valid, precise
biomarkers. Present literature on this topic is reviewed in
A. T. Diplock et al.
section 3. An account is given in section 4 of the human
epidemiological and interventional evidence that links a
high intake of dietary antioxidants with a low risk of
degenerative disease. If it should be proven with reasonable
certainty that antioxidants do indeed lower the risk of
human degenerative disease, it is essential to be sure that
intervention with antioxidants in the diet, by fortification or
supplementation of foods, is entirely free from harmful sideeffects; this important topic is addressed in section 5, and
the possible contribution of the food industry and food
technologists to this enhancement is discussed in section
6. The 7th and 8th sections assess the �state of the art’ with
respect to the foregoing sections and make recommendations for research in the immediate future.
2. Oxidative damage, antioxidant defence and the role of
prooxidants in disease
2.1. Antioxidant defence system of the human organism
2.1.1. Origins and nature of free-radicals and other
oxidants. The development and existence of an organism
in the presence of O2 is associated with the generation of
reactive oxygen species (ROS), even under physiological
conditions. ROS are responsible for the oxidative damage of
biological macromolecules such as DNA, carbohydrates and
proteins (Halliwell & Gutteridge, 1989; Sies, 1991; Halliwell,
1996). These processes are discussed as pathobiochemical
mechanisms involved in the initiation or progression phase of
various diseases (Diplock, 1994; Wiseman & Halliwell, 1996).
Some of the most relevant ROS are: peroxyl radicals (ROOШ’ ),
the nitric oxide radical (NOШ’ ), the superoxide anion radical
2 ), singlet oxygen ( O2 ), peroxynitrite (ONOO ), and
hydrogen peroxide (H2 O2 ). ROS are either radicals (molecules
that contain at least one unpaired electron) or reactive nonradical compounds, capable of oxidizing biomolecules.
Therefore, these intermediates are also called oxidants or
prooxidants (Halliwell & Gutteridge, 1989; Sies, 1991).
There are various sources for specific ROS in the human
organism. However, the superoxide radical anion appears to
play a central role, since other reactive intermediates are
formed in reaction sequences starting with O2Ш’В№ . It is
generated by enzymic one-electron reduction of O from
xanthine oxidase (EC, NADPH oxidase, or by
leakage of the respiratory chain. It has been estimated that
about 1–3 % of the O2 we utilize is converted to Oؒ¹
(Fridovich, 1986; Halliwell, 1996).
H2 O2 is a non-radical reactive species and can easily
diffuse between living cells. It is efficiently converted to
water by the enzyme catalase (EC, a process which
determines its half-life. Recent evidence suggests that H2 O2
is involved in signal transduction regulating the expression
of genes through the nuclear factor kB and apoprotein-1
pathways (Schreck & Baeuerle, 1994; Sen & Packer, 1996).
The most reactive species is the hydroxyl radical with
an estimated half-life of about 10В№9 s. It might be formed
in vivo on high-energy irradiation (e.g. X-rays) by homolytic cleavage of body water or from endogenous H2 O2 in
metal-catalysed processes (Fenton reaction: Fe-catalysed
Haber–Weiss reaction). u.v.-Light is insufficiently
energetic to split water but it can cleave H2 O2 to yield
two molecules of the hydroxyl radical. The high reactivity
of this radical implies immediate reaction at the place where
it is generated.
The peroxyl radical (ROOШ’ ) is relatively long lived
(seconds) with a considerable diffusion pathlength in biological systems. It can be generated in the process of lipid
peroxidation which is initiated by the abstraction of an H
atom from polyunsaturated fatty acids (PUFA); the hydroxyl radical is capable of starting this reaction sequence
(Esterbauer et al. 1992; Reaven & Witzum, 1996).
Further products generated in lipid peroxidation are
alkoxyl radicals (ROШ’ ) and organic hydroperoxides
(ROOH). The latter might rearrange to endoperoxide intermediates which are cleaved to yield aldehydes. The reaction
of aldehydes with amine groups of proteins has been
discussed as a mechanism involved in the modification of
the protein part of lipoproteins.
Singlet molecular oxygen (1 O2 ) is another non-radical
ROS which is suggested to be formed in vivo in lightexposed tissue. Its half-life has been estimated to be 10В№6 s
depending on the nature of the surrounding matrix. 1 O2 can
interact with other molecules either by transferring its
excitation energy or by combining chemically. Preferential
targets for chemical reactions are double bonds; e.g. in
PUFA or guanine in DNA bases (Kanofsky, 1989; Stahl &
Sies, 1993; Cadet et al. 1994).
An interesting ROS which has attracted attention within
the past few years is the nitric oxide radical (NOШ’ ). It is a
signalling compound formed enzymically from arginine and
relaxes smooth muscles in blood-vessel walls resulting in
lowered blood pressure. It is also produced by activated
macrophages contributing to the primary immune defence.
An excess of NOШ’ is cytotoxic. It might react directly with
biomolecules or combine with OШ’В№
2 to form peroxynitrite
(ONOOВ№ ). Peroxynitrite is capable of inducing lipid peroxidation in lipoproteins but might also interfere with
cellular signalling by nitrating tyrosine residues in proteins
(Beckman, 1996; Packer, 1996).
The ROS described here, and also the biological pathways for their endogenous formation, are examples of a
whole class of reactive intermediates and their ways of
generation. It should further be noted that the organism is
also exposed to ROS from external sources. With the diet
many compounds of prooxidant nature, such as quinones
capable of redox cycling, are delivered to the organism. Also
an array of radicals are inhaled with cigarette smoke; ozone, of
which increasing levels are reported due to air pollution, is an
ROS which can oxidize lipids (Pryor et al. 1995).
ROS are also produced in the organism as a part of the
primary immune defence. Phagocytic cells such as neutrophils, monocytes, or macrophages defend against foreign
organisms by synthesizing large amounts of O2Ш’В№ or NO as a
part of their killing mechanism. Several diseases are accompanied by excessive phagocyte activation resulting in tissue
damage which is at least in part due to the activity of ROS.
2.1.2. Enzymic and non-enzymic defence systems in vivo.
To counteract the prooxidant load a diversity of antioxidant
defence systems are operative in biological systems
including enzymic and non-enzymic antioxidants. An
antioxidant has been defined as �any substance that, when
present in low concentrations compared to that of an
Defence against reactive oxidative species
oxidizable substrate, significantly delays or inhibits the
oxidation of that substrate’ (Halliwell & Gutteridge, 1989;
Sies, 1993; Halliwell, 1995).
The major enzymes directly involved in the detoxification
of ROS are superoxide dismutase (EC, scavenging
2 , as well as catalase and glutathione peroxidases (EC which reduce H2 O2 and organic hydroperoxides
respectively. Several subtypes of glutathione peroxidase are
Se-dependent. In animal studies an elevated intake of Se
was associated with protective effects against cancer. Its
preventive effects in man are still under investigation
(Levander & Burk, 1996). Indirect antioxidant functions
are mediated by enzymes that restore endogenous antioxidant levels; e.g. GSH levels are replenished on reduction
of GSSG by glutathione reductase (EC Further,
reactive intermediates produced in reactions of prooxidants
and biological molecules (e.g. epoxides) are conjugated by
phase II detoxification enzymes such as glutathione-Stransferases (EC to favour their excretion. Another
strategy to prevent the formation of ROS is the control of the
levels of free Fe or Cu ions. Metal-binding proteins responsible for the transport of these ions bind them tightly, thus
preventing the initiation of lipid peroxidation or DNA
damage. Some of the most relevant metal-binding proteins
are ferritin, transferrin, and caeruloplasmin.
Various endogenous low-molecular-mass compounds are
also involved in antioxidant defence. GSH, the major
cytosolic thiol, serves as a cofactor for several detoxifying
enzymes (glutathione peroxidases, glutathione-S-transferases), is involved in the reduction of protein disulfides
and additionally scavenges ROS, being oxidized to GSSG.
Other endogenous compounds such as ubiquinol-10, urate,
or bilirubin also exhibit antioxidant activities (Jacob &
Burri, 1996).
2.1.3. Dietary antioxidants; nutrient and nonnutrient. The human diet contains an array of different
compounds that possess antioxidant activities or have been
suggested to scavenge ROS based on their structural
properties. The most prominent representatives of dietary
antioxidants are ascorbate (vitamin C), tocopherols (vitamin
E), carotenoids, and flavonoids. Apart from vitamin C, each
group of these antioxidants consists of a number of
structurally different compounds; e.g. more than 600
different carotenoids have been identified to date and
about fifty of them might occur in the human diet (Sies &
Stahl, 1995; Rice-Evans & Miller, 1996; Rock et al. 1996).
In the diet, there may be synergistic effects of these various
dietary compounds which are difficult to assess at present.
Indeed, the diet may be considered as an orchestra where
interactions between constituents may bring about effects
which are not the necessary properties of the individual
Vitamin C is considered to be one of the most powerful,
least toxic natural antioxidants (Bendich et al. 1986; Weber
et al. 1996). It is water-soluble and is found in high
concentrations in many tissues; human plasma contains
about 60 вђ®mol ascorbate/l. On interaction with ROS it is
oxidized to dehydro-ascorbate via the intermediate ascorbyl
free radical. Dehydro-ascorbate is recycled back to ascorbic
acid by the enzyme dehydro-ascorbate reductase. Thus,
dehydro-ascorbate is found in only very low levels
compared with ascorbate. As a scavenger of ROS ascorbate
has been shown to be effective against the superoxide
radical anion, H2 O2 , the hydroxyl radical, and singlet
oxygen. In aqueous solutions vitamin C also scavenges
reactive nitrogen oxide species efficiently, preventing the
nitrosation of target molecules. The major sources of
ascorbate in the diet are fruits, especially citrus fruits,
kiwi fruit, cherries and melons, and vegetables such as
tomatoes, leafy greens, broccoli, cauliflower, Brussels
sprouts, and cabbage; its content might exceed 100 mg
ascorbate/100 g fresh weight. At low dose levels (100 mg)
the bioavailability values for vitamin C from synthetic and
food sources are very similar (Mangels et al. 1993a); the
efficacy of absorption decreases with increasing dose levels
(Levine et al. 1996). There is evidence from studies in vitro
that vitamin C is capable of regenerating tocopherol from
the tocopheroxyl radical which is formed on inhibition of
lipid peroxidation by vitamin E (Niki et al. 1982, 1985).
This process would allow for the transport of a radical load
from a lipophilic compartment to an aqueous compartment
where it is taken care of by efficient enzymic defence
systems. It should be noted, however, that ascorbate might
also act as a prooxidant in vivo. In the presence of free
transition metal ions (Fe and Cu) and ascorbate the hydroxyl
radical can be generated and initiation of lipid peroxidation
may occur. However, the amounts of free transition metals
in vivo are very small because they are efficiently bound to
proteins. Vitamin C has additional well-established biological functions including cofactor-activity for several
important enzymes (Levine et al. 1996).
The term vitamin E is a generic description for all tocols
and tocotrienol derivatives which exhibit the biological
activity of a-tocopherol (Parker, 1989; Eldin & Appelqvist,
1996; Sokol, 1996; Traber & Sies, 1996). This group of
compounds is highly lipophilic, and operative in membranes
or lipoproteins. Their most important antioxidant function
appears to be the inhibition of lipid peroxidation, scavenging lipid peroxyl radicals to yield lipid hydroperoxides and
a tocopheroxyl radical. The latter is less reactive than
peroxyl radicals towards neighbouring PUFA and acts as a
chain-breaking antioxidant. The tocopheroxyl radical might
be either reduced by ascorbate and GSH or further oxidized
to the respective quinone. Since only small amounts of
tocopheryl quinone are detectable in human blood and
tissues, the regenerative pathway in vivo appears to be
favoured. In comparison with other lipophilic antioxidants,
a-tocopherol is probably the most efficient in the lipid phase
(Niki, 1987). It contains shielding methyl groups adjacent to
the phenolic hydroxyl group and it is optimally positioned in
membranes by its phytyl side-chain, which is located in the
hydrophobic region of the membrane structure. In addition
to its peroxyl-radical scavenging properties, further interactions with ROS have been described, including quenching of
singlet oxygen and interaction with peroxynitrite. The
richest sources of vitamin E in the diet are vegetable oils
(soyabean, maize, cottonseed, and safflowerseed), and products made from these oils such as margarine and mayonnaise. Further, wheat germ, nuts, and some green leafy
vegetables contribute considerable amounts to the vitamin
E supply (Parker, 1989). Vitamin E plasma levels in man are
about 22 вђ®mol/l; the compound is also found in tissues such
A. T. Diplock et al.
as liver, kidney, fat and adrenals. In the liver the RRRisomer of a-tocopherol is preferentially incorporated into
VLDL which are further catabolized in the circulation.
Thus, RRR-a-tocopherol is the major form of vitamin E
in LDL (Traber & Sies, 1996).
Carotenoids are natural colourants with pronounced antioxidant activity (Stahl & Sies, 1993; Olson & Krinsky,
1995). Their chemical properties are closely related to the
presence of an extended system of conjugated double bonds
which is substituted with various endgroups. ROS which are
efficiently scavenged by carotenoids are 1 O2 and peroxyl
radicals (Palozza & Krinsky, 1992). Two different pathways
are operative with respect to the deactivation of 1 O2 :
physical and chemical quenching (Truscott, 1990). Physical
quenching implies the deactivation of 1 O2 by energy transfer from the excited oxygen species to the carotenoid,
yielding a triplet excited carotenoid. The energy of the
excited carotenoid is dissipated through vibrational interactions with the solvent to recover ground state carotenoid.
The carotenoid remains intact in this process and might
undergo further cycles of deactivation. Chemical quenching
contributes less than 0.05 % to total 1 O2 -quenching by
carotenoids but is responsible for the eventual destruction
of the molecule. Carotenoids are the most efficient naturally
occurring quenchers for 1 O2 with quenching rate constants
of about 5–12 × 109 /mol per s. Carotenoids were reported to
scavenge peroxyl radicals by chemical interaction (Kennedy
& Liebler, 1992). It is suggested that carotene radical
intermediates are formed in this process which finally
leads to the destruction of the molecule. Like vitamin E,
carotenoids belong to the group of lipophilic antioxidants
present in lipoproteins such as LDL and HDL. It has been
shown that they are consumed when isolated LDL is
exposed to the process of lipid peroxidation. Their contribution to the antioxidant defence system of LDL is not clear,
since no regeneration pathways for oxidized carotenoids are
known at present. A variety of structurally different carotenoids are present in fruits and vegetables. Some of the major
sources are carrots (a-carotene, b-carotene), tomatoes
(lycopene), citrus fruits (b-cryptoxanthin), spinach
(lutein), and maize (zeaxanthin) (Mangels et al. 1993b).
The absorption and transport processes of carotenoids are
quite complex. Several factors influencing carotenoid bioavailability from food, such as co-ingestion of fat or fibre,
cooking or food processing, have been identified (Erdman et
al. 1993).
Flavonoids are a large group of polyphenolic antioxidants
that occur in several fruits, vegetables, and beverages such
as tea, wine and beer mainly as O-glycosides. They are
efficient antioxidants capable of scavenging radical species
(peroxyl radicals, hydroxyl radical, OШ’В№
2 ) forming a phenoxy
radical (Rice-Evans et al. 1995; Rice-Evans & Miller,
1996). The term flavonoids summarizes a number of
structurally different subgroups including flavanols (catechin, epicatechin), flavonols (quercetin, myricetin, kaempherol), flavanones (naringenin, taxifolin), flavones
(apigenin, hesperetin), isoflavones (genestein), or anthocyanidins (cyanidin, malvidin). Several criteria for optimal
radical scavenging properties of flavonoids have been
postulated based on pulse radiolysis studies. These include
the presence of the 30 ,40 -dihydroxy structure in ring B, the
presence of the 2,3-double bond in conjugation with the 4oxo-group in ring C, and the presence of a 5-hydroxyl group
in ring A with a 3-hydroxyl group and a 4-oxo function in
the C-ring. The antioxidant properties of flavonoids have
been investigated in various studies in vivo and in vitro. It
should be mentioned, however, that the bioavailability of
these compounds is rather poor. They are rapidly conjugated
in phase II detoxification reactions and levels of free
flavonoids in human plasma are very low. Further phenolic
compounds with antioxidant activity are derivatives of
cinnamic acid; e.g. caffeic acid, chlorogenic acid, and
ferulic acid (Rice-Evans & Miller, 1996).
In addition to the flavonoids, a number of other phenolic
compounds of potential interest occur in foods. Thus, olive
oil contains a number of phenolic substances, notably the odiphenol tyrosol, which may contribute to the antioxidant
content of diets rich in olive oil (Kiritsakis, 1990). Similarly,
plants of the Lamiaceae family, notably rosemary, oregano,
sage, mint and thyme, contain a range of potential antioxidants such as carnosol, rosemanol and carvacrol, which can
contribute to the antioxidant potential of the diet (Lagouri &
Boskou, 1996). As with the flavonoids, however, little is
known of the human absorption and tissue distribution of
these compounds.
Several other dietary constituents might also be involved
in the antioxidant defence system either by direct action as
antioxidants or by effects related to the induction of detoxifying enzymes. Enzymes such as glutathione peroxidase
and superoxide dismutase, which require a dietary supply of
Se, and of Cu and Zn respectively, contribute to the overall
oxidative defence mechanism. Some endogenous substances such as urate also add to the antioxidant potential
of living cells, although their significance is only speculative. Enhancement of dietary intake of the minerals identified
may be beneficial when their content in the diet is low.
2.2. Oxidative damage to bodily functions and its
implications in disease
ROS are suggested, or known, to be involved in pathogenic
processes of numerous diseases (Esterbauer et al. 1992; Luis
& Navab, 1993; Diplock, 1994; Sies, 1997) such as
cardiovascular disease, some forms of cancer, cataract,
age-related macular degeneration, rheumatoid arthritis and
a number of neurodegenerative diseases. Oxidative damage
to important biomolecules is a deleterious pathway, but also
influences of ROS on gene regulation or the immune
system might impair bodily functions. There is increasing
evidence from clinical and intervention studies, as well
as from basic research that antioxidants might prevent
or delay the development of disease states. There may
also be particular population groups that will benefit from
enhanced antioxidant intake, such as pregnant women,
neonates and children, senior citizens and, perhaps,
2.2.1. Coronary heart disease. The primary cause for
most cardiovascular diseases is thought to be arteriosclerosis, a multifactorial disease of the artery wall. It is
suggested that in the early stages of arteriosclerosis lipid
deposits, so-called fatty streaks, are formed in the
subendothelial space. There is increasing evidence that
Defence against reactive oxidative species
oxidative stress, particularly oxidation of LDL, is a risk
factor and plays a role in the pathogenic pathway (Berliner
& Heinecke, 1996). LDL oxidation is due to a lipid
peroxidation reaction initiated by free radicals. Separate
investigations of the lipid and protein parts of oxidized LDL
demonstrated that oxidative modifications of both contribute to the proatherogenic properties of oxidized LDL.
Several biochemical mechanisms underlying this effect
have been discussed. These include the formation of foam
cells on the uptake of oxidized LDL via the scavenger
receptor by macrophages resident in the subendothelial area,
release of cytotoxic lipid peroxidation products from
oxidized LDL, or chemoattractant properties of the oxidized
lipoprotein. LDL oxidation is efficiently inhibited by
lipophilic antioxidants of which a-tocopherol appears to
be the most important. Epidemiological studies suggest
preventive effects towards atherogenic lesions to be
associated with an increased uptake of lipophilic antioxidants such as vitamin E or carotenoids (Rimm et al. 1993).
Additional effects of RRR-a-tocopherol, independent of
its antioxidant activity, have been related to the protective
properties of this compound. An early event in the onset of
arteriosclerosis is the migration of smooth-muscle cells
from the media to the intima of the arterial wall followed
by proliferation of these cells. There is increasing evidence
that RRR-a-tocopherol acts as a negative regulator of
smooth-muscle cell proliferation via modulation of protein
kinase C activity. Protein kinase C is an important element
in the signal transduction cascade mediated by growth
factors such as platelet-derived growth factor which are
involved in the control of cell proliferation. It should be
noted that these effects are limited to RRR-a-tocopherol;
RRR-b-tocopherol does not inhibit protein kinase C (Azzi et
ВЁ zer et al. 1995).
al. 1995; O
2.2.2. Carcinogenesis. Carcinogenesis is a complex
multistep process including initiation, promotion and
progression. The generation of ROS is thought to be
linked to tumourigenesis at different levels. Oxidative
damage to DNA has been demonstrated in vitro and in
vivo leading to DNA single or double strand breaks and
DNA cross linking, as well as to chromosomal aberrations
such as breakage or rearrangement. Modified DNA bases
(e.g. hydroxythymidine, or hydroxyguanine) have been
determined after exposure of cells to situations of oxidative
stress. The modification of DNA bases might result in point
mutations, deletions, or gene amplification as a first step of
carcinogenesis. Further, ROS are capable of deactivating
detoxifying enzymes responsible for the scavenging of
potent carcinogens. Data from epidemiological studies
support the idea that antioxidants are preventive in
carcinogenesis by scavenging ROS (Flagg et al. 1995).
Carotenoids exhibit further biological functions which
are not related to their antioxidant activities but might be of
importance with respect to their cancer preventive effects
(Gerster, 1995). It has been shown that provitamin A and
non-provitamin A carotenoids are capable of inhibiting the
growth of transformed fibroblasts (Bertram & Bortkiewicz,
1995). There is increasing evidence that growth arrest is due
to the stimulation of gap-junctional communication
between transformed and surrounding normal cells. These
findings suggest that carotenoids or carotenoid-derived
retinoids play a role in intercellular signalling involved in
growth control. Inhibitory effects of b-carotene and lycopene on cell proliferation have also been described for
several human cancer cell lines (Sharoni & Levy, 1996).
2.2.3. Cataract and age-related macular degeneration.
Oxidative damage and impaired vision have been discussed
in the context of two ophthalmological diseases of the elderly,
cataract and age-related macular degeneration (AMD)
(Schalch, 1992; Taylor, 1993). Senile cataract indicates the
opacity of ocular lenses. Lens proteins are extremely longlived and often show oxidative damage. This is not surprising,
since they are subjected to chronic exposure to light and O2 ,
which is likely to be responsible for the formation of ROS
which might react with lens proteins. As a consequence, the
damaged proteins may aggregate and precipitate, thus losing
their regular function. Supplementation studies support the
hypothesis that a higher intake of vitamins including vitamin
C and vitamin E prevents or delays the development of
cataracts (Seddon et al. 1994b).
AMD is the major cause of visual impairment in Western
countries and affects the anatomical region of the retina with
the highest degree of visual activity. The macular pigment
(yellow spot) represents a colour filter through which light
must pass before detection. The carotenoids lutein and
zeaxanthin are the predominant pigments in this area (Landrum, 1997). Carotenes (hydrocarbon carotenoids) are not
present in the yellow spot. The function of the macular
pigment has not been unequivocally identified but it might
protect against photo-oxidation by blue light, mediated by
excited triplet state molecules, 1 O2 , or superoxide. There are
hints from food-frequency questionnaires that an increased
consumption of food rich in lutein and zeaxanthin is
associated with a diminished risk of AMD (Seddon et al.
1994a). Carotenoids are the most efficient natural compounds scavenging 1 O2 and excited triplet state molecules.
2.2.4. Neuronal diseases. Growing data from experimental models and human brain studies add evidence that
oxidative stress might play a role in the development of
neuronal degeneration related to diseases such as Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease (Kondo, 1996; Simonian & Coyle, 1996).
ROS are capable of inducing both necrosis and apoptosis.
As a consequence of lipid peroxidation membrane rupture
might occur or ion gradients, operative over compartments
which are separated by membranes, might be disturbed.
Neurons might undergo necrotic cell death as has been
demonstrated in cell culture following depletion of
intracellular GSH, the major endogenous antioxidant thiol.
NO has been hypothesized to be an important mediator of
neuronal death under pathological conditions. The ultimate
species responsible for NO toxicity may be peroxynitrite
which is formed by the reaction of the NO-radical with the
superoxide radical.
Beyond the classical aspects of oxidative damage to
biologically relevant molecules as pathological mechanisms
underlying several diseases, and the protective effects of
antioxidants, new fields of research in this area are rapidly
developing. This includes effects of prooxidants and antioxidants on immune functions (Bendich, 1990) and antioxidant and redox regulatory properties on gene expression.
Both mechanisms may be involved in the development of
A. T. Diplock et al.
disease states while protection might be provided by
antioxidants via these pathways.
2.3. Conclusions
An imbalance between ROS and antioxidant defence systems may lead to chemical modifications of biologically
relevant macromolecules like DNA, proteins or lipids which
are possible pathobiochemical mechanisms in the initiation
or development of several disease states. Experimental data
provide evidence that dietary antioxidants scavenge ROS
and are useful in the prevention of these diseases. Epidemiological studies clearly show a correlation between the
increased consumption of food rich in antioxidants and a
decreased risk of several diseases. Thus, an increased intake
of fruits and vegetables can be recommended. Data on
antioxidant supplementation are contradictory. Further
research is necessary to establish whether supplementation
beyond dietary intake levels is of benefit.
3. Available methodologies for evaluating and
quantifying ex vivo damage to DNA, lipids and proteins
by prooxidants in vivo
3.1. Oxidative damage to DNA
The most abundant base alteration induced in DNA by ROS
is the formation of 8-oxo-7,8-dihydro-20 -deoxyguanine (8oxodG). In vivo this DNA base alteration is repaired by
excision and the resulting product 8-oxodG is excreted
unchanged, and independently of diet, into the urine.
Thus, the rate of excretion of 8-oxodG (as given by the
appearance of the metabolite in urine with time) serves as a
biological marker of the integrated rate of oxidative DNA
damage in the whole body.
DNA damage is usually measured in lymphocytes isolated from blood or in urine. Baseline levels of DNA
damage are considered to be important because repair
may be incomplete (one damaged base per 106 bases); the
actual measurement may therefore provide an estimate of
the balance between damage and repair, so the time window
is a crucial consideration here. From studies in vitro it is
known that when DNA is exposed to the activated oxygen
species the product is specific to the oxygen species
involved, thus:
O2 ; ROOШ’ в†’ guanine oxidized
OH в†’ multiplicity of changes to all four bases
2 ; H2 O2 в†’ no base changes
ONOO в†’ xanthine, hypoxanthine, 8-nitroguanine.
There are two types of measurement of oxidative DNA
damage. First, steady-state damage can be measured when
DNA is isolated from human cells and tissues and analysed
for base damage products: it presumably reflects the balance
between damage and DNA repair. Hence a rise in steadystate oxidative DNA damage (e.g. as has been reported in
some human cancerous tissues; Malins & Haimonot, 1991;
Olinski et al. 1992) could be due to increased damage and/or
decreased repair. Second, several DNA base damage products are excreted in human urine, including the nucleoside
8-hydroxy-deoxyguanosine (8-OHdG), 8-hydroxy-adenine
and 7-methyl-8-hydroxyguanine (Ames, 1989; Stillwell et
al. 1989) but the one most exploited is 8-OHdG, usually
measured by a method involving HPLC with electrochemical detection (Ames, 1989; Shigenaga et al. 1994).
The validity of these urinary measurements of oxidative
DNA damage must be considered. The level of 8-OHdG in
urine is presumably unaffected by the diet since nucleosides
are not absorbed from the gut. The question of whether any
8-OHdG is metabolized to other products in man has not
been rigorously addressed. Additionally, it is possible that
some or all of the 8-OHdG excreted in urine may arise not
from DNA, but from deoxyGTP in the DNA precursor pool
of nucleotides. An enzyme has been described which
hydrolyses deoxyGTP containing oxidized guanine to prevent its incorporation into DNA (Mo et al. 1992; Sakumi et
al. 1993). These uncertainties require clarification.
3.1.1. Measurement of guanine damage products in DNA
by HPLC and gas chromatography–mass spectrometry
(GC–MS). As mentioned earlier, 8-hydroxyguanine (8OHG) and 8-OHdG are the products most frequently used as
indicators of oxidative DNA damage. Analysis of 8-OHdG
using HPLC coupled to electrochemical detection (Floyd et
al. 1986), is a highly sensitive technique that is frequently
used after release of 8-OHdG from DNA, usually by
enzymic hydrolysis. GC–MS with selective ion monitoring
has also been used to characterize oxidative DNA base
damage by the identification of a spectrum of products
(Dizdaroglu, 1993a), including 8-OHG, after formic acid
hydrolysis of DNA and derivatization (often by trimethylsilylation) to generate volatile products. When GC–MS is
used to measure modified DNA bases, a quantitative
analysis of these bases in a DNA sample can be achieved
by adding a suitable internal standard to that sample at an
early stage of the analysis, such as before the hydrolysis of
the DNA (Dizdaroglu, 1993b). Stable-isotope-labelled
analogues of the modified bases can also be used as internal
standards (Dizdaroglu, 1993b).
One advantage of the GC–MS approach is that measurement of a wide range of base damage products allows more
accurate quantification of DNA damage and can help to
identify the ROS and reactive nitrogen species (RNS)
that caused the damage (Malins & Haimonot, 1991);
O2 selectively attacks guanine whereas OHШ’ attacks all
four DNA bases. However, the levels of 8-OHdG measured
in DNA by HPLC with electrochemical detection are often
(Halliwell & Dizdaroglu, 1992) (but not always; Lunec
et al. 1994; Herbert et al. 1996) less than the levels of
8-OHG measured by GC–MS with selective ion monitoring. HPLC could underestimate the real amount of 8OHdG in DNA if the enzymic hydrolysis was incomplete;
the action of the exonucleases and endonucleases used to
hydrolyse the DNA may be affected by the modification of
the bases (Halliwell & Dizdaroglu, 1992; Turk &
Weitzman, 1995) and the acid pH often used for nuclease
digestions might induce hydrolysis of 8-OHdG to 8-OHG,
resulting in the loss of HPLC-detectable material. In
contrast, GC–MS might overestimate 8-OHG (and perhaps
other base damage products) as a result of their artifactual
formation during the heating step involved in classical
silylation-based derivatization procedures (Halliwell &
Defence against reactive oxidative species
Dizdaroglu, 1992; Ravanat et al. 1995). A �cold’ derivatization
procedure has been developed that should avoid this
problem (Hamberg & Zhang, 1995). The important factor
is that any necessary heating stages should be done
anoxically: heating DNA bases in the presence of O2
inevitably results in oxidation. Hence some of the claimed
artifacts (Hamberg & Zhang, 1995; Ravanat et al. 1995),
are possibly due to failure to remove O2 . However, it is
difficult to remove O2 completely. Indeed, a major problem
to be considered in all these techniques is the possibility
that DNA is oxidatively damaged during its isolation from
cells and tissues, particularly if phenol-based methods are
used, since oxidizing phenols generate ROS (Claycamp,
1992; Finnegan et al. 1996). However, rigorous control of
isolation procedures and avoidance of phenol in many laboratories (e.g. by studying isolated chromatin or by using
different DNA isolation methods) does not abolish oxidative
damage detected in isolated DNA (Halliwell & Dizdaroglu,
1992; Dizdaroglu, 1993a; Harris et al. 1994; Shigenaga et al.
1994; Finnegan et al. 1996), strongly supporting the view that
there is a low steady-state DNA damage in vivo. Indeed the
presence of a DNA repair enzyme system and the excretion of
base damage products support the view that oxidative damage
really does occur in vivo.
As an alternative means of avoiding possible problems
with derivatization an HPLC method with electrochemical
detection has been developed that allows measurement of 8OHG and three of the other oxidized base products in acidhydrolysed DNA, thus avoiding the need for derivatization.
Liquid chromatography–mass spectrometry techniques are
under development in several laboratories: this is another
approach to avoiding derivatization problems if sufficient
sensitivity can be achieved.
3.2. Oxidative damage to lipids
3.2.1. Lipid peroxidation. There is a range of methods
available for measurement of markers of lipid peroxidation
and products of peroxidation in vivo that can be measured in
blood and urine as indicators of oxidative stress. There are,
however, a number of problems that need to be resolved
before it is possible to be confident that measurements made
are valid and reproducible, and inter-laboratory studies
using the same reference material are urgently needed to
resolve the remaining difficulties. These include variability
of the standards used, and small differences in the technique
employed which can have a marked effect on the result
achieved; furthermore, no single method can, by itself,
provide an unambiguous indicator of levels of lipid
peroxidation, whether by measuring lipid hydroperoxides,
or degradation products therefrom.
Evidence for damage to lipids in vivo is derived from
measurement of peroxides or isoprostanes in blood and
urine. Such indicators of peroxides in vivo are important
vis-a`-vis, for example, the relationship of plasma peroxides
to vessel wall oxidation of LDL in the context of atherosclerosis. The major problem which has yet to be addressed
with some consolidated approach is the differentiation in
identification of peroxides formed as a consequence of in
vivo oxidative stress and those ingested from dietary
Peroxide levels in cells and tissues present a balance
between peroxide formation and peroxide metabolism or
decomposition, i.e. they are essentially a �steady-state’
With respect to measurement of levels of lipid hydroperoxides, a number of methods are available. Ex vivo
measurement of the lipid hydroperoxide products directly
is best achieved by HPLC determination following partitioning of the hydroperoxide into a polar solvent, which
achieves a primary separation between less polar triacylglycerol and cholesterol hydroperoxides and the more polar
free fatty acids and phospholipid hydroperoxides (RiceEvans et al. 1991). Chemiluminescence-based detection
has proved very satisfactory in providing a reliable assay
procedure (Yamamoto, 1994); an alternative is luminolchemiluminescence.
The steady-state levels of peroxides in human body fluids,
such as blood plasma, appear very low, usually < 100 nmol/l.
These data come from assays that measure �real’ lipid
peroxides (Holley & Slater, 1991; Akasaka et al. 1995)
viz by HPLC with chemiluminescence detection rather than
notoriously-unspecific methods such as diene conjugation
or the simple thiobarbituric acid (TBA) test (Halliwell &
Chirico, 1993). HPLC-based TBA tests can, however,
record comparably-low values, provided that butylated
hydroxytoluene is added with the TBA reagents (Halliwell
& Chirico, 1993).
Several other different approaches have been used for
measurements of the lipid hydroperoxide products of
peroxidation of PUFA. The most simple method conceptually involves direct iodometric determination of lipid
hydroperoxide. A further alternative is provided by the
ferrous oxidation in xylenol orange (FOX) method in its
two variants which provide methods for measuring low
levels of soluble hydroperoxides in the aqueous phase, or
lipid hydroperoxides derived from membranes of lipoproteins in the lipid phase (Wolff, 1994). Assays of human
tissues and body fluids by simple �peroxide-determinations’
such as those involving xylenol orange (Jiang et al. 1992) or
iodometric methods (Thomas et al. 1989) could measure
protein peroxides; this could conceivably explain why
levels of alleged �lipid peroxides’ measured by such techniques in human body fluids tend to be higher (often in the
вђ®M range) than those revealed by the more-specific techniques for measuring lipid peroxides that were discussed
earlier (see section 3.2.3).
A more recently introduced type of assay concerns
measurement of isoprostanes which are derived from
PUFA by a non-cyclooxygenase-mediated free-radicalcatalysed mechanism. Formation of the arachidonic acidderived compounds involves formation of four positional
peroxyl radical isomers of the fatty acid which undergo
endocyclization to prostaglandin (PG)G2 -like compounds
that are then reduced to PGF2 -like compounds. Four F2 isoprostane isomers are formed, each of which can, in
theory, comprise eight diastereoisomers. Quantification of
F2 -isoprostanes represents a reliable and useful approach to
assessment of lipid peroxidation and oxidant stress in vivo
(Morrow & Roberts, 1994).
Human body fluids also contain low levels of F2 -isoprostanes, compounds isomeric to prostaglandins that appear to
A. T. Diplock et al.
arise by free-radical oxidation of phospholipids containing
arachidonic acid (Morrow & Roberts, 1994; Morrow et al.
1995). Isoprostanes appear to exist in human plasma largely
esterified to phospholipids rather than �free’, and sensitive
assays to measure them have been described (Morrow &
Roberts, 1994; Wang et al. 1995). Isoprostanes and their
metabolites can be measured in human urine (Morrow &
Roberts, 1994; Morrow et al. 1995) by GC–MS and this
may prove a valuable assay of whole-body lipid peroxidation if a confounding effect of diet can be ruled out. These
compounds are useful �markers’ of lipid peroxidation and
can be measured in plasma (35 (SD 6) pg/ml) and urine
(1600 (SD 600) pg/mg creatinine) of healthy volunteers,
indicative of ongoing lipid peroxidation even in healthy
human subjects (Halliwell, 1996).
3.2.2. Can some measure of �total’ peroxidation be
obtained? Approaches to measurements of �total-body’
lipid peroxidation have been by measurements of urinary
TBA-reactive substances (TBARS), using the HPLC TBA
method (Chirico & Halliwell, 1994), measurements of
hydrocarbon gas excretion and by measurements of F2 isoprostanes in urine. Urinary TBARS measurements have
been found to be confounded by a multiplicity of urinary
constituents that react with TBA, and this problem is further
complicated by contributions from dietary constituents,
particularly cooked meats. Most of the lipid-related TBARS
appearing in urine seem to arise from lipid peroxides or
aldehydes in ingested food, which are presumably largely
generated during cooking (Dhanakoti & Draper, 1987;
Brown et al. 1995). Hence urinary TBARS is not a suitable
assay to assess whole-body lipid peroxidation in response to
changes in dietary composition, although it could theoretically be used to look at effects of supplementary
antioxidants in individuals on a standardized �fixed diet’
(Dhanakoti & Draper, 1987). In any case, HPLC must be
used to separate the real (TBA)2 malondialdehyde adduct
since the majority of the TBARS in urine are not even lipidderived (Gutteridge & Tickner, 1978) or derive from a wide
variety of aldehydes other than malondialdehyde.
Measurement of hydrocarbon gases (alkanes and
alkenes), degradation products of lipid peroxidation in
vivo (Burk & Ludden, 1989; Springfield & Levitt, 1994),
can be confounded by interference from air pollutants and
from the products of gastrointestinal bacterial metabolism.
They are unreliable also because of the low level of the
metabolites to be measured, which challenges the sensitivity
of the assay, and the possibility of metabolism of the
metabolite before excretion, so that the measured amount
represents only a portion of the true level; co-elution of
other metabolites which are indistinguishable from the
products of interest is a further problem; the method is
therefore not considered further here.
There appears to be a general consensus that the most
reliable assay procedures available are the chemiluminescence-linked HPLC determination of lipid hydroperoxides
(Yamamoto, 1994) and the HPLC-linked TBA measurement (Yamamoto, 1994). The isoprostane assay is gaining
momentum and has been excellently reviewed recently
(Morrow & Roberts, 1996).
3.2.3. LDL oxidation. Particular techniques have been
developed to determine the oxidation of LDL and a wide
range of methods is now available (Esterbauer et al. 1992).
A critical review of practical approaches to LDL oxidation
has also appeared recently (Rice-Evans et al. 1996).
The xylenol orange assay or FOX assay describes a
sensitive spectrophotometric system for detecting authentic
peroxides in LDL and has been successfully applied to the
measurement of lipid hydroperoxides in LDL. Hydroperoxides oxidize ferrous to ferric ions in dilute acid and the
resultant ferric ions are determined using ferric sensitive
dyes as an indirect measure of hydroperoxide concentration.
Xylenol orange [o-cresolsulfonephthalein 30 300 -bis (methylimino) diacetic acid sodium salt) binds ferric ions with high
selectivity to produce a coloured (blue-purple) complex
with an extinction coefficient of 1:5 Г— 104 /mol per cm and
absorbance maximum of 560 nm. The method compares
favourably with the iodometric assay, TBA assay and
conjugated diene measurement. No extraction step is
required for analysis of lipoprotein in the 900 ml/l
methanol–25 mM-H2 SO4 environment in which the assay
is performed.
Triphenylphosphine is used as a specific reductant of
hydroperoxides, converting them to the corresponding alcohol. This allows the measurement of authentic hydroperoxides reacting in the assay and removes any background
signal generated. Each sample is therefore measured with
and without triphenylphosphine, the difference between the
two being the lipid hydroperoxides in the sample. Background values for plasma have been reported to be high. The
mean value obtained for native LDL is reported to be 13.3
(SD 8.8) nmol/mg LDL (Rice-Evans et al. 1996). Lipid
hydroperoxides can also be measured easily in LDL using
the tri-iodide assay (El-Sadaani, 1989). The lipids of LDL
are dispersed by the detergent used in an enzymic cholesterol assay kit and the hydroperoxides oxidize IВ№ to I2 which
is detected spectrophotometrically. The values given for
native LDL for the iodometric method correspond to
25 nmol/mg LDL protein (El-Sadaani, 1989), 18.6 (SD
9.4) nmol/mg LDL protein (Esterbauer et al. 1992) and in
the range of 10–20 nmol/mg LDL protein (O’Leary et al.
1992). This sort of range was deemed to be at the borderline
of the detection limit for the iodometric assay (Esterbauer et
al. 1992).
The HPLC procedure for detecting lipid hydroperoxides
has the advantage that the identity and mass of specific lipid
peroxides may be determined down to very low concentrations, well below that available by the colorimetric methods.
This sensitivity is dependent on the availability of chemiluminescence detection (Kritharides et al. 1994; Stocker et
al. 1991). It has recently been reported (Kritharides et al.
1994) that LDL freshly isolated from healthy subjects was
free from detectable amounts of cholesterol-ester hydroperoxides and phospholipid hydroperoxides as measured by
HPLC with post-column chemiluminescence detection,
suggesting that if lipid hydroperoxides are present at all,
the levels must be below 1 nmol/mg LDL protein. This
method of detection does require considerable dedication of
technical resources and is unlikely ever to be suitable for
any routine assays in large-scale clinical research. In some
cases, u.v. detection can be used but with lower sensitivity.
A comment should be made on the wide-ranging differences
between the detected endogenous peroxide levels in LDL
Defence against reactive oxidative species
applying the HPLC–chemiluminescence method compared
with the spectrophotometric assays. (A direct comparison of
the methods has yet to be carried out in a single laboratory
on the same LDL samples.) No one is sure whether the FOX
and El-Sadaani (1989) methods are determining additional
unidentified components (which might include protein
hydroperoxides) or whether the HPLC assay is missing
some contributing features. On the other hand, it may
relate to the methods applied for isolating the LDL; for
example, it has been reported that applying the FOX assay
after rapid isolation procedures gives 3 nmol/mg LDL
protein, compared with 13.3 nmol/mg LDL using the
sequential isolation methods (Kritharides et al. 1995).
3.3. Oxidative damage to proteins
Oxidative damage to proteins is of particular importance in
vivo both in its own right (affecting the function of receptors, enzymes, transport proteins etc. and perhaps generating
new antigens that provoke immune responses), and because
it can contribute to secondary damage to other biomolecules, e.g. inactivation of DNA repair enzymes and
loss of fidelity of DNA polymerases in replicating DNA.
The chemical reactions resulting from attack of ROS or
RNS on proteins are complex. Free-radical attack can
generate protein peroxides, which can decompose in complex ways (Ambe & Tappel, 1961; Fu et al. 1995).
Most use has been made of the carbonyl assay, a general
assay of oxidative protein damage (Levine et al. 1995) to
assess steady-state protein damage in human tissues and
body fluids. The carbonyl assay is based on the fact that
amino acid residues in proteins (particularly histidine,
arginine, lysine and proline) are particularly susceptible to
attack by ROS producing carbonyl functions. Such
increased carbonyl content can be measured after reaction
with 2,4-dinitrophenylhydrazine. The carbonyl assay has
become widely used and many laboratories have developed
individual protocols for it (Levine et al. 1994, 1995).
Sometimes the assay procedures used in a particular laboratory are not precisely specified in published papers and even
when they are, they often differ from those used originally
by the group of Stadtman (Oliver et al. 1987; Levine et al.
1994, 1995). This point is important because there is a
considerable variation in the �baseline’ levels of protein
carbonyls in certain tissues, depending on how the assay is
performed (Cao & Cutler, 1995; Lyras et al. 1996).
Contrariwise, broadly comparable values for protein carbonyls in human plasma, of < 1 nmol/mg protein have been
reported by most groups, so plasma protein carbonyls should
be a useful marker of oxidative protein damage for nutritional
studies. More work needs to be done to identify the molecular
nature of the carbonyls, namely, which amino acid residues
have been damaged and in which proteins they reside.
Western-blotting assays based on the use of antidinitrophenylhydrazine antibodies have also been developed
in an attempt to identify oxidatively-damaged proteins in
tissues and body fluids (Keller et al. 1993; Levine et al.
1994). A cautionary note is the covalent binding of certain
aldehyde end-products of lipid peroxidation to proteins, generating �carbonyls’. Indeed, many oxidized molecules contain
carbonyls which will interfere in the protein carbonyl assay.
Several in vitro assays for damage to specific amino acid
residues in proteins have been developed including assays
of 3-hydroxy-L-tyrosine (L-DOPA) (produced by tyrosine
hydroxylation) (Giseg et al. 1993), valine hydroxides
derived from valine hydroperoxides (Giseg et al. 1993),
ring-opening products of tryptophan oxidation (Griffiths et
al. 1992; Maskos et al. 1992) 8-oxohistidine (Uchida &
Kawakishi, 1993, 1994), dityrosine (Giulivi & Davies,
1993) and ortho and meta-tyrosines, products of attachment
of OH: to phenylalanine (Karam et al. 1991; Wells-Knecht
et al. 1993). The levels of any one (or, preferably, of more
than one) of these products in proteins could, in principle, be
used to assess the balance between oxidative protein
damage and the repair or (more likely) hydrolytic removal
of damaged proteins. The only products exploited to date
have been the hydroxylated phenylalanines (Wells-Knecht
et al. 1993).
Attack of various RNS (ONOOВ№ , NOШ’2 and possibly some
other species) on tyrosine (both free and in proteins) leads to
production of 3-nitrotyrosine, which can be measured
immunologically or by HPLC or GC–MS techniques
(reviewed by Wells-Knecht et al. 1993). Reduction of
nitrotyrosine to aminotyrosine increases the sensitivity of
measurement, since the latter compound can be measured
using highly-sensitive electrochemical detection. Nitrotyrosine is also excreted in human urine (Oshima et al.
1990), although the possible confounding effect of dietary
nitrotyrosine (if any) and of dietary nitrate and/or nitrite is
yet to be evaluated.
For measures of total ongoing protein damage, urinary
nitrotyrosine (Oshima et al. 1990) might be useful as a
generalized index of attack by RNS. Very little research has
been carried out on the presence of oxidized amino acids
and their metabolites in urine, except that bityrosine has
been detected and can be measured by HPLC with fluorescence detection. More work needs to be done in this area,
and the possible confounding effects of oxidized proteins
and amino acids in the diet (e.g. in irradiated foods;
Halliwell & Chirico, 1993) must be considered.
3.4. Measurement of antioxidant nutrients, carotenoids and
flavonoids extracted from human plasma
Several antioxidants are routinely measured in plasma: atocopherol (and g-tocopherol) whose antioxidant roles are
well clarified in vivo; b-carotene, lycopene and other dietary
carotenoids for which there is, as yet, little evidence of
antioxidant activity in vivo; ascorbic acid, the most efficient
reducing agent in vivo, its redox potential defining its central
role as an aqueous phase antioxidant. There is, as yet, little
information as to the importance of dietary flavonoids as
antioxidants in vivo, nor evidence for such activity in vivo,
although these polyphenols are highly efficacious freeradical scavengers in vitro. Furthermore, it is only recently
that it has become possible to detect and identify flavonoids
(and other glycosides) in human plasma, in non-supplemented
Table 1 indicates the most favourable systems for detection, identification and quantification of the antioxidants by
HPLC. Plasma samples can be stored at –70Њ.
A. T. Diplock et al.
Table 1. The most favourable systems for detection, identification and quantitation of antioxidants by HPLC
a-Tocopherol and g-tocopherol
HPLC with fluorescence detection
(lex 296 lem 340)
HPLC with diode array detection
HPLC with diode array detection
Solvent system: hexane–methyl-t-butyl ether (92 : 8, v/v)
Column: Novapak Silica 150 Г— 4:6 mm (4 вђ®m)
Internal standard: d-tocopherol
Detection limit: in plasma, 1.1 вђ®mol/l
Solvent system: acetonitrile–methanol (90 : 10, v/v)
Column: Supelco PKB 100, 5 вђ®m
Solvent system: acetonitrile–methanol–dichloromethane–
hexane (gradient)
Column: Merck lichrocart man-fix, 5 вђ®m
Detection at: 455 nm (b-cryptoxanthin)
468 nm (canthaxanthin)
474 nm (lycopene)
454 nm (b-carotene)
Solvent system: 1 ml/l HCl in 20 % of aqueous
methanol and acetonitrile (gradient)
Column: Nova-Pak C18 250 Г— 4:6 mm (4 вђ®m)
Internal standard: salicylic acid
Detection: 280 nm (selective)
lex and lem , excitation and emission wavelengths.
3.5. Conclusions
There is still a long way to go until validated, precise
biomarkers of oxidative damage become routinely
Concerning oxidative damage to DNA, both HPLC and
GC–MS have their relative merits but also their limitations.
Sight must not be lost of the potential for oxidative damage
to DNA during its isolation from cells and tissues.
Nevertheless, artifacts arising during derivatization are
also problematical and HPLC with electrochemical detection is one proposed route which circumvents the need for
derivatization. Liquid chromatography–mass spectrometry
techniques are also under development. An initiative by the
British Ministry of Agriculture Fisheries and Food (MAFF)
is seeking to coordinate, within the MAFF Antioxidants in
Foods Research Programme, validation work at a number of
centres within Europe, concerned with biomarkers of oxidative damage to DNA. The agenda seeks to have validated
agreed biomarkers available for a new generation of human
studies within 2–3 years.
With regard to ex vivo markers of lipid oxidation in vivo,
the measurement of circulating isoprostanes and lipid
hydroperoxides is the best approach for plasma, the latter
applying HPLC with chemiluminescence detection. Where
this detection system is not available, the HPLC–TBA
method has been shown to be an alternative, although
lacking the high precision of the former. While the principle
of isoprostane analysis is highly promising, this is still in its
early infancy as there is a dearth of information in the peerreviewed literature as yet, but it is rapidly gaining momentum. For urinary markers of lipid oxidation as an indicator
of whole-body lipid peroxidation, the development of the
isoprostane analytical techniques will be an important
advance, since hydrocarbon gas exhalation has too many
confounding variables to be applicable to studies on freeliving human subjects.
The potential for the application of methods for ex vivo
detection of in vivo protein oxidation is promising. The
outcome, of course, is the balance between damage and
repair but to date very few products of oxidative damage to
proteins have been exploited in this context.
There is also a lot of excitement and activity in the areas
of evidence for the formation of RNS in vivo. A major
stumbling block here in the accumulation of evidence for
oxidative damage in vivo is the confounding effects of the
presence of oxidized proteins and amino acids in the diet. A
lot more work needs to be done in this area.
4. Nutritional options modulating oxidative damage and
antioxidant defence systems
4.1. Introduction
The body’s antioxidant defence system is capable of being
altered by dietary means. A first strategy to balance
oxidative damage and antioxidant defence of human cells
and tissues would be to enhance the antioxidant capacity by
optimizing the dietary intake of antioxidants. A second
approach may be to neutralize oxidative compounds in the
diet. Crucial to these strategies is knowledge of the required
level of relevant antioxidants in the diet to provide
protective effects. Another prerequisite is accurate information about food sources, content and bioavailability of
Epidemiological studies are necessary to quantify the
impact of antioxidants on disease aetiology. Intervention
trials formally test the efficacy of enhancing intake of
antioxidants. In evaluating these health benefits preferably
hard end-points (disease incidence, or recurrence and
mortality) should be used. Alternatively intermediate endpoints may be effective, provided that they are genuinely
predictors of the disease of interest. In research on
functional foods, the development and application of biomarkers is extremely important. In the causal pathway of
disease occurrence one can distinguish biomarkers of
exposure (dietary intake), biomarkers of biological response
and of (subclinical) disease, and biomarkers of susceptibility. For antioxidants, all types of markers have clear
relevance. For example, blood levels of vitamin E (an
Defence against reactive oxidative species
exposure marker) may be studied in relation to oxidation
resistance of LDL (a biological response marker) or to
carotid artery wall thickness (a disease marker), in subjects
with familial hypercholesterolaemia, or specific genotype
(both susceptibility markers). Although biomarkers have the
potential for improving validity and reducing bias, several
problems are encountered. Biomarkers of exposure should
accurately reflect relevant dietary intake or body status and
early disease markers should have predictive value for the
hard end-point. Since chronic diseases have long latency
periods requiring large initial numbers to evaluate health
effects, biomarkers of intermediate end-points may, in
certain circumstances, legitimately be used more efficiently.
An effective nutritional strategy will require knowledge
of the type of antioxidants in the diet, their food sources,
bioavailability and required levels of intake for protective
effects. Protective effects of antioxidants have been found in
mechanistic studies in vitro and in vivo, and epidemiological
studies and certain intervention studies have provided useful
information. However, it is appropriate to consider the
totality of the evidence from basic science, epidemiology
and intervention studies, rather than to rely on the evidence
from any one type of study.
4.2. Dietary antioxidants
4.2.1. Sources of dietary antioxidants. There are both
nutrient and non-nutrient antioxidants. Non-nutrient antioxidants include flavonoids (found for example in tea, red
wine, onions, and apples), polyphenols and terpenes. The
focus here will be on nutrient antioxidants, in particular
vitamin C, vitamin E and carotenoids, although possible
effects of non-nutrient antioxidants must be borne in mind
in reaching complete understanding. Thus, it is important to
ascertain whether non-nutrient antioxidants are genuinely
bioavailable, in the particular sense of whether they are
delivered under normal circumstances to tissues where they
might be expected to be effective. Their activity as
antioxidants themselves, or whether they participate in a
cyclical fashion with other antioxidants, also remains to be
The flavonoid content of beverages has been of great
interest recently (Hertog et al. 1993). There is a large
variation in the quercetin and myricetin content of red
wines, which appears to depend on the type of grape and
the vineyard of origin. Quercetin, kaempferol and myricetin
are present in black and green teas, so that, together with
fruit juices (mainly quercetin), these beverages can provide
substantial amounts of flavonoids in the human diet.
Vitamin C is found in citrus fruits, peppers, potatoes and
other fruits and vegetables. The principal sources of vitamin
E are vegetable oils and wheat germ. Other sources are nuts,
seeds and leafy green vegetables. Of the over 600 carotenoids, b-carotene has been the most extensively studied.
Good sources of b-carotene are yellow or orange fruits
and vegetables such as carrots, sweet potatoes, apricots, and
mangoes, as well as dark green leafy vegetables such as
spinach. There is now increasing interest in other major
dietary carotenoids including lycopene (tomatoes), lutein
(spinach, broccoli, maize), zeaxanthin (maize), a-carotene
(carrots), and b-cryptoxanthin (citrus fruits). Chemical
analysis of food products is steadily improving and until
recently, there was a lack of reliable data for the food
content of carotenoids other than b-carotene. However,
the recently released carotenoid food composition database
of the US Department of Agriculture has included analysis
of a-carotene, b-cryptoxanthin, lutein, zeaxanthin, and
lycopene. A number of epidemiological studies of b-carotene and risk reduction of certain diseases are now being
re-evaluated using information derived from this source on
the other carotenoids.
4.2.2. Antioxidant intake and status. Several crosssectional surveys in a variety of population groups have
been conducted. From these studies it can be concluded that
according to the recommended daily allowances (RDA), the
intake of antioxidants is adequate in healthy subjects. Lower
levels have been observed in smokers, the elderly, and in
patients with specific diseases or risk factors and several
studies have demonstrated that intake of a number of
antioxidants may be suboptimal in certain populations. In
countries such as France and Italy antioxidant intake is
largely adequate due to the abundant supply of fresh fruits
and vegetables. However, in other countries where the
selection of products is limited or more seasonal, a number
of population groups are not able to meet the minimum
requirements for vitamins E, C or b-carotene. At particular
risk are the less affluent and the elderly.
The RDA, which are not established for carotenoids, are
defined to prevent nutrient deficiencies and do not take into
account the reduction in risk of chronic diseases. The levels
of antioxidant nutrients that are effective in the reduction in
risk of chronic diseases generally lie higher than the RDA.
Since antioxidants may play important roles in the prevention of chronic diseases, the question is what would be the
optimal range of intake to recommend. Lachance (1996)
distinguishes the following categories: �experimental protective intake’ �amount in optimal menus’, �desirable blood
level’, �calculated intake necessary to achieve desirable
blood levels’. For example, for vitamin E (current RDA:
10 mg men, 8 mg women) the following values have been
proposed: experimental protective intake > 23–100 mg;
amount in optimal menus 23 mg; desirable blood level
23 вђ®mol/l; calculated intake necessary to achieve desirable
blood levels 23 mg. For carotenoids the following values
have been suggested: > 4 mg, 5.7 mg, 0.4 вђ®mol/l, 3.2 mg
respectively. Diplock (1994) reviewed epidemiological
studies of antioxidants and disease and suggested that the
following daily intakes were associated with a reduced risk
of cancer and cardiovascular disease; 150 mg vitamin C,
40–60 mg vitamin E, and 9–12 mg b-carotene. Lachance
(1996) has estimated optimal daily antioxidant intakes to be
145 mg vitamin C, 23 mg vitamin E, and 3.2 mg carotene.
Biomarkers of antioxidant intake reflecting internal status
(blood, adipose tissue, nails etc.) have been used successfully but need much further development, and these also
need to be non-invasive. Conceptually it is important to
know the biological relevance and exposure timeframe of
the biomarker. It is not only the dietary intake but other
exogenous factors such as smoking and alcohol intake, and
endogenous factors, that affect antioxidant status measured
by a biomarker. For many bio-active compounds intake data
are hard to get and, thus, more reliance will have to be
A. T. Diplock et al.
placed on biomarker data. It is thus imperative that the
biomarkers that are used can be shown to be relevant to the
outcome of the balance between oxidant and antioxidant in
the majority of the population.
4.2.3. Bioavailability of antioxidants. Bioavailability of
antioxidants depends on several food and host-related
factors, as recently summarized by de Pee & West (1996)
for carotenoids. A well-recognized food-related factor is
the amount of antioxidant in a meal; for nutrients which are
absorbed by a process of passive diffusion, the proportion
of antioxidant absorbed decreases with increasing amounts
in the food. The molecular forms of antioxidants in
foods, for example, for which isomers or molecular
linkages such as esters exist, are also important
determinants of bioavailability. In addition, the food
matrix in which antioxidants are located often influences
availability of the nutrient. For example, b-carotene is
organized in a pigment–protein complex in green vegetables, but found in lipid droplets in other vegetables and
fruits. The b-carotene can be released more readily, and is
thus more biovailable, from a fat droplet than from a protein
Host-related factors influencing bioavailability include
genetic factors, nutrient status and absorption modifiers.
Absorption modifiers for fat-soluble vitamins and carotenoids,
are lipids in the diet. To ensure efficient absorption, sufficient
fat must be present in the meal and diet.
A recent epidemiological study on lycopene intake and
prostate cancer illustrates the importance of bioavailability
in functional food research. Intake of tomatoes, tomato
sauce, and pizza were significantly related to lower risk of
prostate cancer. Tomato juice was not associated with a
protective effect (Giovannucci et al. 1995). The lycopene
from tomato juice has a low bioavailability, but cooking
tomatoes in an oil-based medium substantially enhances
intestinal absorption.
4.2.4. Fat intake and antioxidant status. Consumption
of reduced-fat products in order to reduce energy intake, or
of products with enhanced PUFA content (Sarkkinen et al.
1993), may also affect the antioxidant status. It was shown
that vitamin E intake was significantly lower among
subjects who had an increased intake of reduced-fat
products; however, no effect was observed for b-carotene
or lycopene, or for the antioxidant enzyme activities
(superoxide dismutase, catalase and glutathione peroxidase)
(Velthuis-te Wierik et al. 1996). There is, however,
evidence that plasma antioxidant levels may not be a
reliable index of body status; it was found that erythrocyte
levels were unaffected in a study in men who had
significantly lowered plasma levels of vitamin E (Haddad
& Blankenship, 1985). Inhibitors of dietary fat absorption,
which may be used to counteract obesity, appear to have fatsoluble vitamin-lowering properties (Melia et al. 1996).
Besides a reduced antioxidant intake, fat substitutes like the
sucrose polyester-based products may also reduce the
absorption of fat-soluble antioxidants, although this only
occurs when the fat intake is below 20 % of total energy.
However, published data do not suggest a major impact on
the absorption of antioxidants when the level of inclusion of
the fat substitute in the diet is low, such as would be the case
when snack foods are prepared using the fat substitute. In
general, diets low in PUFA do result in lower intake of
vitamin E. On the other hand, PUFA-rich diets may affect
the antioxidant–prooxidant balance requiring higher intake
of antioxidants.
4.3. Epidemiological studies on protective effects of
4.3.1. Cardiovascular disease. Several scientific reviews
have addressed the role of antioxidants in cardiovascular
disease (van Poppel et al. 1994). As discussed earlier, the
primary role of antioxidants in reducing the risk of
cardiovascular disease is through inhibition of peroxidation
in LDL, although they may also influence other cardiovascular disease processes.
The epidemiological evidence for a protective role of
antioxidants in cardiovascular disease is strongest for
vitamin E. Three large-scale epidemiological studies
demonstrated a relation between vitamin E intake and
CHD. The Nurses Health Study, conducted in 87 245
women, found a significant 34 % reduction in CHD in
women who had consumed vitamin E supplements containing more than 67 a-tocopherol equivalents (a-TE) daily for
more than 2 years (for a definition of a-tocopherol equivalents see p. S95). The Health Professionals Study of 39 910
men showed vitamin E to be associated with a 41 %
reduction in risk of CHD. Again, the greatest risk reduction
was found with intakes of supplements of more than 67 aTE for 2 years or more.
Kushi et al. (1996) reported a significant 62 % reduction
in mortality from CHD in women consuming foods containing more than 6.46 a-TE/d. Surprisingly, there was no risk
reduction seen with use of vitamin E supplements. This may
be due to the lack of information on duration of supplement
use, since from the other studies it appears that benefits of
supplements are evident only after 2 years.
Several epidemiological studies have examined the association between vitamin C and cardiovascular disease. A
European cross-cultural study found a significant inverse
relationship between CHD mortality and serum vitamin C
levels. The first National Health and Nutrition Examination
Survey (NHANES I) results showed a 50 % reduction in
cardiovascular mortality associated with a daily consumption of greater than 50 mg vitamin C. Gale et al. (1995)
reported a significant protective effect against stroke of
vitamin C intakes more than 45 mg. However, neither the
Nurses’ Health Study nor the Health Professionals Study
described earlier found a significant protective effect of
vitamin C.
A recent study found that use of both vitamin E and C
supplements was significantly more protective against cardiovascular mortality than use of vitamin E supplements
alone, or no use of supplements (Losonczy et al. 1996).
The epidemiological evidence for a role of carotenoids in
cardiovascular disease prevention has been reviewed by
Kohlmeier & Hastings (1995). A number of studies consistently showed a decreased risk of CHD among subjects
with high b-carotene intake or serum levels. In the Massachusetts Health Care Panel study of 1299 elderly people,
Gaziano et al. (1995) found a 75 % reduction in risk of fatal
myocardial infarction in subjects in the highest quartile of
Defence against reactive oxidative species
carotene intake. A study of 25 802 subjects showed a
significant doubling of the risk of myocardial infarction in
subjects with low serum b-carotene levels (Street et al.
1994). Several studies have, however, shown increased risk
of CHD among subjects with low b-carotene status. The risk
seems to be confined to current smokers. However, bcarotene intake contributes about 25 % of total carotenoid
intake, thus carotenoids other than b-carotene as well as
other components in fruits and vegetables may be
responsible. The sparse data on individual carotenoids do
not allow any firm conclusions. Several non-nutritive bioactive compounds may be of relevance in the aetiology of
cardiovascular disease. Epidemiological studies on
flavonoids are promising, but evidence of benefit is
still fragmentary (Hertog, 1994; Muldoon & Kritchevsky,
4.3.2. Cancer. Numerous epidemiological studies have
shown that individuals who regularly consume fruits and
vegetables have a decreased risk of cancer. A protective
effect of fruit and vegetable consumption was found in 128
of 156 dietary studies (Block et al. 1992). High intake of
fruits and vegetables (in the upper one-fourth of the
population) is associated with an approximately 50 %
reduced risk of cancer, depending on the tumour site. The
most consistent evidence of risk reduction associated with
fruits and vegetables has been seen in the epithelial cancers
of the respiratory and gastrointestinal tract (Steinmetz &
Potter, 1991). Evidence is strong for lung cancer, with
significant risk reduction found in twenty-four of twentyfive studies, as well as for stomach and pancreatic cancer
with protection in twenty-six of thirty studies. For
oesophageal, laryngeal and oral cancer, twenty-eight of
twenty-nine studies showed a significantly reduced risk with
fruit consumption. Cancers of the cervix, ovary and
endometrium were associated with a significant risk
reduction in eleven of thirteen studies.
Although there is, therefore, indirect evidence that antioxidants (carotenoids, vitamin C, vitamin E and possibly
non-nutritional antioxidants) may be beneficial (TaylorMayne, 1996), it has to be shown whether, and to what
extent, this is true for individual antioxidants and which
specific components are responsible. To illustrate the complexities: plasma b-carotene in the normal physiological
range is inversely related to lung cancer incidence. Other
carotenoids such as a-carotene, which is strongly correlated
with b-carotene, may however be the relevant factor. In
addition, other bioactive components in fruits and vegetables or other aspects of lifestyle, including diet, may play a
causal role. Focusing, in epidemiological studies, on food
items which mainly contribute to the daily intake of a
specific bioactive compound, e.g. tomatoes and tomato
products in the case of lycopene, may be a promising
research strategy to identify the responsible factor.
In attempts to identify the components in fruits and
vegetables associated with risk reduction of cancer, epidemiological studies focusing on intake or serum levels of
specified antioxidants have been undertaken. In epidemiological studies of the protective effect of vitamin E on
various cancer sites, vitamin E was associated with slightly
reduced risk of lung cancer in studies involving low exposure to tobacco smoke. Several studies of oral, pharyngeal
and cervical cancer have also found a relationship between
vitamin E status and cancer risk. The evidence for stomach
and pancreatic cancers has not been consistent, and no
association with breast cancer has been found. Diets high
in vitamin E intake have been less consistently shown to be
associated with cancer protection. Moreover, observational
studies of vitamins C and E consumed in supplements
provide little support for a strong protective role against
As discussed earlier, vitamin C inhibits the formation of
carcinogenic nitrosamines, stimulates the immune system,
protects against chromosomal breakage, and regenerates
vitamin E as part of the antioxidant defence system. The
epidemiological evidence for a risk-reducing role of vitamin
C in cancer is not as strong as for fruits and vegetables.
However, an extremely strong and consistent protective
effect of vitamin C was found in seventeen of nineteen
studies of stomach, oesophageal, oral and pharyngeal cancers (Block et al. 1992). Additional studies showed that
subjects with low serum levels of vitamin C have a 50 %
increased risk of gastric metaplasia or chronic gastritis,
which are both precancerous lesions.
The most consistent body of epidemiological evidence
showing protective effects of an antioxidant nutrient on
cancer is for b-carotene. The strongest results are seen for
lung cancer. Of twenty-five studies which investigated bcarotene and lung cancer risk, twenty-four showed a significant reduction in risk with high b-carotene intakes or plasma
levels (van Poppel & Goldbohm, 1995). For stomach cancer,
of fifteen studies, eight found a significant risk reduction
associated with b-carotene, and six showed a non-significant
risk reduction. Only one study did not find a protective effect
of b-carotene against stomach cancer. No consistent associations were found with colorectal, prostate or breast cancer.
4.3.3. Other age-related diseases. Cataract and AMD
are eye disorders which show increasing incidence among
the elderly. Cataracts result from glycosidation of lens
proteins initiated by u.v. light, which leads to opacification
of the lens. It has been shown that in vitro lens proteins may
be protected against oxidative attack by carotenoids and
vitamins C and E. Of ten epidemiological studies, nine
showed strong inverse relationships with at least one
antioxidant nutrient. Observations are most consistent for
vitamins C and E.
Macular degeneration is the leading cause of irreversible
blindness among persons older than 65 years. The carotenoids lutein and zeaxanthin are present in the retina as
pigments to protect against the damaging effects of light;
strong inverse relationships were found between intakes of
b-carotene, lutein and zeaxanthin and risk of AMD. Consumption of spinach, which is a good source of lutein and
zeaxanthin, was also associated with significantly reduced
risk of AMD.
Free radicals have also been implicated in the development of neurodegenerative disorders such as Parkinson’s
disease and Alzheimer’s disease, and in diabetes, rheumatoid arthritis, and chronic obstructive pulmonary diseases.
These age-related diseases may, therefore, be beneficially
influenced by antioxidant consumption. However, as yet
few epidemiological data exist on the association of antioxidants with these disease risks.
A. T. Diplock et al.
The question whether it is a specific antioxidant or
another component, or mixture, of fruits and vegetables
that exerts the protective effect on disease cannot be
definitively answered by epidemiological studies. Additional information provided by intervention trials is
needed before drawing any final conclusions.
4.4. Human intervention studies of antioxidants
The gold standard for testing the effectiveness of specific
antioxidants is a randomized placebo-controlled
intervention trial, preferably with a hard end-point such as
disease occurrence or cause-specific mortality. Since trials
conducted with clinical end-points of disease incidence or
mortality are exceedingly costly and time consuming,
intermediate end-points or validated biomarkers are increasingly being used. The limitations of intervention trials are
that they can often only be interpreted for the particular
study population, and for the antioxidant dose
provided during the trial. Most of the intervention trials
have focused on cardiovascular disease and cancer; only a
few studies have addressed other age-related disorders, i.e.
4.4.1. Cardiovascular disease. Intermediate endpoints. A number of studies have supplemented
individuals with antioxidants and measured the reduction
in lipid peroxidation. However, indices of lipid peroxidation
have not yet been definitively associated with cardiovascular disease end-points. The only intervention studies
which have evaluated the effect of antioxidants on
cardiovascular disease using intermediate disease endpoints have been with vitamin E. Using arterial narrowing
as a marker for cardiovascular disease progression, the
effect of vitamin E was evaluated in 100 patients
randomized to 804 a-TE or placebo following angioplasty.
The group receiving vitamin E showed significantly less
recurrence of stenosis over the 4-month supplementation
Carotid artery wall thickness, measured by non-invasive
arterial wall imaging, has also been used as a reliable
intermediate end-point for atherosclerosis. A recent study
(Azen et al. 1996) demonstrated that use of vitamin E
supplements larger than 67 a-TE/d reduced the progression
of atherosclerosis.
Disease end-points. Although vitamin E shows promise
in reducing cardiovascular disease, relatively few intervention trials have been conducted. The first of the trials to be
reported was the a-tocopherol b-carotene (ATBC) study
which found no effect on cardiovascular disease mortality
among heavy chronic smokers receiving 50 mg vitamin E
daily (Albanes et al. 1994).
Linxian, China was the site of a study which gave a
multivitamin–multimineral supplement plus 15 mg b-carotene and found a 38 % reduction in stroke incidence in
subjects with oesophageal dysplasia. A non-significant
10 % reduction in stroke incidence was also observed in a
trial involving 35 000 subjects from the general population
of Linxian who received 15 mg b-carotene, 30 mg vitamin E
and 50 вђ®g Se (Blot et al. 1993).
A trial conducted in 161 Japanese subjects, who consumed 3 or 100 mg a-tocopherol/d for 6 years, showed a
significant reduction in the number of coronary disorders in
the 100 mg/d group (Takamatsu et al. 1995). The recently
published Cambridge Heart Antioxidant Study (CHAOS)
found a significant reduction in the incidence of non-fatal
myocardial infarctions in 2002 subjects randomized to 268
or 536 a-TE/d (Stephens et al. 1996). However, there was a
non-significant excess of cardiovascular deaths in the vitamin E-supplemented group.
b-Carotene supplementation resulted in increased cardiovascular disease incidence or mortality in two studies
conducted in high-risk populations of smokers and asbestos-exposed workers, ATBC and the b-carotene and retinol
efficacy trial (CARET) (Albanes et al. 1994; Omenn et al.
1996). In a trial of healthy male US doctors, the Physicians’
Health Study (Hennekens et al. 1996) showed no effect on
cardiovascular disease following 12 years of supplementation with 50 mg b-carotene every second day. This was in
contrast to earlier findings in a subgroup of the study which
found a significant 50 % reduction in secondary coronary
events in subjects randomized to b-carotene (Gaziano et al.
It is known that in vitro vitamin C can regenerate vitamin
E from the vitamin E radical which is formed during the
inhibition of lipid peroxidation. Several large-scale trials are
currently underway to study a combination of vitamins E
and C, and b-carotene, in the prevention, or amelioration, of
risk of cardiovascular disease.
4.4.2. Cancer. Intermediate end-points. Most of the
intervention trials investigating the potential role of
antioxidants in reducing the risk of cancer have focused
on precancerous lesions. Relatively little work has been
done on biomarkers for cancer, such as DNA damage.
Recent reviews have summarized the use of antioxidants in
oral leukoplakia, a precancerous lesion of the oral cavity
(Kaugars et al. 1994; Garewal, 1995). Significant impact on
lesion size or occurrence was found in seven intervention
studies with a supplement of between 30 mg and 180 mg bcarotene. b-Carotene has also been shown to reduce cervical
as well as gastric dysplasia, and there are a number of
intervention studies currently in progress.
Several promising biomarker candidates for cancer have
been suggested. Ideal characteristics of these biomarkers
include: they should appear earlier than, or more frequently
before, tumour development; they should be directly associated with tumour progression; they should be shown to be
reversible; they should have inexpensive, accurate and
simple methods of detection; they should be validated.
Biomarkers of cellular proliferation and differentiation
e.g. colonic adenomatous polyps and premalignant lesions
such as dysplasia have been used to evaluate the preventive
role of nutrient antioxidants. In contrast to observations
using microscopic measures of cellular proliferation, the
major antioxidants vitamins E and C, and b-carotene seem
to be ineffective in decreasing the recurrence of colonic
adenomas. Clarification is needed of changes in biomarkers
that have been shown to occur with progression of
proliferation, and in subsequent steps in the carcinogenic
process, before definite conclusions can be drawn from
these intermediate biomarker data.
The preventive potential of antioxidants has been tested
with intermediate end-points for other tumour sites, such as
Defence against reactive oxidative species
lung, mouth, oral cavity and cervix. It is difficult to
summarize the overall findings. However, until now, no
really strong benefits have been identified. There is a need
for a second generation of chemoprevention trials that
include biomarkers, provided that these markers have
fulfilled the criteria described earlier.
Disease end-point. There have been a number of
intervention trials which studied the potential for b-carotene
to reduce the risk of cancer. The 5-year Linxian trial of
29 584 adults in China found significant reductions in
mortality from total and stomach cancer of 13 % and 21 %
respectively, in the group randomized to 15 mg b-carotene,
30 mg vitamin E and 50 вђ®g Se (Blot et al. 1993). In 3318
adults with oesophageal dysplasia in the same community,
there was a non-significant 8 % decrease in oesophageal
cancer in subjects randomized to a multivitamin, multimineral supplement with 15 mg b-carotene.
The Finnish ATBC study of 29 133 chronic heavy
smokers tested the effects of 20 mg b-carotene, either
alone or in combination with 33.5 a-TE for an average of
6 years. There was a significant increase of lung cancer
incidence (16 %) in the groups which received b-carotene
(ATBC Cancer Prevention Study Group, 1994). A more
detailed analysis of the results revealed that the increased
risk of lung cancer appeared to be restricted to participants
who had smoked more than twenty cigarettes daily over an
average period of 30 years (Albanes et al. 1996).
The CARET study of 18 314 subjects at high risk of lung
cancer (heavy smokers, and asbestos-exposed workers)
evaluated the combination of 30 mg b-carotene and 25 000
IU vitamin A (equivalent to 7.5 mg retinol) over an average
of 4 years. The intervention group had a significantly
increased risk of lung cancer (relative risk 1.36; Omenn et
al. 1996). A reduced risk of lung cancer (relative risk 0.80)
was seen in subjects who were former smokers at the
beginning of the study. Interestingly, participants with
high initial serum b-carotene concentrations had a 31 %
reduction in risk of lung cancer (P Вј 0:003), regardless of
which group they were randomized to. This effect was also
seen in the ATBC study and Physicians’ Health Study which
was conducted over 12 years in 22 071 male physicians who
consumed 50 mg b-carotene every second day. There was
no beneficial influence of b-carotene on cancer incidence.
However, due to the long duration of the trial it is important
to note that there were also no negative effects seen (TaylorMayne, 1996).
4.5. Conclusions
Epidemiological studies support the hypothesis that the
antioxidants vitamin E, vitamin C and b-carotene may
play a beneficial role in reducing the risk of several chronic
disorders. More research is needed on the impact of other
non-nutrient compounds, such as other carotenoids and
flavonoids, on human health.
Human intervention trials testing the efficacy of antioxidants do not allow firm conclusions because of
inconsistent findings, an insufficient number of studies and
the use of varying doses. However, there is some evidence
that large doses of b-carotene may be deleterious to the
health of heavy habitual smokers.
In general, human intervention studies using hard endpoints should be regarded as the gold standard. However, for
diseases with a long induction period, such as cancer and
cardiovascular disease, these types of studies may not be
very feasible because of high costs, and intermediate endpoints need to be sought to overcome this difficulty. The
relationship between the disease and the nutritional factors
that may have been involved at an early stage of its
aetiology is, however, very complex and difficult to interpret. The development of biomarkers may, however, help in
understanding the complexity of degenerative diseases at
their different stages.
5. Potential safety implications related to antioxidant
nutritional enhancement
5.1. Introduction
The substances to be considered are the antioxidant nutrients vitamin C (ascorbic acid), vitamin E (a-tocopherol):
the carotenoids (particularly b-carotene): and the non-nutrient antioxidants, mainly flavonoids, which occur in food and
which may be significant in the overall antioxidative protection afforded by the diet. This benefit may be conferred in
three main ways: (1) as antioxidants in food during storage
and in the gastrointestinal tract; (2) as antioxidants in the
human body in vivo; (3) by providing protection in food
against oxidation as well as acting as true antioxidants in
vivo. From the point of view of safety, the effect of
antioxidants must strictly be concerned with their effects
in vivo; however, the possibility that a given compound may
be converted by chemical reaction, or bacterial action, into a
toxic substance within the gastrointestinal tract, or during
storage of the food that contains it, must also be borne in
mind because such products may themselves be absorbed
and exert their toxicity in vivo.
5.2. Vitamin C
The tolerance and safety of ingested vitamin C in human
subjects has been reviewed several times (Hanck, 1982;
Rivers, 1989; Diplock, 1995). In their review Bendich &
Langseth (1995) consider in detail the fourteen controlled
clinical studies that have reported no side-effects of vitamin
C dosage, consistent with uncontrolled anecdotal reports
which have appeared. In a more recent detailed review
(Bendich, 1997), twenty-two placebo-controlled doubleblind studies are reviewed that indicate no consistent
detrimental side-effects of dosages of vitamin C up to
daily doses of 6 g. This conclusion is supported by the
findings of a further eight less-well-controlled studies. The
fact that very large numbers of people regularly take large
doses of vitamin C without reports of any adverse effects is
anecdotal support for the view that vitamin C is very safe
and free from any adverse side-effects. It is clear that
overload with vitamin C cannot occur in man even at very
high levels of dietary intake (Rivers, 1989). Absorption,
tissue concentration, metabolic pathways in which ascorbate participates and renal elimination are all controlled by
homeostatic mechanisms. The amount of a dose of vitamin
C that is absorbed is inversely proportional to the size of the
A. T. Diplock et al.
dose and saturation was achieved at a Km of 5.44 mM in a
human study using intestinal perfusion. A consistent body
pool size of ascorbate in man of about 20 mg/kg body
weight, which appeared to change little irrespective of
increases in intake to very high levels, has been reported
(Kallner et al. 1979). However, a recent detailed study
(Levine et al. 1996) provides more information; steadystate plasma concentrations were determined in normal
subjects following administration of 30–2500 mg vitamin
C daily. Steady-state plasma concentrations followed
sigmoid kinetics, the steep portion of the curve occurring
between 30 and 100 mg/d, and complete saturation did not
occur until a daily intake of 1000 mg. Different kinetics
were obtained in blood cells.
Possible adverse effects on human health have been
reported from time to time. However, an exhaustive
search of the literature has failed to find confirmation of
this, and in each case evidence exists which refutes the
finding which has led to the suggestion. This is summarized
as follows. (1) The formation of urinary oxalate stones in
subjects ingesting large amounts of vitamin C over a long
period proved to be without foundation. Although human
subjects do metabolize some ascorbate to CO2 , they excrete
considerable amounts of unchanged ascorbate, and a range
of metabolites among which is a small amount of oxalate;
intake of ascorbate at levels in excess of the level required to
maintain plasma levels at about 10 mg/l results in excretion
of the excess ascorbate unchanged (Kallner et al. 1979).
Approximately 35–40 % of the daily excretion of oxalate is
derived from ascorbate, but ingestion of large amounts of
vitamin C results in a very small increase in the excretion of
oxalate. It was shown clearly (Schmidt et al. 1981) that
there is no dose–response relationship between administered vitamin C and excreted oxalate. Part of the explanation
of the difference between this result and that of earlier
workers is that in the early experiments the alkalinity of
urine samples that arises on standing caused conversion of
some ascorbate to oxalate; if steps are taken to avoid
alkalinity then this conversion is minimal (Wandzilak et
al. 1994). Recent work (Levine et al. 1996) indicates,
however, that both oxalate and urate excretion are elevated
beyond a daily intake of 1000 mg. (2) Similar anxieties were
expressed with regard to urate excretion with the possibility
that ascorbate might therefore indirectly exacerbate the
effect of urate on gout. Two studies demonstrate that in
healthy subjects ascorbate ingestion has no effect on the
excretion of urate (Mitch et al. 1981; Schmidt et al. 1981).
When high non-physiological plasma levels of ascorbate
were induced by continuous infusion of ascorbate in gouty,
as compared with normal, subjects (Berger et al. 1977),
there was no effect on the clearance of urate indicating that
it is highly improbable that high dietary intake of ascorbate
has any effect on the urinary excretion of urate in subjects
with gout. (3) Low plasma levels of vitamin B12 were
reported (Herbert & Hacob, 1974) to occur in subjects
taking large doses of ascorbic acid but this was shown to
be explained by analytical error. Erroneously low levels of
plasma vitamin B12 can occur if no cyanide is added to the
assay to liberate protein-bound cobalamins and to stabilize
the cobalamins so released (Newmark et al. 1976; Markus et
al. 1980). (4) High ascorbic acid intake has been shown to
have only a small effect on Fe absorption in healthy Fereplete subjects (see review by Bendich, 1997) which
repudiates suggestions that Fe overload could be a consequence of high ascorbate intake (Cook et al. 1984). It
appears that the regulation of body Fe stores is unaffected
by any increased availability of Fe from the diet that
might be caused by an effect of the excess ascorbate on
Fe. (5) Early reports (Cochrane, 1965; Rhead & Schrauzer,
1971) of rebound scurvy in a small number of subjects
following withdrawal of high vitamin C supplements were
uncontrolled and have not been substantiated. Studies in
guinea-pigs showed no evidence for these claims even when
the study was designed to demonstrate a rebound effect. No
increased catabolism of ascorbate was demonstrated during
high vitamin C dosage nor was there any such increase when
the vitamin C dosage was withdrawn (Norkus & Rosso,
1975, 1981). Although the human data available remain
contradictory, evidence available at present leads one to
conclude that the phenomenon, if it exists at all, does not
constitute a significant health problem. (6) Ascorbic acid
added to cells in vitro in culture increases the rate of
mutagenesis (for review see Rivers, 1989). Detailed reports
exist of increased DNA fragmentation, increased DNA
repair and chromosome aberrations in cells cultured in
media that include added ascorbate. However, these effects
only occur in cultures that contain added Cu2Гѕ or Fe3Гѕ ions
and when steps were taken to ensure very low levels of these
metals in the culture medium, no detrimental effect on DNA
was observed. It can be concluded that in any such system in
vitro the mutagenic effect of ascorbate is probably due to an
ascorbate–metal ion-driven generation of O-derived free
radicals. There is no evidence of ascorbate-induced mutagenicity in vivo so that it is highly improbable that any effect
that depends on metal ion-driven generation of free radicals
caused by ascorbate has any significance. Efficient freeradical scavenging and repair systems protect DNA in vivo
from such effects, and intracellular concentrations of ascorbate, and concentrations of metals, which are efficiently
sequestered on binding proteins, are so low as to be unlikely
to be harmful (Halliwell & Gutteridge, 1989).
The conclusion from an exhaustive survey of the
literature is that oral intake of high (up to 600 mg/d, i.e.
six times the current RDA) levels of vitamin C are safe and
entirely free from side-effects (Bendich, 1997). Very high
levels (up to 2000 mg/d) have not been consistently reported
to result in side-effects, although some reports of low
reliability suggest that minor side-effects may occur.
5.3. Vitamin E
There have been four reliable reviews of the toxicological
safety of oral intake of vitamin E (Bendich & Machlin,
1988, 1993; Kappus & Diplock, 1992; Diplock, 1995). A
problem in comparing studies of this nature has been the
confusion that exists in the literature between different
forms of vitamin E. In particular, IU are often quoted as a
measure of quantity. The IU was abandoned by WHO in
1957 (cited by Diplock, 1985) and studies which cite this
measure are often ambiguous because it is not possible to
determine the precise amount of vitamin E that was
Defence against reactive oxidative species
The international standard was based on 2-ambo-a-tocopheryl acetate, which was an early sample of vitamin E
which contained RRR-a-tocopheryl acetate with an
unknown content of �2-epi-a-tocopheryl acetate’ (i.e.
S,R,R-a-tocopheryl acetate). Because of the uncertainty as
to the precise composition of this standard, the IU was
abandoned, and in 1983 WHO recommended the use of
precise descriptions of pure compounds. Despite this, the IU
has continued to be used for labelling purposes, particularly
in the US and Canada. There is no description of the various
stereoisomers present, which may have widely differing
biological activity, and this causes serious confusion.
Vitamin E activity is expressed in the present paper as mg
RRR-a-tocopherol equivalents (a-TE) wherever this is
possible. To estimate the total a-TE the number of mg of
tocopherols present is multiplied by a factor as follows:
RRR-a-tocopheryl acetate
RRR-a-tocopheryl succinate
all rac-a-tocopherol
all rac-a-tocopheryl acetate
Г— 1 :0
Г— 0:91
Г— 0:81
Г— 0:74
Г— 0:67:
Study of conventional aspects of the toxicity of vitamin E
in animals was undertaken by many workers over a long
period of time (Demole, 1939; Weissberger & Harris, 1943;
Levander et al. 1973; Dysma & Park, 1975; Krasavage &
Terhaar, 1977; Abdo et al. 1986). There is no evidence of
any detrimental effect attributable to vitamin E, and similar
conclusions are possible with respect to the teratogenicity
and reproductive toxicity of vitamin E in animals at even
large levels of intake of the vitamin (Hook et al. 1974;
Krasavage & Terhaar, 1977). The possibility that vitamin E
might have anticlastogenic and mutagenic effects has been
studied extensively, and its potential mutagenicity was
tested in several different ways (Shamberger et al. 1979;
Beckman et al. 1982; Gebhart et al. 1985). Vitamin E was
conclusively demonstrated to have no mutagenic properties,
and indeed appears to have some effect in reversing the
mutagenic effects of other compounds. Although early
literature suggested that impure fractions containing
vitamin E had tumour-promoting capability, there is a
very large body of evidence that refutes this claim in studies
where pure compounds were used (Yang & Desai, 1977;
Weldon et al. 1983). Even at very high levels of inclusion in
the feed (up to 25 000 IU/kg feed), vitamin E was not shown
to have any carcinogenic activity.
Many reports purport to deal with the toxicity in human
subjects of vitamin E, but there are different levels of
reliability that can be attributed to them. Published reports
include those that describe single observations on one
subject, and planned toxicological studies which may or
may not include placebo groups, but which often include
bias because they lack blinding. There are fewer highly
reliable studies which have been planned and carried out
with all necessary rigour, which include sufficient numbers
of subjects to enable proper statistical evaluation of the
results; they use placebo groups and careful double-blinding, so that the results obtained are valid and reliable.
Sufficient such studies have been undertaken to enable an
authoritative view of the human toxicity, or lack of toxicity,
of vitamin E. There are a few studies (Farrell & Bieri, 1975;
Corrigan, 1982; Ernst & Matrai, 1985) which do not have
adequate controls but are nevertheless of some interest. No
consistent adverse effects of vitamin E emerge from these
uncontrolled studies. However, the study of Corrigan (1982)
indicates that vitamin E supplementation may aggravate
vitamin K deficiency induced by warfarin anticoagulant
therapy (see next paragraph).
With regard to controlled double-blind studies of vitamin
E toxicity in man, several reports show conclusively that
vitamin E has very low toxicity in human subjects with no
consistent adverse effects being reported in most subjects
(Anderson & Reid, 1974; Gillian et al. 1977; Inagaki et al.
1978; Tsai et al. 1978; Stampfer et al. 1983; Bierenbaum et
al. 1985; Kitagawa & Mino, 1989). However, some adverse
effects were observed on prothrombin time, or on other
factors associated with blood clotting. This question was
reviewed carefully (Kappus & Diplock, 1992; Diplock,
1995); in several studies no effect was observed on blood
clotting whereas in other studies there was a marked effect
of vitamin E on some aspects of blood-clotting mechanistics. Following a study of all the reports available it was
concluded that vitamin E at a high level of intake may affect
the coagulation variables if a low level of vitamin K is also
present. Therefore, it is clear that vitamin E cannot be
recommended for administration under these conditions.
Vitamin E does not, by itself, cause coagulation abnormalities in persons who have no pre-existing coagulation
abnormalities, and in individuals who are the majority of
the population, vitamin E supplementation is entirely free
from these adverse effects on blood-clotting. This view has
been challenged recently (Kim & White, 1996). In a doubleblind study in twenty-five patients given warfarin therapy,
vitamin E (either 536 or 804 a-TE/d) was given for 4 weeks
without any measurable effects on blood coagulation variables. However, in view of the short time span of this study
and the small number of subjects (four per experimental
group) it would be unwise to overturn the consensus view
that vitamin E therapy is contra-indicated in subjects with
coagulation abnormalities.
The question has been raised (Steinberg, 1993) of the
safety of long-term ingestion of amounts of vitamin E in
doses of 100 mg/d which were found (Rimm et al. 1993;
Stampfer et al. 1993) to confer significant protection against
the risk of coronary artery disease. Higher doses than this
(up to about 500 mg vitamin E daily) have also been
recently shown to confer benefit in apparently causing
improvement in subjects with angiographically proven
cardiovascular disease (Stephens et al. 1996). Results
(Takamatsu et al. 1995) of a trial in which 100 mg d-atocopheryl acetate was administered to human subjects for 6
years indicate that there were no adverse effects of the
treatment during clinical follow-up. In another study about
250 mg vitamin E was given daily for 5 years without any
side-effects (Greenberg & The Polyp Prevention Study
Group, 1994). While it is not possible at present to state
categorically that oral ingestion of large amounts of vitamin
E is entirely safe for long periods, because no one has been
able to test the toxicity of the vitamin for very long periods
of time, application to vitamin E of the usual criteria of
A. T. Diplock et al.
safety applied to any drug suggests that not only will longterm supplementation prove to be free from harmful sideeffects but that this may also convey considerable health
The following conclusions, which were reached earlier
(Kappus & Diplock, 1992; Diplock, 1995) with respect to
the safety of oral intake of vitamin E by human subjects, can
be endorsed here. (1) The toxicity of vitamin E is very low.
(2) Animal studies show that vitamin E is not mutagenic,
carcinogenic or teratogenic. (3) Reported increases in serum
lipids in human subjects following high oral dosage are
inconsistent and of little significance. (4) In double-blind
human studies, oral dosage resulted in few side-effects, even
at a dosage as high as 3.2 g/d. (5) Dosage up to 1000 mg/d is
considered to be entirely safe and without side-effects. (6)
Oral intake of high levels of vitamin E can exacerbate the
blood coagulation defect of vitamin K deficiency: high
vitamin E intake is contra-indicated in these subjects.
5.4. Carotenoids
Present knowledge of the human toxicology of carotenoids
derives almost exclusively from work on b-carotene; the
assumption that other carotenoids have similar toxicology to
b-carotene may not be justified. For instance the absorption,
uptake and tissue distribution may differ among the different carotenoids, some of which are bioavailable and others
of which are not. Reviews about the safety of b-carotene
(Bendich, 1988; Diplock, 1995) are supported by a review
(Wang, 1994) on its absorption and metabolism. Use of bcarotene as a food and cosmetic colourant and as a drug and
nutrient has necessitated extensive reliable toxicity studies
done using a range of techniques (Bagdon et al. 1960;
Heywood et al. 1985). The Ames test revealed no mutagenicity, which was confirmed by studies using the mouse
bone-marrow micronucleus test. Embryotoxicity was not
found in rats and rabbits, and in a multiple-generation study
in rats given up to 1 g/kg per d orally, reproductive function
was normal, and there was no interference with embryonic
morphology. A study conducted over a 2-year period in
dogs revealed no tumourigenicity or chronic toxicity of any
kind and in a mouse carcinogenicity study b-carotene was
without any tumourigenicity. In several organs of dogs and
mice given high doses of b-carotene vacuolated cells were
seen due to the formation of fat storage cells; this was not
dose-related and was thought to be harmless. These toxicity
trials led to b-carotene being placed in the US Food and
Drug Administration category of �foods generally recognized
as safe’ for use as a food colourant, in drugs and cosmetics
and as a dietary supplement and nutrient (Office of Life
Sciences Research, 1979). In addition b-carotene has been
used for 30 years to treat patients with genetically inherited
photosensitivities; in this context the ingestion of large
amounts of pure b-carotene has not produced toxic sideeffects (Matthews-Roth, 1986). Some individuals taking
supplements of >30 mg/d may experience hypercarotenaemia but this disappears quickly after discontinuing the
treatment, and it is a benign condition without permanent
adverse effects. Anecdotal reports, linked to b-carotene, of
leukopaenia, reproductive disorders, increased prostatic
cancer incidence, retinopathy, and allergic reactions have
not been substantiated in proper clinical trials. A short-term
phase I toxicity trial of supplemental b-carotene in a small
number of human volunteers (Xu et al. 1992) demonstrated
a progressive statistically significant decrease in serum
vitamin E concentration during supplementation for 9
months with 15, 30, 45 and 60 mg b-carotene/d. However,
other studies have demonstrated no such interaction (Willett
et al. 1983; Albanes et al. 1992; McLarty, 1992; Goodman
et al. 1994; Nierenberg et al. 1994; Ribaya-Mercado et al.
1995). There is no satisfactory explanation available for the
results of Xu et al. (1992), but the balance of probability is
that there is no likelihood of any interaction between bcarotene and a-tocopherol that would alter the nutritional
availability of vitamin E to human subjects. Careful
monitoring of nutrient interactions should become part of
all long-term intervention studies.
It can be concluded that supplementation of normal
individuals in the population with moderate amounts of bcarotene can be undertaken safely. The safety of this
practice by heavy smokers, who are at high risk of developing lung cancer, has been put in question by two recent
studies (ATBC Cancer Prevention Study Group, 1994;
Omenn et al. 1996), details of which are given in section
4. The increase in incidence of lung cancer (18 % and 28 %
respectively in the two studies) has no easy explanation.
Both studies were conducted among subjects who had a high
risk of developing lung cancer and may have been at an
advanced precancerous state when the b-carotene administration was commenced. Observational epidemiological
evidence suggests that subjects who are not in this state
may benefit from b-carotene administration which is likely
to exert a protective role at an early stage of the cancer
process. Until further work clarifies the situation in heavy
smokers with respect to taking supplements, larger doses
should be avoided by such individuals. However, it should
be stressed that for normal subjects who do not smoke, bcarotene supplementation is entirely safe.
5.5. Non-nutrient antioxidants (flavonoids and other related
Few data exist on absorption, metabolism and possible
adverse effect of flavonoids and flavonoid-related compounds, such as genistein, daidzein or phenolic acids.
Since these compounds are present in our daily diet they
are believed to carry no health problems. Obviously, reliable
comprehensive safety data, practically non-existent today,
will be required, should it appear that increased intakes of
specific flavonoids and other phenolic compounds would
confer significant health benefits. As concentrations of
phenolic compounds in edible plants, fruits and vegetables
vary considerably, estimates of human daily intakes range
from approximately 100 mg to 1000 mg (Kuhnau, 1976),
depending on the diet.
5.5.1. Absorption. It appears that two major metabolic
routes operate in man in the absorption of dietary
flavonoids. Intestinal micro-organisms hydrolyse the
flavonoid glycosides (nearly all the flavonoids are present
as glycosides) to their constituent aglycone and sugar. Most
of the aglycones are subsequently metabolized by microorganisms. A minor portion is absorbed as aglycones
Defence against reactive oxidative species
(Hackett, 1986). A study on the absorption of quercetin in
volunteers found no detectable amount of quercetin or of its
metabolites in plasma or in urine (Gugler et al. 1975).
In a more recent study less than 0.25 % of ingested
quercetin was found to be excreted, either unchanged or
as conjugates, in the urine of healthy ileostomy volunteers
(Hollman et al. 1995). In contrast, 0.1–1.4 % 14 C-labelled
catechin was excreted in urine in the form of unchanged
compound. However, 55 % of the ingested dose was
excreted as its methylated derivatives (Hackett et al.
1983). There appear to be no human studies available on
the absorption and metabolism of anthocyanins.
5.5.2. Possible adverse effects. When tested in vitro in
cell cultures, flavonols and especially quercetin have a high
cytotoxicity (Babich et al. 1993). However, their toxicity in
vivo was shown to be remarkably low when tested in rats
(National Toxicological Program, 1992). Most of the
flavonols including quercetin are also mutagenic in the
Ames test and other short-term tests in vitro (Sugimura et al.
1977; International Agency for Research on Cancer, 1983).
However, other tests in vitro have shown quercetin to be a
potent inhibitor of mutagenic activity of food carcinogens,
such as heterocyclic aromatic amines (Stavric et al. 1990).
The same anti-mutagenic activity was found in the livers of
mice when quercetin was added to their feed (Stavric, 1994;
Stavric et al. 1990). Another test on mice showed the
mutagenic effect of heterocylic aromatic amines to be
enhanced by quercetin (Rowland, 1993).
Studies on three flavonols, quercetin, myricetin and
kaempferol carried out by the molecular toxicology
branch of the US Food and Drug Administration point to
the prooxidant properties of these compounds and �suggest a
dual role for these flavonoids in mutagenesis and
carcinogenesis’. The authors stress that these polyphenolic
flavonoids are generally considered to be both antioxidants
and anticarcinogens (Sahu, 1992; Sahu & Gray, 1993,
1994); these studies were performed in isolated rat liver
nuclei under aerobic conditions.
A study on quercetin conducted by the US National
Toxicological Program (1992) concluded that there was
�some evidence of carcinogenic activity’ in male rats
receiving 40 000 mg quercetin/kg diet for 2 years; there
was no evidence of carcinogenicity in the lower dose
groups. It is important to note that the statistical evaluation
of the National Toxicological Program study was not
without controversy (Ito, 1992) and a number of previous
long-term, well-controlled feeding experiments with rats,
mice and hamsters did not show any carcinogenic activity of
quercetin (Hirono et al. 1981; Ito et al. 1989). It should also
be emphasized that quercetin is one of the most abundant
flavonoids, being present in most common vegetables and
fruits with an average daily intake estimated to be approximately 25 mg/person (National Toxicological Program,
1992). The consumption of quercetin from a regular diet
appears not to induce health problems. The observed very
low bioavailability of quercetin in human subjects may
explain the apparent contradiction between the results of
the tests carried out in vitro and its apparent beneficial
effects in man, although the available evidence for beneficial effects of quercetin needs further confirmation.
Whereas flavonoids are not mutagenic, anthocyanins have
not been tested regarding their potential mutagenic properties. No carcinogenic effects have been reported for other
Tannins, which may exert anticarcinogenic effects by
acting as free-radical scavengers, have also been reported to
be associated with an increased incidence of oesophageal
cancer (Mortin, 1989), but there is no definite evidence to
support this assertion. The acute toxicity of tannins was
studied in rats, mice and rabbits. Median lethal dose (LD50 )
values for a single dose of orally administered tannins range
from 2.25 to 6.00 g/kg body weight (Singleton & Kratzer,
1989). These results are difficult to apply to the human diet
since in food the chemical structures and the molecular
masses of tannins vary considerably. Anthocyanin extracts
are already widely used by the food industry. Anthocyanins
obtained by extraction of vegetables and edible fruits have
been approved worldwide as food colours (see, for Europe,
European Parliament and Council Directive, 1994). They
are most commonly based on the following anthocyanidins:
peonidin, malvidin, delphinidin, petunidin, pelargonidin,
cyanidin. There are two reviews (World Health Organization, 1982; Timberlake, 1988) of the limited toxicological
data concerning anthocyanidins. These include data on
mutagenicity, reproductive toxicity, and teratogenicity and
conclude that anthocyanin-containing extracts are of a very
low order of toxicity. The only negative effects were
reduced organ and body weights associated with reduced
energy intakes at the highest dose, probably reflecting
reduced palatability (Clifford, 1996). An average daily
intake of 2.5 mg/kg body weight per d was allocated for
anthocyanin colour from grape skin extracts (World Health
Organization, 1982), the composition of which may vary
depending on grape variety and extraction process. Anthocyanin colours from grape skin extracts also contain other
flavonoids and some tannins. Recently epigallocatechin
gallate and green-tea extract have been shown to prevent
gastrointestinal carcinogenesis in volunteers; no adverse
effects were reported (Yamana et al. 1996).
In conclusion, it is not possible to provide a definitive
statement as to the toxicity of flavonoids and other nonnutrient antioxidants, because the literature is either
controversial or lacking. A key question that needs to be
resolved is the uptake and tissue distribution of non-nutrient
substances, which has been hampered by the lack of suitable
methodology. Recently a reliable and reproducible method
has been published which shows that flavonoids are indeed
absorbed in human subjects and enables the measurement in
human plasma of flavonoids as their glycosides at concentrations of 0.5–1.6 mmol/l (Paganga & Rice-Evans, 1997).
It seems advisable therefore to test rigorously well-defined
flavonoids in toxicological programmes for possible adverse
effects if new scientific evidence confirms potential
beneficial effects on human health.
6. Role of food technology in nutritional and safety
aspects of antioxidants
6.1. Introduction
Our food supply is safer and offers more variety today than
it ever has in the past and this can be largely attributed to the
A. T. Diplock et al.
applications of food technology to food raw materials. Food
processing preserves foods safely so that they can maintain
high nutritional and organoleptic values during storage and
achieve a wide distribution.
After microbial spoilage, oxidation is the second most
important cause of food spoilage, even in those products
which might be considered low in oxidizable substrates,
such as potato flakes. Important nutrients such as unsaturated lipids, vitamins and proteins can be lost through
oxidation. This has been known for a long time and therefore attention to the control of oxidative processes in foods
has been one of the highest priorities of the food industry.
Until recently antioxidants have been viewed as tools in this
fight against oxidation but now, because of growing recognition that antioxidants are themselves important for the
maintenance and optimization of health, special efforts may
be required to protect the antioxidant nutritional value of
O2 is the enemy of antioxidants. This is evident but bears
mentioning because the protection of foods from interaction
with O2 is the basic principle on which antioxidant
protective technologies are based. Many of these have
been drawn from experience with lipid oxidation. These
technologies can be conveniently broken down into two
sections: physical and chemical.
6.2. Physical processes
6.2.1. Structural integrity. The structural integrity of the
food plays an important role in protecting antioxidants from
contact with O2 and thus potential oxidative destruction. In
its whole food form, the antioxidant is encased in its
protective liposome or cell membrane structures and is out
of contact with both O2 and oxygenases. For example, intact
oilseeds are quite stable but once they are crushed, extracted
and heated in the refining process, resistance of the oils to
oxidation decreases. Thus, one nutritional consequence of
this loss of structural integrity is negative in that some
antioxidants are lost to oxidation. However, the positive
nutritional consequence is that antioxidants become more
It has been known for some time that in human subjects
the gastrointestinal absorption of carotenoids from vegetables is inversely proportional to particle size (Rodriguez &
Irwin, 1972). Heat treatment also improves absorption:
more lycopene appeared in plasma when human subjects
drank heated tomato juice compared with the same dose of
raw tomato juice (Stahl & Sies, 1992). In another recent
study, ascorbic acid was more bioavailable from cooked
broccoli than it was from the raw form (Mangels et al.
1993a). This appears to be a human attribute: studies in
other species such as rats (Sweeney & Marsh, 1974) and
preruminant cows (Poor et al. 1993) have shown,
respectively, no differences or only small differences in
absorbability of cooked v. uncooked vegetables. Therefore
it may not be appropriate to extrapolate information
obtained using animal models to man.
6.2.2. Moisture content. The moisture content of foods
should be judiciously chosen and carefully controlled if the
antioxidant content of the food is to be optimized since
oxidation shows a U-shaped curve, i.e. rapid rates of
oxidation when moisture content is low as well as when it
becomes too high. Most dehydration processing will destroy
lipoxidases but if water content falls below a level which
permits the formation of a protective monolayer of water
over the surface of the food increased auto-oxidation can
result (Labuza, 1971).
According to Labuza (1971), a higher level of water can
act as a solvent which mobilizes catalysts and reactants to
sites of oxidation. Water may also interact chemically or by
H-bonding with other molecular species.
6.2.3. Temperature. Thermal treatment holds an important place in food processing because of the many benefits it
brings to food preservation. It also has both a negative and a
positive impact on antioxidants. The positive effects include
inactivation of oxidases, and breakdown of food structures
leading to improved bioavailability. These topics have been
dealt with earlier.
Degradation. Thermal treatment also has an important
negative impact on antioxidants in foods. Carotenoids and
anthocyanins are sensitive to heating: in many studies of
foods and food models, these compounds progressively
degrade as time and temperature increase. With radical
treatments this effect can be radical too. For example at 100РЉ
for 5 min, only 0.93 % b-carotene was lost in a glycerol
model, but 97.8 % was lost when the thermal treatment was
carried on for 4 h at 210РЉ (Onyewu, 1985). In palm oil
thermal destruction of b-carotene doubles with every 20РЉ
rise in processing temperature (Jideani, 1992). However,
technologies exist to protect b-carotene, tocopherols and
tocotrienols. Using a modification of the refining method
one Malaysian supplier guarantees specifications of 400 mg
b-carotene/kg and 800 mg/kg minimum of total tocopherols
and tocotrienols. This is quite a good level given that most
crude palm oils contain on average only 500–750 mg/kg
(Hood, 1995). Water-soluble antioxidants such as ascorbic
acid are also sensitive to heating. Blanching of green beans,
spinach, broccoli, or peas before freezing resulted in,
respectively, losses of 0, 20, 20 and 33 % of this vitamin
(Unilever, 1995).
Isomerization. Another effect of heating is to increase
isomerization of carotenoids in foods. In one study, thermal
processing of guava juice increased cis-lycopene levels by
5-fold and decreased trans-lycopene, although in this food,
b-carotene remained unchanged (Padula & Rodriguez,
1987). Supplemental b-carotene added to wholewheat
breads and crackers before baking showed significant
trans to cis isomerization (4–15 % for bread and 18–23 %
for crackers) (Ranhota et al. 1995). More isomerization was
observed in the crackers because of the more severe thermal
treatment, and less water was left in the final product
compared with the bread product. The processing associated
with canning is sufficient to cause isomerization of carotenoids: 18–30 % of b-carotene is converted to cis isomers by
usual treatment of typical fruits and vegetables (Quackenbush,
1987). The nutritional significance of this is that the cis isomer
of b-carotene is markedly less well absorbed by the
gastrointestinal tract and markedly less well transported in
the body (Gaziano et al. 1995). Thus, cis and trans isomers
of carotenoids are not biologically equivalent.
Storage temperature. Losses of antioxidants from fresh
fruits and vegetables during storage can be quite significant.
Defence against reactive oxidative species
At ambient temperature 90 % of vitamin C was lost from
spinach leaves within 3 d. At refrigerator temperatures this
loss was greater than 50 %. In one recent study, freezing
effectively halted any decrease in vitamin C losses in peas,
broccoli, green beans and spinach over 3 months (Unilever,
6.2.4. Minimizing oxygen. There are some processes
which can eliminate or minimize the presence of O2 in the
foods. This is quite important as, for example, the rate of
degradation of L-ascorbic acid in orange juice (Kennedy et
al. 1992) and in canned, sterilized green beans (Bloeck et al.
1986) depends on O2 present in the sample. Fruit juices
containing entrapped air are deaerated by being sprayed
into a vacuum deaerator which minimizes the potential
for destructive changes to ascorbic acid and other oxidizable
components due to O2 . Other products e.g. dried red
peppers (Lee et al. 1992) can be prepared or packaged
under a N2 atmosphere with successful retention of
One of the newer approaches is the potential to use
enzymes as antioxidative agents in food systems. Enzymes
can act by removing O2 , ROS such as H2 O2 and superoxide
radicals or by reducing lipid hydroperoxides. The enzymes
presently under most study are glucose oxidase (EC, thiol oxidase, galactose oxidase (EC,
pyranose oxidase and hexose oxidase in conjunction with
catalase, superoxide dismutase and glutathione transferase
and glutathione peroxidase (for review see Meyer &
Isaksen, 1995). These systems are under study but are
not currently of commercial significance in the food
6.2.5. Protection from light. At wavelengths of less than
500 nm, light is an important generator of lipid oxidation
and vitamin destruction. Improving packaging by adding
colour (red, brown, black) or obscuring material is one of
the most effective strategies for protecting foods from light.
Some components of foods act as photosensitizers:
riboflavin, chlorophyll, myoglobin, haemoglobin pigment,
pheophytin and certain conjugated double-bond systems are
examples. Sandmeier (1996) has recently shown that
curcumin and curcumoid compounds, which are often
present in curry mixes, have rather potent photosensitizing
properties. Removing these photosensitizers or incorporating food-grade quenchers such as b-carotene, and atocopherol, together with chelators of trace metals, can
also be effective means of controlling the damaging effects
of light.
6.2.6. Irradiation. The effects of irradiation are somewhat variable depending on the food being irradiated, the
nutrient under study and the strength and length of time of
irradiation. The irradiation of peppers demonstrated no
change in vitamin C content in doses up to 300 Gy (Mitchell
et al. 1992). In fact, post-irradiation storage resulted in
increases in total vitamin C in intact fruits and vegetables
(Mitchell et al. 1992).
In one study of chicken meat, b- and a-tocopherol
decreased linearly with irradiation in direct relationship to
dose. At 3 kGy, which is the maximum recommended by the
Food and Drug Administration for chicken, 15 % b-tocopherol
and 30 % a-tocopherol were lost (Lakritz & Thayer, 1992).
However, a newer study by the same authors (Lakritz &
Thayer, 1994) showed different results: only a 6 % reduction
in a-tocopherol and no significant change for b-tocopherol
in chicken meat at 3 kGy and 2РЉ. Irradiation with a low dose
(0.010 kGy) had no effect on the polyphenol content of herb
teas (Katusin et al. 1988).
6.3. Chemical processes
6.3.1. Enzymes. The control of polyphenol oxidases has
been a priority in food science for a long time because
oxidized polyphenols are responsible for undesirable brown
colours in cut fruits and vegetables, and for the development
of off-colours and off-flavours in frozen foods over time.
Inactivation of these enzymes can be achieved by rapid
high-temperature heat treatment (blanching) while the fresh
character of the food can still be retained. Rapid inactivation
of the oxidases after pressing apples is a prerequisite for the
long-term stability of apple juice (Anonymous, 1993).
Extrusion processes, because of their conditions of high
temperature and pressure, destroy oxidases and increase
lipid binding to protein and carbohydrate elements in the
food which acts to protect from lipid oxidation (Artz et al.
Cloudy fruit juices are more stable than clear juices
because of their content of polyphenols and their glycosides.
These latter compounds are not substrates for polyphenol
oxidase (EC (Baruah & Swain, 1959). For the
preparation of clear juices such as apple or raspberry,
pectinases are often used. These enzymes hydrolyse the
glycosidic side-chains of flavonoids which are then less
oxidatively stable in the juice (Rommel & Wrolstad,
1993a,b). However, glycosides of flavonols and isoflavones
are poorly absorbed by the small intestine compared with
their aglycones (Brown, 1988). Therefore the nutritional
impact of using pectinases is difficult to gauge: on the one
hand the polyphenols become more bioavailable, and therefore potentially capable of having an impact on health, but
on the other hand there may be fewer of them in the food, as
being less stable, they are oxidized.
6.3.2. Supplementation. Supplemental antioxidant
nutrients can be added to foods for technological reasons.
Ascorbic acid is widely used to increase the resistance to
oxidation of many foods. For example, it is used as a dip for
cut fruit and vegetables as it is preferentially oxidized
instead of the catechol–tannin compounds and effectively
inhibits enzymic browning reactions (Potter & Hotchkiss,
1995). In hydrophobic food matrices, vitamin E is also
frequently used to provide antioxidant protection. Since
vitamin C recycles vitamin E, it is desirable to use both of
these antioxidants together. However, since vitamin C is
hydrophilic and vitamin E lipophilic, it is difficult to use
them together effectively in food systems. One of the
solutions is to use ascorbyl palmitate, a hydrophobic
vitamin C which has been shown to be useful in food lipid
systems (Klaui & Pongracz, 1981).
A mixture of vitamin E, vitamin C and phospholipids is
an effective antioxidant partly because the phospholipid acts
as an emulsifier, allowing the two vitamins to be in contact
with one another and also because the phospholipid itself
actively participates in the antioxidant process (Loeliger et
al. 1996). Both vitamins can be incorporated into liposomes
A. T. Diplock et al.
which can be used as a delivery system in oil-in-water
emulsions (Pothakamury & Barbaosa-Canovas, 1995).
Sometimes higher levels of antioxidants can be incorporated
indirectly into a foodstuff. Feeding chickens supplemental
vitamin E increased a-tocopherol levels in the meat and this
was associated with greater stability (less formation of
volatiles) of the meat when it was later irradiated (Patterson
& Stevenson, 1995).
Supplementation with nutrient antioxidants is, thus, a
reasonable strategy in many situations for the improvement
of the stability and nutritional value of a processed food.
Indiscriminate supplementation with antioxidants, as with
any nutrient, would however be quite undesirable as there
are many unknown factors concerning potential interrelationships in vivo between different antioxidants and
eventual long-term effects. To illustrate this point, in a
recent study, b-carotene doses resulted in less lycopene in
LDL suggesting that the two related hydrocarbons compete
for a similar absorption or transport mechanism (Gaziano et
al. 1995).
Non-nutrient antioxidants such as SO2 can be used to
advantage to protect those with known or suspected nutritional benefit. For example, the phenolic and procyanidin
composition of grape juice processed with SO2 is
consistently higher than that of grape juice processed without SO2 addition (Spanos & Wrolstad, 1990), although there
appears to be no effect on quercetin. SO2 will also prevent
oxidation of carotenoids but causes bleaching of anthocyanins (Sian & Soleha, 1991).
Supplemental antioxidants from natural sources such as
herbs and spices are effective in conserving foods from
oxidation. Many plant extracts have antioxidant activity:
rosemary, sage, thyme, oregano, ginger, turmeric, cloves
and bay leaves all are active in descending potency (Loliger
et al. 1996). Different compounds have very different
activities. For example three components of thyme extract,
thymol, carvacrol and p-cymene-2,3-diol, were decreasingly resistant to thermal stress in a lard model (Ternes et
al. 1995). Mixtures of antioxidants can be obtained from
natural sources, for example, by mixing with oil and pressing (Aeschbach & Wille, 1993), or by using supercritical
CO2 extraction of antioxidants. This latter technique has
aroused interest because the solvent involved (CO2 ) is nontoxic, non-flammable and of low cost (Tsuda et al. 1995).
O2 is almost eliminated from the system. Polyphenols can
be recovered through membrane technologies, for example
from the waste water from olive oil processing (TraВЁgardh,
1995). It is presently poorly understood whether compounds
from this or other fruit and/or vegetable sources have
antioxidant ability in vivo similar to that they demonstrate
in vitro and therefore could potentially contribute direct
health benefits.
6.4. Conclusions
The food industry has long experience in the control of
oxidative damage in foods and this experience can be used
to advantage for the protection of food antioxidants which
are beneficial. Some of these, such as vitamins C and E and
b-carotene are known, and strategies for their protection in
foods are already exploited by many food technologies.
However, there are many compounds with antioxidant
activity which may, or equally may not, have biological
activity. Since oxidation cannot be eliminated completely,
efforts must be made to identify those antioxidants which
are important to health, and in what form they are most
useful. This requires that more information must be
obtained in the much neglected field of their bioavailability
as well as on their bioactivity. With this kind of information,
food technology strategies for the preservation of those
antioxidants which are beneficial to health could then be
applied in a cost-effective manner.
7. Critical assessment of the science base and conclusions
7.1. Identification of criteria
The foregoing sections have surveyed the science base that
underpins the argument that oxidative damage is a significant causative factor in the development of human diseases;
that antioxidants are capable of preventing or ameliorating
these disease processes; that the administration of antioxidants to human subjects is safe; and that food technology
can adapt to meet the needs for nutritional quality and safety
in the use of antioxidants in foods. It is now necessary to
evaluate clearly what the impact of this survey is on the
question of whether an identified specific antioxidant nutrient can be said to positively affect function and have a riskreducing role. This must involve consideration of whether a
cause–effect relationship can be established between a
dietary antioxidant and a health benefit, and what is the
optimum level of intake of the antioxidant in conferring that
There are four questions the answers to which are
considered to be fundamental to providing the required
critical assessment.
(1) What is needed to establish that free-radical events are
involved in detriment to health which eventually can be
associated with the pathology of an identified disease?
(2) What is needed to establish that antioxidants have
specific health benefits?
(3) What is needed to make a claim about the functional,
nutritional or health benefits of antioxidant nutrients?
(4) What is needed to bring an antioxidant product to the
The technology base is very relevant because it is
important, in bringing a product to the market, that the
active ingredients in the food are still active when the
consumer consumes them. A further matter also needs
careful consideration. There is a clear difference between,
on the one hand optimizing health, by improving intake of
food constituents, and on the other hand preventing disease,
although the boundaries between these two objectives are
not clear-cut. Furthermore, the difference between an effect
of a nutrient as a food, and its pharmacological effect,
usually at a higher level of intake, needs to be borne in
mind. It is important to distinguish between healthy
products which optimize well-being and health, and those
which may be capable of preventing disease. A single
functional food is unlikely by itself to prevent disease; if
it were to do so it would be at a very high level of dietary
Defence against reactive oxidative species
intake and questions of toxicity might become important.
The disease is the paradigm, the �experimental model’, by
means of which it is possible to determine the importance to
human subjects of the nutrient. Antioxidants appear to be
useful for optimizing well-being, such as in ageing and in
simple stress situations, not preventing disease. The future
emphasis for functional foods that contain antioxidants must
be in providing a suitable level of intake that enhances wellbeing and health. A physician examines a patient by a set of
established signs, as a result of which the patient is pronounced �well’. There is, however, a category beyond this
which may be referred to as �well-being’; this can be defined
by objective criteria (McDonald & Newell, 1996; Spilker,
1996). The optimal level of intake of an antioxidant might
be demonstrated to be higher than that which is capable of
being delivered by conventional food, in which case the
�functional food’ might have the level of the antioxidant
enhanced during manufacture by fortification or some other
The eventual goal of all this is to determine whether
specified foods can be considered as �functional foods’ in
that they may be able to confer health benefit following their
consumption. For the purposes of this discussion, a functional food is considered to be a food which delivers a
physiological benefit, and description of it should convey
unambiguous information that is without deception to the
consumer about physiological or health benefit. In order that
a claim about health benefit can be made, in an ideal world
information should be available, which can be critically
substantiated from the literature, which establishes the
following criteria, each of which, although inter-related
with the others, should be individually satisfied.
Criterion 1. A plausible and validated basic science
rationale for: (a) the involvement of free radicals in those
biochemical and cellular processes which have been shown
to lead to detriment to well-being and health, and to
specified human diseases; and (b) the involvement of dietary
antioxidants in the prevention of these free-radical-involving biochemical processes. This should include results
from studies carried out in vitro, in cells in culture, in in
vitro/ex vivo models, and in animal models.
Criterion 2. The existence of human population epidemiological data which demonstrate a statistically validated inverse relationship between intake (or preferably
serum concentration), of individually specified antioxidants
and the risk of, or mortality from, particular diseases.
Criterion 3. The existence of prospective statistically
validated epidemiological evidence that links intake (or
serum concentration), of identified antioxidants at an early
stage of the disease process, with risk of human disease
which may develop some time after the exposure to the
antioxidants. In such studies both intermediate end-points
that have been clearly shown to predict subsequent disease,
as well as final end-points, may be used.
Criterion 4. The existence of biomarkers for evaluating
free-radical events in human subjects, and the modulating
effect on them of antioxidants, which are of interest in
maintaining well-being as well as the balance between
health and disease. This will include validation of biomarkers, which must preferably be methodologically uncomplicated, by inter-laboratory studies of the same material
which has been shown, when investigated in different
laboratories, to give the same answers, preferably by more
than one method. A further requirement is that the chosen
biomarker must be directly relevant, that is it must have
functional significance; the biomarker must have been
shown by unequivocal techniques to have some significance
to both function and the health maintenance or disease risk
to which it is linked.
Criterion 5. The existence of statistically validated interventional human evidence in large groups of human subjects
(a) which clearly shows that enhancement of intake of
specified antioxidants is associated with improvement in a
valid index of well-being and health, or a lowered risk of
subsequent disease; (b) which demonstrates optimal levels
of intake of antioxidants, derived from indices measured by
chosen biomarkers. This would imply that such biomarkers
have been established which demonstrate a valid relationship between intake of the antioxidant and the index
evaluated by the chosen biomarker(s).
Criterion 6. The existence of clear evidence that the
intervention that is proposed with an antioxidant nutrient
is safe. This will include evidence that a conclusion as to
safety applies with equal force to all groups in the population, including those that are indulging in behaviour which
might be expected to increase the risk of the disease
These are clearly extremely stringent criteria. What is
required in coming to a conclusion as to the health benefit of
antioxidants is to evaluate the �state of the art’ with respect
to them, and to come to a conclusion as to how and to what
extent these criteria are satisfied at present.
7.2. Critical evaluation of the present knowledge base
Each of the foregoing sections 2–6 has examined in depth
one aspect of the science database that exists concerning the
discussion here. The following conclusions are derived from
those given in detail in the appropriate section.
7.2.1. Conclusions from section 2. An imbalance of
ROS and antioxidant defence systems may lead to chemical
modifications of biologically relevant macromolecules. This
imbalance provides a logical pathobiochemical mechanism
for the initiation and development of several disease states.
Experimental data obtained in vitro provide evidence that
antioxidants function in systems that scavenge ROS and that
these are relevant to what occurs in vivo. The relevance in
vivo of these observations depends inter alia on knowledge
of the uptake and distribution of the antioxidant within the
human body, and on what tissue levels of the antioxidant
may be expected in relation to dietary levels. Epidemiological studies show a correlation between consumption of
foods rich in antioxidants and a decreased risk of several
diseases. In particular, diets rich in fruit and vegetables are
associated with low risk of several diseases and antioxidants
are among the responsible constituents of such a diet. Data
on antioxidant supplementation are contradictory and
further research is necessary to establish whether supplementation beyond normal dietary intake levels is of benefit.
7.2.2. Conclusions from section 3. There is some way to
go until validated precise methods are available for
measuring biomarkers of oxidative damage in human
A. T. Diplock et al.
subjects in vivo under minimally invasive conditions. With
respect to oxidative damage to DNA, HPLC and GC–MS
methods have both merits and limitations. Oxidation
artifacts also arise in sample preparation and in derivatization of semi-purified samples. Lipid oxidation products in
plasma are best measured as isoprostanes or as lipid
hydroperoxides using specific HPLC techniques. Development of isoprostane measurement will advance specificity
and precision and will enable measurement of lipid
peroxidation products in urine, which will give a measure
of whole-body lipid peroxidation. Measurement of hydrocarbon exhalation is a technique fraught with artifacts and is
unsuitable for human studies. The measurement of oxidative
damage to proteins has some potential but such methods
have not been effectively exploited. Measurement of
metabolites derived from RNS has potential, but at present
there are many confounding factors which restrict its use.
7.2.3. Conclusions from section 4. Epidemiological
studies support the hypothesis that the major antioxidant
nutrients vitamin E and vitamin C, and b-carotene, which
may or may not be acting as an antioxidant in vivo, may play
a beneficial role in prevention of several chronic disorders.
More research is needed on the impact of other non-nutrient
compounds, such as other carotenoids and flavonoids, on
human health. In general, human intervention studies using
hard end-points are the gold standard. Trials are restricted
mainly to the major antioxidants and do not allow firm
conclusions because of inconsistent findings, an insufficient
number of studies and the use of varying doses. However,
there is evidence that large doses of b-carotene may be
deleterious to the health of certain subgroups of the
population such as heavy habitual smokers. In functional
food research, preventive trials with intermediate end-points
may be of help for testing the efficacy of antioxidants.
Bioavailability studies and dose-finding studies in combination with the development and application of biomarkers are
required for a successful research strategy.
7.2.4. Conclusions from section 5. Vitamin C is safe at
levels of supplementation up to 600 mg/d, and higher levels,
up to 2000 mg/d, are without risk. Vitamin E has a very low
human toxicity and an intake of 1000 mg/d is without risk;
3200 mg/d has been shown to be without any consistent risk.
Large intakes of b-carotene must be viewed with caution
because they have been shown to confer detriment to a
population at high risk of lung cancer when administered
after many years of high-risk (smoking) behaviour. Until
further work clarifies the situation in heavy smokers with
respect to taking supplements, larger doses should be
avoided by such individuals. There is little reliable
information about the human toxicology of flavonoids and
related non-nutrient antioxidant constituents of the diet. A
key question is whether these substances are taken up by
human subjects and distributed to the tissues in quantities
sufficient to confer biological effect.
7.2.5. Conclusions from section 6. The food industry
has long experience in the control of oxidative damage in
foods and this experience can be used to advantage for the
protection of food antioxidants which are beneficial. Some
of these, such as vitamins C and E and b-carotene, are well
known, and strategies for their protection in foods are
already exploited by food technologies. There are, however,
many compounds with antioxidant activity which may, or
equally may not, have biological activity. Since oxidation
during manufacture cannot be eliminated completely,
efforts must be made to identify those antioxidants which
may be important to health and to discover in what form
they are most useful. This requires that more information
must be obtained in the much neglected field of their
bioavailability as well as of their bioactivity. With this
information, food technology strategies for the preservation
of those antioxidants which have been shown to be
beneficial to health can be applied in a cost-effective
7.3. Evaluation of criteria
By using what is revealed in section 7.2, it is now possible to
evaluate the criteria set out in section 7.1.
Criterion 1. There is excellent scientific evidence, derived
from several kinds of investigation, for the involvement of
free-radical events, either as initiating cause of, or at a later
stage in, biochemical and cellular processes that lead to
health detriment. Antioxidants that are consumed in the
human diet are involved in modulating these free-radicalinvolving events and, thus, they promote health. Valid
animal models exist for some forms of cancer and these
have been used to demonstrate an apparent preventive effect
of some antioxidants against the development of cancer,
which gives general support to the contention of health
benefit and cancer prevention by antioxidants. There is no
acceptable animal model for arteriosclerotic cardiovascular
disease. However, there are models which reproduce some
aspects of atherosclerosis and limited studies with these
support the view that vitamin E may have a role with other
factors in delaying the arteriosclerotic process.
Criterion 2. There is human epidemiological evidence for
an inverse association between intake of antioxidant
nutrients, or the fruits and vegetables that contain them,
sometimes supported by measures of serum concentration,
and risk of specified diseases in the population. Although
there are some instances of lack of such correlations being
reported, most studies support this conclusion. The validity
of many studies would have been greatly strengthened by
inclusion of measures of serum concentrations of the antioxidants under study, and the validity of the conclusion
varies in strength for the individual antioxidants under
Criterion 3. There is abundant prospective epidemiological evidence for a clear correlation between dietary
intake of fresh fruit and vegetables and a lowered
subsequent risk of cancer. Some data also show a similar
correlation with vascular disease. This is a widely accepted
view and a claim that increased fruit and vegetable intake is
associated with lowered incidence of some forms of cancer
and atheromatous cardiovascular disease is entirely valid.
Although it is generally thought that this health benefit is
conferred by the antioxidant nutrient and non-nutrient
substances in fruit and vegetables, no conclusive proof of
this contention exists and much confusion has been caused
by statements to this effect; data that show an association
between high serum levels of specified antioxidants and
lowered disease incidence provide no answer to the question
Defence against reactive oxidative species
because the antioxidant may merely be a marker for some
active agent derived from fruit and vegetables, or a lifestyle
difference associated with high fruit and vegetable intake.
The use of �cancer’ or �cardiovascular disease’ as biomarkers of free-radical causation, or antioxidant efficacy,
are also unreliable because both diseases are multifactorial
and develop over a long period of time with the possibility
of multiple inputs into their aetiology, in which antioxidants
may be only one relevant factor. There is some evidence,
particularly with respect to cardiovascular disease, that links
very high intakes of vitamin E, derived from food supplements, with a dramatically lowered incidence of disease.
However, the likely level of intake in this case is beyond
what might be feasibly obtained from the diet, and at normal
dietary levels of intake there appears to be little health
benefit in this respect. Such an effect could, however, be
obtained by the consumption of a functional food fortified to
contain a high level of vitamin E.
Criterion 4. At the present time biomarkers exist for
evaluating oxidative damage to DNA and PUFA; some
biomarkers for oxidative damage to proteins also exist.
However, there are many anomalies with respect to these
biomarkers in that there are both internal inconsistencies in
measurement of the same material by different methods, and
external inconsistencies in that some laboratories report
values that differ widely from those obtained elsewhere
using apparently similar methods. There is a great need for
validation studies in which methodology is tested within the
same laboratory, but also most importantly between different laboratories which must be able to obtain measurements
that are consistent with the results of others using the same
material. The predictive value of the biomarker in evaluating its relevance to the aetiology of a particular disease also
requires validation. Only when this kind of validation has
been done and all experts agree as to methodology will it be
possible to utilize the methods in a new generation of human
studies, which will assess the quantitative relationship
between antioxidant intake and health benefit in human
Criterion 5. The results of intervention studies have been
very mixed and at present the situation is confused. There
are intervention studies in specified populations from which
some conclusions can be drawn. However, it is not valid,
and indeed may be highly dangerous, to extrapolate to other
populations. For example, intervention studies in China
have used end-points which relate to a particularly high
natural incidence locally of a particular cancer; the observed
data are of interest insofar as they refer to a particular cancer
in a specific location. The fact that the incidence of that
cancer elsewhere is very low implies that there are other
factors involved which may prevent widening the conclusions from China to other locations. Second, the trials
involving b-carotene supplementation in smokers should
only be perceived as applying to heavy smokers and must
not be applied to other sections of the population who do not
smoke or who only smoke moderately. A further disadvantage of present interventional evidence is the fact that,
because cost considerations have driven experimental
design, individual antioxidants have often not been examined but instead a cocktail of several has been employed.
There are no valid studies that demonstrate optimal levels of
antioxidant intake in human subjects, and no data exist
concerning dose–response relationships of nutrient intake
and biological efficacy. Thus, this criterion may prove to be
too stringent, although it is important to identify some kind
of �gold standard’ in this respect. In order to give informed
advice as soon as possible about satisfaction of this criterion
it is necessary to shorten the time that is required to conduct
and evaluate human studies because those that rely on final
end-points are very long and extremely costly to carry out.
The development and use of intermediate end-points that
give genuine information about the final end-point must,
therefore, be an important consideration.
Criterion 6. There is excellent evidence that antioxidant
nutrients are safe and can be included in the food of human
subjects up to quite high levels without discernible risk. It is
necessary to cite some specific cautions, for example, in
administering large levels of vitamin E to persons with
blood coagulation disorders, and b-carotene to heavy
smokers; such caveats are, however, rare, specific and
easily identified. There is less certainty with respect to
non-nutrient antioxidants and, if these substances are
shown to have potential functional health benefit, then it
will be necessary to devote attention to their safety.
7.4. Final conclusions
(1) There is evidence that mechanisms that involve free
radicals are implicated at some stage of the development of
human diseases, and that the maintenance of well-being
depends on the supply through the diet of antioxidant
nutrients and b-carotene which modulate free-radical processes in vivo. If it is shown that non-nutrient antioxidants
are biologically available then they too may contribute to
the total antioxidant effects of the diet in vivo, as well as
contributing antioxidant potential during processing,
storage and in the gastrointestinal tract.
(2) Present epidemiological evidence is incomplete but in
general it supports the basic hypothesis that antioxidant
nutrients contribute to well-being and health. There is no
evidence to the contrary which would negate this conclusion. There is minimal similar evidence with respect to the
non-nutrient antioxidants.
(3) The development of biomarkers for use in human
studies is well advanced but biomarkers that are becoming
established need critical evaluation before they can be used
in a new generation of human studies to examine and
evaluate a quantitative cause-and-effect relationship
between antioxidant intake and health benefit.
(4) There are few reliable human intervention studies
which establish cause–effect relationships, and there is no
evidence which shows clearly the optimal amounts of
nutrient and non-nutrient antioxidants that are needed in
the human diet. The identification and development of
intermediate end-points for evaluating the effect of
intervention needs further careful work.
(5) Increasing human dietary intake of antioxidants is safe
and without undesirable side-effects except in rare welldefined instances.
(6) There is a need to ensure that measures adopted by
food technologists in the processing of foods maintain the
antioxidant content of the food, and that the antioxidants are
A. T. Diplock et al.
still active at a suitable level when the food is consumed by
the public. If it can be shown with certainty that a certain
level of antioxidants in the food is associated with health
benefit it may be necessary for food producers to consider
means of enhancing the content or form of the antioxidant
up to or beyond that usually found in the natural food.
8. Recommendations for future research
8.1. Introduction
The foregoing section 7 has compared the available
scientific database with the evidence that is required to
achieve scientific consensus as to what may be needed in
order to make a specific claim about the role of antioxidants
in maintenance of health and well-being. This enables the
identification of areas where further research is required.
Improvement in dietary antioxidant intake in human populations is expected to result in lowering of the risk of a
number of degenerative diseases. While desirable as an
ultimate objective per se, the impact on public health and
the resultant decrease in health-care costs make it imperative that substantial sums of money should be spent on
research in this important area.
8.2. Specific recommendations
Specific recommendations for further research are
warranted in each of the areas covered by the foregoing
sections 2 to 5. With regard to section 6 which deals with the
role of food technology, there is no particular direction that
research needs to take at the present time. Developments in
food technology will be based on, and adapt to, nutritional
recommendations resulting from biologically driven work.
For example, if it is found that the bioactivity of certain
flavonoids is high, then steps will have to be taken to
preserve, or even enhance, the amounts of these compounds
in food products. Similarly, if glycosides of certain
compounds prove to be more bioavailable than the free
antioxidant then similar considerations will apply to the
handling of glycosidic derivatives by food technologists to
maximize their content in the food.
8.2.1. Oxidative damage and antioxidant defence systems
of the human organism. It is axiomatic to the present
considerations that prooxidants cause, or are implicated in
the development of, human disease. Direct measurement of
prooxidants in vivo is difficult or impossible. It is imperative
to establish which are the critical free-radical �hits’ that are
the relevant ones; for cancer, which of the multiple
modifications of DNA caused by oxidative insult are those
that lead to detriment; for atherogenesis, what is the relative
relevance of protein and lipid modifications to LDL which
contribute to the process, and what is their relationship to
the overall multifactorial process of arteriosclerosis? Are
the processes examined really relevant to the disease
causation? For cancer, there is strong evidence that
prooxidants are involved in the initiation stage of the
complex process that leads to disease. But what is the
relevance of the different forms of oxidative damage to the
process? Investigation of this should clarify the role of
different antioxidants in controlling carcinogenesis. We
need simpler and more specific methods to track ROS both
in the initiation and progression of the cancer process.
Studies on free-radical involvement in atherosclerosis need
to focus more clearly on the relevance of oxidative damage
to other factors involved, and on the relative importance of
each to the overall complex biochemical process. Noninvasive techniques need to be developed to study these
phenomena in human subjects in vivo and this applies
particularly to the field of eye diseases which do not readily
lend themselves to direct investigation. We need to identify
which are the important antioxidants in terms of the
maintenance of health, and what is their relationship to
one another. We need to clarify whether it is the antioxidant
role of the substance that is important, or whether it is some
other function, and the possible non-antioxidant effects of
antioxidants, in particular with respect to modulation of
gene expression, also need further research. We need to
know whether there are combined effects of synergy or
antagonism both with respect to the antioxidants themselves
and to other food components. The primary aim is to
identify the active components in the overall system that
promotes health. With respect to single antioxidants there is
a need for more information regarding regeneration of
vitamin E from its radical under cellular conditions, and the
role of b-carotene in apparently exacerbating the cancer
process, when given at a late stage to heavy smokers, needs
8.2.2. Ex vivo methodologies for quantitating and validating damage in vivo to biological macromolecules. Before
meaningful work can proceed on providing evidence of the
level of antioxidants needed to maintain health and well-being,
it is necessary to refine and validate methods that are already
available for measurement of oxidative damage in human
subjects in a non-invasive manner. This will enable real
measurement of variables related to oxidative damage and its
attenuation by antioxidants to be made. With respect to
biomarkers for oxidative damage to DNA, methods available
need to be developed further to prevent oxidation during workup, and formation of artifacts during derivatization of samples,
and careful validation studies in a number of different
laboratories need to be undertaken using identical test material
and methodology. Studies of mitochondrial, as opposed to
nuclear, DNA may prove to be of value because there is some
evidence that suggests that mitochondrial DNA oxidation
correlates with other variables of oxidative damage. Similarly,
for biomarkers of lipid oxidation the relatively new isoprostane
method needs development and comparison with older
established, but less reliable, methods, and this needs to be
examined both in plasma and urine as well as other accessible
biological material. Validation in many centres by measurement of oxidative damage in the same biological material
needs to be undertaken using the same methodology. There is a
somewhat longer-term need for development of techniques to
be used ex vivo as measures of protein oxidation in vivo, which
will, however, probably be able only to reflect the difference at
a steady state between damage and repair. It is possible that the
present early indications of biomarkers of RNS may be capable
of development into useful methodology.
8.2.3. Nutritional options modulating oxidative damage.
For proper epidemiological research, as well as for human
intervention studies, it would be desirable to put special
Defence against reactive oxidative species
emphasis on the following. (1) Chemical analysis of the
antioxidant content of foods so that more realistic food
composition tables can be compiled; ideally this should take
account of agricultural practices, industrial processing and
food preparation. (2) Studies of bioavailability of antioxidants from the diet, and the factors that influence the
absorption, distribution and tissue uptake of the compounds
and the likely impact of the antioxidants on metabolic
processes. This will include studies of the metabolism of
antioxidants and the possible metabolic interactions
between them. (3) Development and validation of biomarkers of intermediate end-points, both biological response
markers and early disease markers, and emphasis on the
relevance of the biomarker to the disease end-point as well
as the disease process. (4) Application of the validated
biomarkers of intermediate end-points in randomized
controlled trials testing the efficacy of antioxidants in
functional foods for the maintenance of health and wellbeing.
8.2.4. Safety implications of nutritional enhancement of
antioxidants. The detailed evidence that is already available which demonstrates that vitamin C and vitamin E are
safe at quite high levels of inclusion in the human diet,
means that it is unnecessary to recommend further work in
this area. The safety of b-carotene was not questioned
before the results of the Finnish and American intervention
studies, which showed an apparent exacerbation in the
incidence of lung cancer in heavy smokers who were given
supplements of b-carotene. This observation needs urgent
clarification, and this should also include work on the safety
of other carotenoids which have been shown to have
biological activity in human subjects. With respect to the
flavonoids and other polyphenols, it is likely that their
bioactivity will be explored, and the key question of their
bioavailability clarified, in the near future. It will be
necessary to examine the safety of such bioflavonoid, and
other similar compounds, which may be shown to have
bioactivity in human subjects, as information becomes
available about their bioactivity.
8.3. Priorities for the recommendations made
The most urgent requirement for further research is the
validation of available biomarkers of oxidative damage.
Much useful work has been done already but several
anomalies remain and new questions have emerged.
Before these biomarkers can be used, in particular as
biomarkers of intermediate end-points, in a new generation
of human studies, which especially need to assess the level
of inclusion of antioxidants in the diet that is optimal for
health and well-being, it is necessary to engage in a
programme of validation of the biomarkers that are available. These validation studies will also necessitate the
inclusion of studies on the analysis of antioxidants and
their metabolites. Three types of validation are essential:
first there is a need for comparison of results obtained in the
same laboratory on identical material using different but
complementary methodology so that any numerical
differences in the results obtained can be eliminated or at
least minimized. Different methods sometimes appear to
give different results for what at first sight should be the
same measurement by different means. Where numerical
differences remain following evaluation, there must be clear
reasons given as to the scientific explanation of the difference; for example, the different methods may be measuring
slightly different aspects of the same oxidative damage.
Second, there is a need for comparison of results obtained in
as many different laboratories as possible of identical
material which is exchanged between participating laboratories, each of which should use as many agreed methods
as possible to assess the degree of oxidative damage in the
samples employed. Third, there is a need for a different kind
of validation, which is that measurements made must be
shown to be clearly linked to those phenomena which give
rise to disease in human subjects. For example, is the DNA
oxidative damage that is measured a real indicator of the
involvement of such damage in mutagenesis and eventual
carcinogenicity? Is the oxidation of LDL a reliable indicator
of atherogenesis and eventual vascular disease? The development of a disease must be the ultimate paradigm by which
the relevance of a biomarker is judged.
As a logical second stage of the work described in the
foregoing paragraph, the validated and accepted biomarkers
will be used in a new generation of human studies. These
need not be as lengthy or time-consuming, and thus not as
expensive, as previous prospective epidemiological intervention studies, because the use of intermediate end-points
should enable answers to key questions to be obtained
considerably more quickly than in earlier studies whose
end-points were disease phenomena occurring many years
after the initiation of the study. These new studies will
provide, for the first time, rigidly controlled evidence of the
benefit to be gained from antioxidants in the human diet, and
will enable quantitation of the optimal levels of intake of
antioxidants. In this connection, care must be taken to
ensure that the importance of the antioxidant contribution
of the whole diet, as distinct from that of each individual
antioxidant, will be evaluated.
The proposals for further research set out in section 8.2
will need to be addressed as and when funds are available,
but this should be done at the same time as the major
programme of research elaborated in the first two paragraphs of this section. The proposed work set out in the first
paragraph could be set in motion with minimal delay and it
is anticipated that preliminary results could be available
within 2 years of its inception, with reliable final results
being available shortly thereafter. The interventional studies
in human subjects that are proposed in the second paragraph
could commence at the end of 3 years and results would
begin to accumulate within 5 years of the initiation of the
Although it is possible to be quite specific with respect to
the objectives and proposed methodology of the research
that is envisaged, and the time-frame within which specified
outcomes may be expected to be achieved, it is beyond the
capabilities of those concerned with this report to attempt to
evaluate the costs that might be involved. Research costs are
a highly volatile quantity and there is considerable variation
in cost that might be expected even within different institutions of a single Member State of the European Union. Cost
comparisons between different Member States are even
more difficult because labour costs, and cost of delivery of
A. T. Diplock et al.
research, differ very widely. It would be invidious to
attempt to identify locations where the research might be
carried out more cheaply than in another, and it is the
considered view of this group that money identified by the
European Union for supporting this research should be
made available on the usual competitive basis, with invitation of specific proposals from researchers concerned with
their own specific areas of expertise. These can then be
assessed both with respect to their scientific merit, as well as
their cost, and appropriate awards of money made on this
basis, which follows accepted practice in all Member States
of the Union.
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# Nutrition Society 1998
British Journal of Nutrition (1998), 80, Suppl. 1, S113–S146
Functional food science and the cardiovascular system
G. Hornstra1*, C. A. Barth2, C. Galli3, R. P. Mensink4, M. Mutanen5, R. A. Riemersma6, M. Roberfroid7,
K. Salminen8, G. Vansant9 and P. M. Verschuren10
Department of Human Biology, Maastricht University, PO Box 616, NL-6200 MD, Maastricht, The Netherlands
ВЁ ffentlichen Rechts, Arthur-Scheunert-Allee 114-116,
German Institute for Human Nutrition, Stiftung des O
D-14558 Bergholz-RehbruВЁcke, Germany
Institute of Pharmacological Sciences, University of Milano, Via Balzaretti 9, I-20133 Milan, Italy
Nutrition Research Centre, Department of Human Biology, Maastricht University, PO Box 616, NL-6200 MD, Maastricht,
The Netherlands
Department of Applied Chemistry and Microbiology, University of Helsinki, PO Box 27, SF-00014 Helsinki, Finland
Cardiovascular Research Unit, Hugh Robinson Building, University of Edinburgh, George Square, Edinburgh EW8 9XF, UK
UCL, Ecole de Pharmacie, Tour Van Helmont, Avenue E. Mounier, B-1200 Brussels, Belgium
Research and Development, Valio Ltd, PB 390, SF-00101 Helsinki, Finland
Laboratory voor Experimentele geneeskunde endocrinologie (LEGENDO), Katholieke Universiteit Leuven, Gasthuisberg,
B-3000 Leuven, Belgium
Unilever Research Laboratory, Olivier van Noortlaan 120, NL-3133 AT Vlaardingen, The Netherlands
1. Some aspects of coronary heart disease (CHD)
1.1. Lipoprotein metabolism
1.2. Arterial thrombosis
1.3. Immunological interactions
1.4. Hypertension
1.5. Insulin resistance
1.6. Hyperhomocysteinaemia
2. Dietary components and serum lipoproteins
2.1. Effects of dietary components on fasting
lipid and lipoprotein concentrations
2.1.1. Fatty acids
2.1.2. Fat replacers
2.1.3. Soyabean protein preparations
2.1.4. Mono- and disaccharides
2.1.5. Resistant starch
2.1.6. Ethanol
2.1.7. Dietary cholesterol
2.1.8. Fibre
2.1.9. Phytosterols
2.1.10. Tocopherols and tocotrienols
2.1.11. Garlic
2.1.12. Other components
2.2. Postprandial effects
2.3. Gene–diet interaction
2.4. Possible mechanisms of dietary fats
2.4.1. Concept of Spady and colleagues
2.4.2. Concept of Hayes and colleagues
3. Some diet effects on arterial thrombotic processes:
platelet and endothelial cell functions, blood
coagulation and fibrinolysis
Arterial thrombosis and cardiovascular
3.2. Platelet function as a marker for CHD
3.3. Some diet effects on arterial thrombogenesis and
platelet function
3.3.1. Fatty acids
3.3.2. Antioxidants and platelet function
3.4. Endothelial cell function
3.4.1. Dietary fatty acids and endothelial cell
3.5. Coagulation and fibrinolysis
3.5.1. Effect of dietary factors on coagulation
and fibrinolysis
4. Immune-mediated processes underlying CHD
4.1. Immunocompetent cells involved in the
atherosclerotic lesion
4.1.1. Endothelial cells
4.1.2. Smooth-muscle cells
4.1.3. Immunocompetent leucocytes
4.1.4. Mechanisms for the recruitment of blood
cells in arterial lesions
4.2. The immune system response modulates
atherosclerosis progression
4.3. Nutrition and the immunological aspects of
4.3.1. Effects of n-3 fatty acids on cellular
immune response and inflammatory events
in atherogenesis
4.3.2. Antioxidants
5. Diet, hypertension and heart function
5.1. Aetiology of hypertension
Abbreviations: ALA, a-linolenic acid; apo, apolipoprotein; AT-III, antithrombin-III; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FVIIc, factor
VII coagulant activity; ICAM, intracellular adhesion molecule; Lp(a), lipoprotein(a); MI, myocardial infarction; MTHFR, 5,10-methylenetetrahydrofolate
reductase; NIDDM, non-insulin-dependent diabetes mellitus; oxLDL, oxidized LDL; PAI-1, plasminogen activator inhibitor-1; PG, prostaglandin; P : S,
polyunsaturated : saturated fatty acid ratio; SMC, smooth-muscle cells; TGF-b, transforming growth factor-b; t-PA, plasminogen activator; Tx,
thromboxane; VCAM, vascular cell adhesion molecule; vWf, von-Willebrand factor.
*Corresponding author: Dr G. Hornstra, fax +31 43 367 0976, email G. [email protected]
G. Hornstra et al.
5.1.1. Membrane function
5.1.2. Role of humoral mediators
5.1.3. Insulin resistance
5.1.4. Environmental factors
5.2. Strategies to reduce CHD by lowering blood
5.2.1. Intervention trials
5.2.2. Individual v. population approach
5.3. Dietary fatty acid composition and hypertension
5.4. Heart function
5.4.1. Effect of diet
5.5. Function of the ischaemic heart
5.5.1. Dietary fatty acids and arrhythmia
6. Insulin resistance, obesity and non-insulin-dependent
diabetes mellitus
6.1. Insulin resistance, cardiovascular disease and
cardiovascular risk factors
6.1.1. Lipid abnormalities and insulin resistance
6.1.2. Hypertension and insulin resistance
6.2. Nutritional aspects
7. Hyperhomocysteinaemia and cardiovascular risk
7.1. Causes of hyperhomocysteinaemia
7.2. Athero-thrombotic mechanisms of
7.2.1. Interaction with lipoproteins
7.2.2. Smooth muscle cell proliferation
7.2.3. Endothelial functions
7.2.4. Function of blood platelets
7.2.5. Coagulation and natural anticoagulants
7.2.6. Fibrinolysis
7.2.7. Altered gene expression
7.3. Hyperhomocysteinaemia or reduced B-vitamin
status of primary importance in cardiovascular
7.4. Dietary B-vitamins lower plasma homocysteine
8. Critical assessment of the science base
8.1. Identification of criteria
8.2. Evaluation of the present knowledge base with
respect to food functionality
8.2.1. Plasma lipoproteins
8.2.2. Arterial thrombosis
8.2.3. Immunological interactions
8.2.4. Hypertension
8.2.5. Insulin resistance
8.2.6. Hyperhomocysteinaemia
9. Conclusions and recommendations for further
9.1. Plasma lipoproteins
9.2. Arterial thrombosis
9.3. Immunological interactions
9.4. Hypertension
9.5. Insulin resistance
9.6. Hyperhomocysteinaemia
Cardiovascular disease has a multifactorial aetiology, as is illustrated by the existence of
numerous risk indicators, many of which can be influenced by dietary means. It should be
recalled, however, that only after a cause-and-effect relationship has been established between
the disease and a given risk indicator (called a risk factor in that case), can modifying this factor
be expected to affect disease morbidity and mortality. In this paper, effects of diet on
cardiovascular risk are reviewed, with special emphasis on modification of the plasma lipoprotein
profile and of hypertension. In addition, dietary influences on arterial thrombotic processes,
immunological interactions, insulin resistance and hyperhomocysteinaemia are discussed. Dietary lipids are able to affect lipoprotein metabolism in a significant way, thereby modifying the
risk of cardiovascular disease. However, more research is required concerning the possible
interactions between the various dietary fatty acids, and between fatty acids and dietary
cholesterol. In addition, more studies are needed with respect to the possible importance of the
postprandial state. Although in the aetiology of hypertension the genetic component is definitely
stronger than environmental factors, some benefit in terms of the development and coronary
complications of atherosclerosis in hypertensive patients can be expected from fatty acids such as
a-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. This particularly holds for
those subjects where the hypertensive mechanism involves the formation of thromboxane A2
and/or a1-adrenergic activities. However, large-scale trials are required to test this contention.
Certain aspects of blood platelet function, blood coagulability, and fibrinolytic activity are
associated with cardiovascular risk, but causality has been insufficiently proven. Nonetheless,
well-designed intervention studies should be initiated to further evaluate such promising dietary
components as the various n-3 and n-6 fatty acids and their combination, antioxidants, fibre, etc.
for their effect on processes participating in arterial thrombus formation. Long-chain polyenes of
the n-3 family and antioxidants can modify the activity of immunocompetent cells, but we are at
an early stage of examining the role of immune function on the development of atherosclerotic
plaques. Actually, there is little, if any, evidence that dietary modulation of immune system
responses of cells participating in atherogenesis exerts beneficial effects. Although it seems
feasible to modulate insulin sensitivity and subsequent cardiovascular risk factors by decreasing
the total amount of dietary fat and increasing the proportion of polyunsaturated fatty acids,
additional studies on the efficacy of specific fatty acids, dietary fibre, and low-energy diets, as
well as on the mechanisms involved are required to understand the real function of these dietary
components. Finally, dietary supplements containing folate and vitamins B6 and/or B12 should be
tested for their potential to reduce cardiovascular risk by lowering the plasma level of
Cardiovascular system
1. Some aspects of coronary heart disease (CHD)
Major health risks with respect to the cardiovascular system
are CHD and hypertension. In addition, cardiovascular
complications of diabetes mellitus are important in this
respect. The main aetiological processes involved comprise
disturbances in lipoprotein metabolism, a prothrombotic
shift in the arterial thrombogenic balance, derangements
of the immunological system, insulin resistance and hyperhomocysteinaemia. In the present paper, the potential
effects of nutritive and non-nutritive food components on
these processes will be reviewed. Epidemiological studies
have provided important information on the factors
involved in the aetiology of CHD, which has been used as
a basis for preventive strategies. Nethertheless, only
approximately 50 % of the incidence of cardiovascular
disease can be explained by the major risk factors, leaving
space for substantial, largely unexplored, contributing
mostly present in an inactive form and become activated
as a result of vascular injury. Platelets undergoing a series of
biochemical and morphological changes express proteins
and cell receptors, adhere and form aggregates, and bind to
neutrophils and monocytes. Similarly, endothelial cells
express intercellular adhesion molecules after stimulation.
1.3. Immunological interactions
The atherosclerotic lesion is associated with multiple interactions between immuno-competent cells in the blood
(monocytes, T-lymphocytes and platelets) together with
the two major cell types in the artery wall, endothelial
cells and smooth-muscle cells (SMC). Thus, circulating
blood monocytes and T-lymphocytes interact with
�damaged’ endothelium, enter the subendothelial space of
the artery wall and release bioactive molecules. SMC
can produce and secrete proteoglycans and facilitate the
formation of connective tissue and, thereby, contribute to
the formation of advanced atherosclerotic lesions which are
also promoted by a thrombotic process (McGill, 1984).
1.1. Lipoprotein metabolism
The pathological changes in the coronary arteries that lead
to the development of atherosclerotic plaques are now better
understood. One of the earliest changes may be endothelial
dysfunction (Healy, 1990) followed by the development of
fatty streaks due to the formation and accumulation of
oxidized lipoprotein particles in the subendothelial space
(Steinberg et al. 1989). A critical role for antioxidant
vitamins, such as a-tocopherol, ascorbic acid and (perhaps)
b-carotene in the prevention of endothelial dysfunction and/
or LDL oxidation has been hypothesized (Gey et al. 1993).
Many studies have found an association between serum
lipoprotein concentrations and the risk of CHD. Associations, however, do not necessarily reflect a causal relationship; such a relationship can only be established by wellcontrolled intervention studies. Formally, causality has only
been proven for the positive relationship between LDLcholesterol levels and the risk of CHD (Frick et al. 1987).
However, strong evidence also exists that a high concentration of HDL-cholesterol or a low LDL- (or total) : HDLcholesterol ratio protects against CHD (Shaten et al. 1991;
Castelli et al. 1992). Further, raised fasting triacylglycerol
(Hokanson & Austin, 1996) and lipoprotein(a) (Lp(a))
concentrations (Bostom et al. 1996), as well as the presence
of small LDL particles (Austin, 1992) and postprandial
lipidaemia (Karpe et al. 1994) may be positively associated
with an increased CHD risk.
1.2. Arterial thrombosis
Arterial thrombosis starts within seconds after vascular
damage and involves the participation of blood platelets
and leucocytes, and of coagulation and fibrinolysis. The
process results in the formation of mural, embolizing and,
ultimately, occlusive thrombi, thereby promoting the progress of atherosclerotic disease, tissue and organ infarction
and sudden death (Fuster et al. 1990). Under normal
physiological conditions, the cellular components and proteins involved (e.g. proenzymes and procofactors) are
1.4. Hypertension
CHD is strongly related to both systolic and diastolic blood
pressure in a graded fashion as demonstrated in an analysis
of nine large prospective studies (MacMahon et al. 1990).
There are multiple causes for primary hypertension with a
strong genetic component. Treatment of hypertension and
isolated hypertension (without an increased diastolic blood
pressure) results in a reduction in coronary disease-related
events (Collins et al. 1990).
Increased blood pressure per se appears to increase
atherosclerosis, presumably by promoting the entry of
LDL into the subendothelial space (Curmi et al. 1990).
Haemodynamic factors appear to play a role and it is a wellknown fact that certain arteries and sites near vessel bifurcations have a predilection to develop atherosclerosis.
1.5. Insulin resistance
Although the phenomenon of insulin resistance has been
known for a long time (Himsworth, 1936), the link with
atherogenesis was first hypothesized by Stout & VallanceOwen (1969). This theory was revived by Reaven (1988)
when he proposed the existence of a syndrome characterized
by obesity, hypertension, dyslipidaemia and glucose intolerance, in which insulin resistance was the common link
(metabolic syndrome X). Since then, several excellent
reviews regarding the link between insulin resistance,
metabolic abnormalities and diseases have been published
(Ferrannini et al. 1991; Elliott & Viberti, 1993; Laws &
Reaven, 1993; Despre`s & Marette, 1994).
1.6. Hyperhomocysteinaemia
Compelling evidence is now available suggesting that
homocyst(e)ine is implicated in cardiovascular disease.
This view is based on a large number of epidemiological
studies, recently summarized by Boushey et al. (1995) and
by Malinow (1996) and supporting the hypothesis that the
G. Hornstra et al.
plasma homocysteine concentration is an independent
graded risk indicator for arteriosclerotic vascular diseases
(coronary, cerebral, and peripheral arterial occlusive diseases, as well as carotid thickening). From the meta-analysis
by Boushey et al. (1995) a total of 10 % of the population’s
coronary artery disease risk was suggested to be attributable
to homocysteine. Recent studies by Tonstad et al. (1996)
demonstrate that a modest elevation in plasma homocysteine level in children is related to premature cardiovascular
death in their male relatives and may partly account for the
contribution of family history to risk of cardiovascular
2. Dietary components and serum lipoproteins
The association between serum lipoprotein concentrations
and the risk of CHD is generally acknowledged. As diet
plays an important role in the modulation of lipoprotein
metabolism, the purpose of this section is to briefly summarize effects of various dietary components on lipoprotein
metabolism. Attention will also be given to the role of the
genetic background of individuals in modulating these
dietary effects.
2.1. Effects of dietary components on fasting lipid and
lipoprotein concentrations
2.1.1. Fatty acids. For the purpose of this discussion, the
fatty acids are categorized into four classes: saturated fatty
acids, monounsaturated fatty acids (mainly oleic acid,
18 : 1n-9), polyunsaturated fatty acids (mainly linoleic
acid, 18 : 2n-6), and trans fatty acids (mainly 18 : 1trans).
In a meta-analysis of twenty-seven well-controlled dietary studies (Mensink & Katan, 1992), it was found that,
relative to an isoenergetic amount of carbohydrates, a
mixture of saturated fatty acids increases LDL-cholesterol
concentrations. Polyunsaturated fatty acids, however, lower
LDL-cholesterol, but to a lesser extent, as estimated by
Keys et al. (1965b). The effect of oleic acid was between
that of carbohydrates and polyunsaturated fatty acids.
Further, it was demonstrated that all fatty acids increase
HDL-cholesterol, but this effect appeared to diminish
with increasing unsaturation of the fatty acid. Therefore,
it was concluded that under isoenergetic metabolic-ward
conditions, the most favourable lipoprotein risk profile
for CHD was achieved if saturated fatty acids were replaced
by unsaturated fatty acids. However, no distinction was
made between the effects of the individual saturated fatty
As already indicated by the studies of Keys et al. (1965b),
the cholesterolaemic effects of the various saturated fatty
acids are not equal. It was therefore suggested that saturated
fatty acids should be divided into those with less than twelve
C atoms, those with twelve to sixteen C atoms (lauric acid,
12 : 0, myristic acid, 14 : 0, and palmitic acid, 16 : 0) and
those with eighteen C atoms (stearic acid, 18 : 0). For
statistical reasons, the data from the meta-analysis do not
allow estimation of the impact of all the various saturated
fatty acids, but it is possible to calculate the separate effects
of palmitic and stearic acids, the two most abundant
saturated fatty acids in the diet. In agreement with the
findings of others, these analyses clearly show that, in
contrast with other saturated fatty acids, stearic acid affects
neither serum LDL- nor HDL-cholesterol levels (Fig. 1).
Other studies suggest that lauric and myristic acids are more
cholesterolaemic than palmitic acid, due to increases in both
LDL- and HDL-cholesterol (Zock et al. 1994; Temme et al.
1996). In a recent study, it was demonstrated that a mixture
of caprylic acid (8 : 0) and capric acid (10 : 0), two mediumchain fatty acids (which are fatty acids with a chain length
between four and ten C atoms) slightly increased LDLcholesterol relative to oleic acid, and had no effect on HDLcholesterol (Cater et al. 1997). Taking these studies
together, it appears that, despite different effects on HDLand LDL-cholesterol, all saturated fatty acids, including
stearic acid, increase the LDL : HDL ratio to a comparable
Fig. 1. Relative effects of palmitic acid (f), stearic acid (,), cis-monounsaturated fatty acids (*) and cis-polyunsaturated fatty acids (j) on fasting
serum lipid and lipoprotein concentrations in human subjects. (From Mensink & Katan, 1992.)
Cardiovascular system
Results from various studies have shown that trans
monoenoic acids increase LDL- and decrease HDLcholesterol relative to oleic acid (Katan et al. 1995; Aro
et al. 1997). The effect of trans polyunsaturated fatty acids,
which can be formed on treatments as mild as deodourization
of vegetable oils, has not been properly examined as yet.
A few recent studies do suggest that in normolipidaemic
subjects palmitic acid is not always an LDL-cholesterolraising saturated fatty acid (Ng et al. 1992; Choudhury et al.
1995). This finding, of course, would be of great practical
significance if it proved to be correct that under certain
conditions palmitic acid can replace oleic acid without
affecting LDL-cholesterol levels. Therefore, these results
need to be confirmed under various experimental conditions, before any solid conclusions can be drawn.
Although linoleic acid is the most abundant polyunsaturated fatty acid in the diet, a small part of the dietary
polyunsaturates is provided by a-linolenic acid (ALA;
18 : 3n-3) and by the very-long-chain fatty acids eicosapentaenoic acid (EPA; 20 : 5n-3) and docosahexaenoic acid
(DHA; 22 : 6n-3) from fish oils. Effects on plasma lipoproteins
seem comparable between ALA and linoleic acid (Chan et al.
1991). Fish oils, however, have a hypotriacylglycerolaemic
effect and have, in normolipidaemic subjects, no effects on
LDL and HDL levels. In hyperlipidaemic subjects, fish oils
also lower triacylglycerols, but they can increase LDL- and
HDL-cholesterol concentrations (Harris, 1997). Controversy
still exists with respect to the question of whether EPA is the
major triacylglycerol-lowering component of fish oil
(Rambjor et al. 1996; FrГёyland et al. 1997), or whether
Лљ gren et al. 1996; Davidson
DHA has the same property (A
et al. 1997; Grimsgaard et al. 1997).
The structure of dietary triacylglycerols may also affect
serum lipid levels. Each natural triacylglycerol has a unique
distribution of the three fatty acids over the glycerol
molecule. However, the fatty acid configuration of dietary
triacylglycerols is sometimes modified to produce fats that
have features desired by food manufacturers and consumers.
As human lipases preferentially remove fatty acids from the
1 and 3 positions of triacylglycerols, it is possible that
changing the positional distribution of fatty acids could
have an effect on serum lipoprotein concentrations. However, results from different studies (Grande et al. 1970;
Nestel et al. 1995; Zock et al. 1995) have demonstrated that
the position of dietary stearic acid or palmitic acid on the
glycerol molecule is not an important determinant of the
fasting serum lipoprotein profile. Nevertheless, animal studies carried out by Kritchevsky et al. (1973) suggested that
randomization of peanut oil may prevent the promoting
effect of peanut oil on cholesterol-induced atherosclerosis.
These effects could not be explained by differences in
absorption or transport of dietary cholesterol (Tso et al.
1984). Randomized butter or lard, however, did not protect
against atherosclerosis (Kritchevsky & Tepper, 1977)
and the interpretation and relevance of these studies for
the human situation are not clear and need further
2.1.2. Fat replacers. Cholesterol absorption is decreased
when human subjects consume diets containing nonabsorbable fat replacers (Jandacek et al. 1990) and therefore these compounds do lower serum LDL-cholesterol
concentrations (Mellies et al. 1983). When fat intake is
decreased due to replacement by these fat substitutes,
HDL-cholesterol concentrations may also be lowered
(Widhalm et al. 1994).
2.1.3. Soyabean protein preparations. Anderson et al.
(1995) have recently published the results of a meta-analysis
concerning the effects of soyabean protein on serum lipid
concentrations in human subjects. It was estimated that a
daily intake of 47 g soyabean protein would lower serum
total cholesterol concentrations by 0.60 mmol/l, which was
mainly explained by a decrease of 0.56 mmol/l in LDLcholesterol. The estimated reduction in serum total
cholesterol concentrations in subjects with total cholesterol
below 6.5 mmol/l was about 4 % (0.20 mmol/l) and about
20 % (1.85 mmol/l) in subjects with cholesterol levels above
8.7 mmol/l. Triacylglycerol levels decreased by 0.15 mmol/l,
while no significant changes were seen in HDL-cholesterol
levels. No difference in effect could be demonstrated between
isolated soyabean protein and/or textured soyabean protein.
However, only the individual results from seven out of thirtyone studies reached statistical significance. In those studies, all
carried out in Italy by four different groups, subjects were
hyperlipidaemic, and textured soyabean protein was used as
the source of soyabean protein. Thus, it cannot be excluded
that some special, unknown, characteristic of the textured
soyabean protein explains the findings, and results cannot be
extrapolated to all types of subjects per se. Further, it remains
to be determined whether possible beneficial effects of
soyabean protein are due to soyabean protein per se or to,
for example, phyto-oestrogens, as suggested by the authors.
2.1.4. Mono- and disaccharides. Blaak & Saris
(1995) have published a comprehensive review on health
aspects of various digestible carbohydrates. It was concluded that, in the majority of studies with normolipidaemic, hypertriacylglycerolaemic or diabetic subjects, monoand disaccharides had similar effects on the serum
lipoprotein profile to those of starch, when consumed in
amounts found in Western diets.
2.1.5. Resistant starch. Resistant starch is not, at least
not entirely, degraded in the small intestine, and reaches the
large intestine. Here, it is metabolized by the action of certain
bacteria. Although it has been suggested that the metabolic
products favourably affect cholesterol metabolism, neither
raw nor retrograded starches appear to have a beneficial effect
on the serum lipoprotein profile (Heijnen et al. 1996).
2.1.6. Ethanol. A moderate alcohol consumption is
negatively related to the risk of CHD. This association,
which may be partly explained by the ability of alcohol to
increase HDL-cholesterol (Choudhury et al. 1994), appears
not to be due to a specific alcoholic drink in particular, but
rather to alcohol per se (Rimm et al. 1996).
2.1.7. Dietary cholesterol. Keys et al. (1965a) suggested that the serum total cholesterol concentration is a
function of the square root of cholesterol intake. A nonlinear relationship between dietary cholesterol intake and
serum total cholesterol concentrations was also proposed by
Hopkins (1992), but Hegsted et al. (1965) suggested that
serum total cholesterol concentrations are linearly related to
the absolute dietary cholesterol intake. Whatever the exact
relationship is between dietary and serum cholesterol
concentrations, lowering dietary cholesterol intake will
G. Hornstra et al.
lower serum total cholesterol concentrations, although this
effect may diminish when saturated-fat intake is low
(Bronsgeest-Schoute et al. 1979). About 75–85 % of this
effect is due to an increase in LDL- and about 15–25 % to an
increase in HDL-cholesterol (Katan et al. 1986; Clarke et al.
2.1.8. Fibre. Based on a meta-analysis of ten trials,
Ripsin et al. (1992) concluded that the daily consumption of
approximately 3 g soluble fibre from oat products lowers
serum total cholesterol concentrations by about 0.15 mmol/l.
This effect was positively related to the initial serum
cholesterol concentration. Other water-soluble fibres have
also been reported to reduce total cholesterol concentrations,
mainly by lowering LDL-cholesterol (Stasse-Wolthuis et al.
1980). Insoluble fibres have a lesser impact on serum total
cholesterol levels (Glore et al. 1994).
2.1.9. Phytosterols. The estimated daily intake of
phytosterols in Western countries is about 160–360 mg, of
which campesterol, sitosterol and stigmasterol are the most
common. These compounds are structurally related to
cholesterol, lower cholesterol absorption, and have long
been recognized as LDL-cholesterol-lowering agents (Miettinen et al. 1995). Saturated phytosterols are more efficient
in reducing serum LDL-cholesterol concentration than
unsaturated phytosterols (Ling & Jones, 1995). Esterification of sitostanols, the saturated equivalent of sitosterols, to
rapeseed oil fatty acids further increases the LDLcholesterol-lowering efficacy of phytosterols. A daily
intake of about 2.0–2.5 g esterified sitostanol lowers
serum LDL-cholesterol concentrations by about 10 % in
hypercholesterolaemic subjects (Miettinen et al. 1995).
2.1.10. Tocopherols and tocotrienols. Tocopherols and
tocotrienols are components with vitamin E activity. Tocopherols are present in most vegetable oils and are more
common in the diet than the tocotrienols, which are found at
relatively high concentrations in palm and rice-bran oils.
Tocopherols do not have any effect on serum lipoprotein
concentrations (Kesaniemi & Grundy, 1982). Some studies
have suggested that tocotrienols lower LDL-cholesterol
concentrations (Qureshi et al. 1995), but other studies
have not found any improvement of the serum lipoprotein
profile after tocotrienol supplementation (Wahlqvist et al.
1992; RP Mensink, AC van Houwelingen, D Kromhout
and G Hornstra, unpublished results). To explain these
differences in findings, it was postulated that effective
tocotrienol preparations should contain less than 150–
200 mg a-tocopherol/g and 450 mg g- plus d-tocotrienol/g
(Qureshi et al. 1996). This suggestion, however, awaits
further confirmation. Also, the very recently reported
potent LDL-cholesterol-lowering effect of a novel
tocotrienol-enriched fraction (TRF25) from rice bran
deserves attention in future studies (Qureshi et al. 1997).
2.1.11. Garlic. Two recent meta-analyses found that
garlic preparations, in amounts approximately equivalent to
half to one clove per day, decreased serum total and LDLcholesterol levels by about 10 % in subjects with elevated
plasma cholesterol concentrations (Warshafsky et al. 1993;
Silagy & Neil, 1994). It was, however, noticed that many of
the studies used had methodological shortcomings, which
were accounted for in two very recent studies (Simons et al.
1995; Adler & Holub, 1997). However, results of these two
studies were conflicting, despite the fact that the same garlic
powder was used and in the same amount. Thus, although
evidence exists that garlic may lower LDL-cholesterol
concentrations, some questions still remain to be resolved.
In addition, it should be emphasized that the cholesterollowering effect may be confined to certain fractions of garlic
only. This emphasizes that each substance or preparation
should be evaluated properly in well-controlled studies at
more than one location, before any firm conclusions can be
2.1.12. Other components. Despite promising results in
the past, very recent well-controlled studies have demonstrated that fermented milk products (Richelsen et al. 1996)
and inulin (Pedersen et al. 1997) are not very likely to have
beneficial effects on the fasting serum lipoprotein profile.
Also, the claimed lipid-lowering effects of oligofructose in
man have not yet been properly demonstrated.
2.2. Postprandial effects
So far, only the effects of dietary components on fasting
lipid and lipoprotein concentrations have been discussed.
However, lipoprotein remnant particles, which circulate in
the blood after a meal, are also atherogenic.
As chylomicrons, precursors of the remnant particles,
mainly transport dietary triacylglycerols (and cholesterol), it
is not surprising that postprandial triacylglycerol concentrations are more pronounced on high-fat diets, even if fasting
triacylglycerol levels are lower. The fatty acid composition
of the habitual diet might also be an important determinant
of the postprandial triacylglycerol response, as this response
appears to decrease when the diet contains highly unsaturated fatty acids from fish oils (Harris, 1997). Hayford et al.
(1979) have also reported that sucrose-containing diets
induced a higher triacylglycerol response than diets containing maize-syrup. Finally, components that interfere with
dietary cholesterol absorption may affect the composition of
the chylomicron and its remnant particles. However, the
extent and importance of these effects are difficult to
quantify and the postprandial effects of diets is certainly
an area that should be investigated more thoroughly in the
very near future.
2.3. Gene–diet interaction
Apolipoprotein (apo) E, an apolipoprotein associated with
chylomicrons, VLDL, HDL and remnant particles, is a
ligand for both the remnant receptor and the LDL-receptor.
There are three common alleles in the population, which are,
in decreasing frequency: E3, E4, and E2. As apoE2 has a
lower affinity for the remnant receptor than the other two
isoforms, subjects with the apoE2 isoform exhibit a delayed
clearance of chylomicrons and chylomicron remnant particles after a fat load. Subjects with the apoE4 isoform,
however, appear to be more responsive to a reduced
cholesterol and saturated fat intake than other subjects
(Ordovas et al. 1995), which might partly be explained
by the higher fractional intestinal cholesterol absorption
in apoE4-subjects (Miettinen, 1991). This would also
explain why apoE4-carriers benefit more from sitostanol
ester intake than non-apoE4 carriers (Vanhanen et al.
Cardiovascular system
1993). Interestingly, Dreon et al. (1995) have also reported
that reducing fat intake caused a shift from large to smaller
LDL particles, which was most pronounced in apoE4-subjects.
Although less extensively studied, it has also been suggested that common polymorphisms for apoA-I (LopezMiranda et al. 1994) and apoA-IV (Mata et al. 1994) may
explain a part of the inter-individual response when dietary
fat and/or cholesterol intake is modified. Associations
between certain polymorphisms of apoB, apoC-III and
lipoprotein lipase (EC and lipid responses to
dietary changes have also been reported, but are not very
consistent (Ordovas et al. 1995). More research is needed
to further assess the genetic impact on diet–lipoprotein
2.4. Possible mechanisms of dietary fats
Theoretically, diet may modify cholesterol metabolism in
several ways at different levels: (1) cholesterol and/or fat
absorption; (2) faecal sterol excretion; (3) cholesterol and/or
apolipoprotein synthesis and excretion; (4) receptordependent and -independent lipoprotein uptake; (5) lipoprotein composition and catabolism; (6) changes in enzymes
and/or proteins, like lipoprotein lipase, cholesterol-ester
transfer protein, and lecithin-cholesterol acyl transferase.
It should be realized, however, that these mechanisms do
not operate in isolation. For example, it can be imagined that
endogenous cholesterol synthesis is increased in order to
compensate for a decreased cholesterol absorption.
To date, most of the studies have focused on the effects of
dietary fatty acids on LDL metabolism and two competing
theories will be summarized briefly.
2.4.1. Concept of Spady and colleagues. A detailed
model to delineate the effects of dietary fatty acids on
lipoprotein metabolism in the hamster has been described by
Spady et al. (1993). According to their hypothesis, dietary
fatty acids change LDL-receptor activity, but not the
production of apoB-100 by the liver or whole-body
cholesterol synthesis. Thus, if the activity of the LDLreceptor is reduced, LDL-cholesterol levels are also
increased because of increased conversion of intermediate
density lipoproteins to LDL. Although the results from this
hamster model are very consistent, it is not known whether
this concept can be extrapolated to man.
2.4.2. Concept of Hayes and colleagues. Hayes and
Khosla (1992) and Hayes et al. (1992) have postulated that
the cholesterol-raising saturated fatty acids increase the
production of apoB-100 by the liver and have no effect on
the activity of the hepatic LDL-receptor. This will result in
an increased VLDL-output, the effect being the strongest for
lauric and myristic acids; palmitic acid has a smaller effect.
As the activity of the LDL-receptor is not increased, LDLcholesterol levels must rise because of increased conversion
of VLDL into intermediate-density lipoproteins and subsequently LDL. Linoleic acid lowers the LDL-cholesterol
concentration, because, according to Hayes’ hypothesis, it
up-regulates the LDL-receptor. This effect of linoleic acid is
maximal already at an intake of 6–7 % of daily energy. It is
now postulated that, at adequate intakes of linoleic acid, the
up-regulated LDL-receptor can counterbalance the relatively small effect of palmitic acid on VLDL production.
Under these conditions, palmitic acid and oleic acid have
similar effects on LDL-cholesterol concentrations. However, in situations that down-regulate the LDL receptors (for
example, dietary cholesterol intake above 300 mg/d or total
serum cholesterol above 6.5 mmol/l), linoleic acid cannot
fully neutralize the effect of palmitic acid on apoB-100
3. Some diet effects on arterial thrombotic processes:
platelet and endothelial cell functions, blood coagulation
and fibrinolysis
3.1. Arterial thrombosis and cardiovascular disease
Evidence that arterial thrombosis contributes to genesis and
complications of cardiovascular disease is mainly based on
pathological and epidemiological studies, showing significant associations between the various thrombotic processes,
or the levels of factors involved in these processes, and
disease morbidity or risks (Fuster et al. 1992; Davies, 1997).
Evidence that modulation of these processes or factors
affects disease risk seems strong for platelet function
(Antiplatelet Trialist’s Collaboration, 1994), is reasonable
for coagulation (Chalmers et al. 1977; Loeliger, 1984;
Smith et al. 1990), and substantial for fibrinolysis (Fibrinolytic Trialists’ Collaborative Group, 1994), but is lacking for
endothelial function.
The protective effect of aspirin on cardiovascular morbidity and mortality has long been considered the major
evidence for a causal relationship between arterial thrombosis (and platelet function in particular) and cardiovascular
disease (Steering Committee of the Physicians’ Health
Study Research Group, 1989), but recent evidence indicates
that the beneficial effect of aspirin may be secondary to its
anti-inflammatory properties (Ridker et al. 1997). So, for
the time being, thrombotic processes and factors should be
considered risk markers for cardiovascular disease, not risk
3.2. Platelet function as a marker for CHD
Although platelet activation is instrumental in arterial
thrombogenesis, too little is known about the predictive
value of platelet function (adhesion, release and aggregation) on the incidence of CHD. It has been clearly shown
that the suppression of platelet activation, either by drugs
(Antiplatelet Trialist’s Collaboration, 1994) or by blocking
the platelet fibrinogen receptor, glycoprotein IIb/IIIa (EPIC
Investigators, 1994) offers protection against myocardial
infarction (MI) and other ischaemic events respectively.
However, rather little is known about the association
between increased platelet activation and CHD morbidity
and mortality. So far, increased platelet aggregation induced
by ADP or thrombin has been shown to be associated with
past MI and electrocardiographic evidence of ischaemia
respectively (Elwood et al. 1990, 1991), and prospective
evidence has been presented of an association between
increased platelet count and ADP-induced platelet aggregability, and long-term incidence of fatal CHD (Thaulow
et al. 1991). However, a nearly twofold difference in the
CHD rate of two Finnish cohorts was not associated with
G. Hornstra et al.
differences in platelet aggregation induced by different
agonists (Salo et al. 1985). Two studies have demonstrated
that platelet activation, as measured by the urinary excretion
of the platelet specific protein b-thromboglobulin (b-TG), is
significantly associated with the risk of CHD (Ghaddar et al.
1995; Gorgels et al. 1995). Platelet volume is increased at
the time of acute MI (Bath & Butterworth, 1996) and there
is compelling evidence that changes in platelet volume are
associated with myocardial risk (Martin et al. 1991; Brown
& Martin, 1994).
The main method to assess platelet function in dietary
studies has been the platelet aggregation test in vitro, with
the help of which it has repeatedly been shown that changes
in dietary fatty acids can modulate the platelet aggregation
pattern. However, the results are far from consistent and
their interpretation in terms of thrombosis tendency is
difficult, if not impossible, since direct comparisons
between platelet aggregation in vitro and arterial thrombosis
in vivo have not been made in man. In the rat, diet-induced
changes in platelet aggregation in vitro appeared to be
negatively related to changes in arterial thrombosis in vivo
(Hornstra et al. 1993).
3.3. Some diet effects on arterial thrombogenesis and
platelet function
3.3.1. Fatty acids. The intake of linoleic acid (18 : 2n-6)
is strongly correlated with the linoleic acid content of
plasma phospholipids, cholesterol esters and triacylglycerols. In addition, platelet total linoleic acid, ALA (18 : 3n-3),
arachidonic acid (20 : 4n-6), EPA (20 : 5n-3) and DHA
(22 : 6n-3) are significantly correlated with the concentrations of these fatty acids in plasma triacylglycerols, plasma
phospholipids and/or adipose tissue. However, the concentrations of unsaturated fatty acids in e.g. adipose tissues do
not predict risk for thrombosis (Kardinaal et al. 1995). In
two Finnish cohorts, platelet aggregation induced by ADP
showed a significant positive correlation with the contents
of linoleic acid in adipose tissue and plasma triacylglycerols, but not with linoleic acid in platelets (Salo et al.
There may be a specific preventive influence of ALA on
CHD, since the intake of ALA (range 0.8–1.5 g/d) was
inversely associated with the risk of MI in the health
professionals follow-up study (Ascherio et al. 1996b).
Also in the lifestyle intervention of de Lorgeril et al.
(1994), among several other dietary changes, about 2 g
ALA/d was estimated to be protective. However, in the
large Multiple Risk Factor Intervention Trial, no effect with
an ALA intake of 1.7 g/d was found (Dolecek & Grandits,
1991). In this study, a positive association was observed
between the dietary linoleic acid : ALA ratio and cardiovascular mortality. Serum ALA concentration appeared to be
negatively associated with the risk of stroke (Simon et al.
1995) and in a prospective study (Miettinen et al. 1982),
serum ALA, EPA, and DHA were all low in MI patients.
Observational studies in Norway during the Second
World War and cohort studies among Greenland Eskimos
and populations in Japan point to a protective effect against
CHD of long-chain n-3 fatty acids from fish or marine
mammals (Hornstra, 1989). Although later observational
studies gave conflicting results (Hornstra, 1989), there are
now five large-scale prospective studies demonstrating a
negative association between fish consumption and cardiovascular mortality (Kromhout et al. 1985; Norell et al. 1985;
Shekelle et al. 1985; Dolecek & Grandits, 1991; Daviglus et
al. 1997). In five other studies of about similar size and
design, however, no significant relationship was found
(Curb & Reed, 1985; Vollset et al. 1985; Lapidus et al.
1986; Morris et al. 1992; Ascherio et al. 1995). This has
been suggested to be due to the rather high habitual fish
consumption in the �low-fish’ group in these latter studies
(Kromhout, 1985). However, since all processes that are
thought to be involved in the cardio-protective effects of fish
(oil) show clear dose–response relationships over a wide
range of fish (oil) intakes, this is a rather unlikely explanation.
Evidence has also been reported for a positive association
between fish consumption and cardiovascular risk. Thus, in
two cohort studies a higher mortality from CHD was
observed in areas with a relatively high fish consumption
as compared with low-fish regions (Simonsen et al. 1987;
Hunter et al. 1988). In two large-scale prospective studies in
Finland, fish consumption was also positively related to
cardiovascular mortality (Salonen et al. 1995; Piettinen et al.
1997). In the study of Salonen et al. (1995) it was suggested
that the high intake of Hg from the freshwater fish may have
caused increased cardiovascular risk by promoting lipid
In conclusion, results of epidemiological studies with
respect to the importance of dietary fish (oil) for the
prevention of IHD are equivocal and not conclusive. Moreover, it should be reiterated that epidemiological studies can
only indicate associations between two phenomena; they
can never discriminate between causal and casual relationships. The final proof for the effectiveness of a fish (oil)enriched diet for the prevention of cardiovascular disease
has to be obtained via long-term, well-controlled, prospective primary intervention trials, which have not yet been
reported. So far, only one secondary intervention study has
been published (Burr et al. 1989), demonstrating that subjects who were advised to eat fatty fish at least twice a week
had a 29 % reduction in 2-year cardiovascular mortality as
compared with volunteers whose diet advice did not include
The effect of cis unsaturated fatty acids on platelet
function including in vitro aggregation data has recently
been reviewed (Mutanen, 1997). From this review it appears
that the results are very inconsistent which, at least in part,
may have methodological reasons.
A promising approach to assess platelet activation in vivo
is the measurement of thromboxane (Tx) metabolites (2,3dinor-TxB2 and 11-dehydro-TxB2 ) in urine, or of the concentration of the platelet-specific protein b-thromboglobulin, released from a-granules. Dietary fish oil or long-chain
n-3 fatty acids lower high basal Tx excretion rate, while
only a modest effect is found at a low basal excretion rate.
Results concerning the effects of other unsaturated fatty
acids on urinary Tx metabolites are almost totally lacking.
Preliminary results indicate that two diets with the same
saturated fat content but differing in their linoleic acid
contents (5 and 12 % energy) similarly increased 2,3dinor-TxB2 in urine; Turpeinen et al. 1997), which indicates
Cardiovascular system
enhanced platelet activation in vivo. High stearic acid and
trans fatty acid diets also stimulated 2,3-dinor-TxB2
excretion (Turpeinen et al. 1998). Furthermore, the results
from these studies indicate that platelet b-thromboglobulin
release in vivo was not affected by changes in dietary fatty
A general shortcoming in most of the studies to explain
the effects of dietary fatty acids has been a lack of information on the fatty acid composition of individual platelet
phospholipids. In addition, little is known about the role of
the baseline diet with respect to the incorporation of fatty
acids into platelets. Platelet membrane fatty acid composition can be changed by dietary means to some extent.
The total amount of a given fatty acid in the platelet is
probably less important than the factors regulating free fatty
acid levels and types in the membrane and in the platelet
interior. Platelet receptor responsiveness to physiological
stimuli and subsequent signal transduction and fatty acid
liberation for eicosanoid synthesis are probably highly
dependent on membrane fatty acid composition. However,
one can only speculate as to the precise underlying
A potentially important second messenger during platelet
activation is protein kinase C, the activation of which can be
modulated by cis unsaturated fatty acids, while saturated
and trans unsaturated fatty acids are inactive (Khan et al.
1995). Six isoenzymes of protein kinase C have now been
identified in human platelets, and these may be involved in
various aspects of platelet activation.
Other mechanisms by which fatty acids, especially n-3
fatty acids, might regulate platelet function involve changes
in TxA2 /prostaglandin(PG)H2 receptor affinity following
changes in membrane phospholipid composition (Bayon
et al. 1995). An alteration of the platelet redox state and
the resulting modulation of the expression of certain
enzymes could also be involved (M Lagarde, F Achard,
M Gilbert, C BeВґnistant, D Lemaitre and E VeВґricel, unpublished results). The enhanced sensitivity of platelets from
hypercholesterolaemic patients indicates that LDL may also
activate platelets. In vitro mildly oxidized LDL (ox-LDL)
has been shown to activate platelets significantly while
purified apoE seems to inhibit this (Weidtman et al. 1995;
Zhao, 1996). In addition, activated platelets release substances, e.g. platelet-derived growth factor, which can
modify LDL and enhance the macrophage uptake of
ox-LDL (Aviram, 1995).
3.3.2. Antioxidants and platelet function. On the basis
of epidemiological studies, dietary antioxidants (tocopherols, carotenoids, flavonoids and Se) have repeatedly been
suggested to reduce CHD risk, but the results of intervention
ВЁ hrval et al. 1996; van de
studies are more equivocal (O
Vijver, 1997).
Platelet function in vitro, and platelet adhesion in
particular, has been shown to be inhibited by high levels
of a-tocopherol which cannot be obtained from dietary
sources alone (Steiner et al. 1995). This mechanism may
partly explain the beneficial efficiency of pharmacological
amounts of a-tocopherol to prevent MI in the Cambridge
Heart Antioxidant Study (CHAOS) (Stephens et al. 1996).
Se supplementation in human subjects with a low Se status
decreases platelet aggregation in vitro, but has no effect on
platelet activation in human subjects with a normal Se
status. No experimental data are available as to the effects
of carotenoids on platelet function in man. Data on the
effects of flavonoids are from in vitro experiments only. The
results indicate an inhibition of platelet eicosanoid synthesis
and platelet aggregation (Goldberg, 1996).
3.4. Endothelial cell function
Damage of the endothelium leads to endothelial dysfunction
which is characterized by enhanced expression of cytokines,
cell adhesion molecules, von Willebrand factor (vWf),
platelet activating factor, and endothelin, and decreased
synthesis of PGI2 (prostacyclin) and transforming growth
factor-b (TGF-b). The level of vWf has been related to the
risk of MI and sudden death in patients with angina pectoris
(Thompson et al. 1995). The soluble form of the vascular
cell adhesion molecule (VCAM) and vWf were both shown
to be raised in stroke patients, while the intracellular
adhesion molecule (ICAM) was raised in patients at risk
of stroke only (Blann et al. 1996). Latent TGF-b in human
vascular SMC is activated by plasmin which is produced
from plasminogen by plasminogen activator (t-PA). In vitro
Lp(a) impairs this activation. Active TGF-b inhibits SMC
migration, proliferation and activation. Suppression of
TGF-b led to increased in vitro expression of ICAM-1 in
endothelial cells incubated with Lp(a) (Grainger &
Metcalfe, 1995). The question of whether cell adhesion
molecules are regulators of platelet function is far from
clear at the moment and requires further study.
3.4.1. Dietary fatty acids and endothelial cell function. Dietary fatty acids are able to regulate prostacyclin
production to some extent. Studies in Greenland Eskimos
(Fischer et al. 1986) and in a Japanese fishing village
(Hamazaki et al. 1989), as well as various intervention
studies with fish or fish oil (for reviews, see Hornstra, 1989;
Hornstra et al. 1990) have led to the conclusion that n-3
fatty acids of marine origin increase both PGI2 and PGI3
production in man. However, the methods used (measurement of major PGI metabolites in urine) are very
complicated and this is probably the reason why data from
various other studies with respect to PGI2 are not consistent
(Knapp et al. 1986) and effects of other dietary fatty acids
have not been reported. The experimental evidence with
respect to the effect of dietary fatty acids on NO synthase
regulation is not clear at present.
In two cohort studies, negative associations were found
between dietary consumption of n-3 fatty acids and plasma
levels of vWf (Shahar et al. 1993). In an intervention study,
a low-fat (28 % energy), low-saturated fatty acid (9 %
energy), and low-cholesterol (215 mg/d) diet for 3 years
resulted in significantly lower plasma vWf levels than the
control diet. Moreover, a negative correlation between
plasma vWf and dietary n-3 and n-6 fatty acids was found
(Blann et al. 1995). In patients suffering from non-insulindependent diabetes mellitus (NIDDM), a diet enriched in
monoenoic fatty acids (30 % energy) decreased plasma vWf
when compared with a diet high in carbohydrate (11 %
energy as monoenes) (Rasmussen et al. 1994).
In human endothelial cell cultures, both DHA and EPA
attenuated the induction of ICAM-1, VCAM-1 or E-selectin
G. Hornstra et al.
in interleukin-1b-activated cells (Collie-Duguid & Wahle,
1996). On the other hand, DHA, but not EPA or arachidonic
acid, was shown to inhibit the cytokine-induced expression
of VCAM-1 (Weber et al. 1995) by blocking the activation
of nuclear factor kB, an inducible transcription factor which
specifically activates transcription of cell adhesion
molecules. Activation of nuclear factor-kB is significantly
enhanced in vitro by linoleic acid (Henning et al. 1996) and
recent results with rabbits suggest that monounsaturated
fatty acids might inhibit VCAM-1 expression in vivo (De
Caterina et al. 1995b).
Availability of arachidonic acid is an important determinant of PGI2 synthesis by endothelial cells but recent results
with respect to the effects of DHA and EPA on PGI2
production suggest that alteration of the expression of the
enzymes responsible for formation of PGI2 may also be
crucial (M Lagarde, F Achard, M Gilbert, C BeВґnistant, D
Lemaitre and E VeВґricel, unpublished results). The regulation of the expression of cell adhesion molecules probably
includes oxidant–antioxidant sensitive mechanisms, since
in vitro VCAM-1 gene expression can be inhibited by
synthetic antioxidants. Conversely, LysoPC, a component
in oxidized LDL, has been shown to upregulate VCAM-1
and ICAM-1 expression in endothelial cells and rapidly
induces P-selectin expression in both platelets and endothelial cells (Ochi et al. 1995; Murohara et al. 1996). This
latter effect is probably the basis of leucocyte deposition.
3.5. Coagulation and fibrinolysis
In the circulation, coagulation and fibrinolysis factors balance each other. The main markers used to evaluate blood
coagulability are fibrinogen, factor VII (and other coagulation factors), antithrombin III (AT-III), fibrinopeptide A
released from fibrinogen by thrombin, and prothrombin
fragment F1Гѕ2 . Today, prothrombin F1Гѕ2 is considered a
sensitive marker of clotting activation. Fibrinolytic potential
is assessed by measuring plasminogen, tPA, its inhibitor
plasminogen activator inhibitor-1 (PAI-1), and cross-linked
fibrinogen degradation products (D-dimers). The latter indicator reflects both coagulation and fibrinolysis.
The early results from the Northwick Park Heart Study
(Meade et al. 1986) indicated strong independent associations between baseline plasma fibrinogen and factor VII
coagulant (FVIIc) activity levels and the risk of CHD. In the
same population, a U-shaped association between ATIII and
the risk for CHD was found. Low fibrinolytic activity
predicted a higher risk for CHD in a later analysis (Meade
et al. 1993). Fibrinogen is also generally accepted as an
independent risk factor for CHD, while the predictive value
of PAI-1 and tPA levels as risk factors is still contradictory
(Hamsten, 1995; Ridker & Vaughan, 1995). In patients with
angina pectoris, the levels of fibrinogen and tPA antigen
have been shown to be independent predictors of subsequent
MI or sudden death (Thompson et al. 1995). Elevated
plasma levels of D-dimers have been shown to be associated
with early atherosclerosis (Salomaa et al. 1995) and
increased risk of future MI (Ridker et al. 1994), although
in the latter study the D-dimer level did not appear to be
an independent predictor. Recent results from 2952 men
clinically free from CHD show that six markers of the
hypercoagulable state (FVIIc, FVII antigen, activated
factor VIII and factor IX, prothrombin fragment F1Гѕ2 , and
fibrinopeptide A) are all positively associated with CHD
risk (Miller et al. 1996).
3.5.1. Effect of dietary factors on coagulation and
fibrinolysis. According to several studies, dietary fatty
acids hardly influence plasma fibrinogen. There is one study
from Denmark (Bladbjerg et al. 1995) showing increased
plasma fibrinogen level after an extremely high stearic acid
(about 15 % energy) diet when compared with a diet high in
myristic and lauric acids. In a recent study, plasma
fibrinogen concentration increased slightly during the
stearic acid (9.3 % energy) diet, but the biological
significance of this is questionable (Mutanen & Aro,
1997). In the population-based cross-sectional atherosclerosis risk in communities (ARIC) study, a negative association
between the intake of long-chain n-3 fatty acids and plasma
fibrinogen levels was found (Shahar et al. 1993). However,
intervention studies with long-chain n-3 fatty acids have
given very inconsistent results (Hornstra, 1992).
Current knowledge about diet and factor VII (FVIIc
activity or FVII antigen levels) indicates that fasting
FVIIc can be reduced by low-fat diets. The fatty acid
composition of the diet, i.e. saturated, monounsaturated or
n-3 and/or n-6 polyunsaturated fatty acid contents, have not
been found to be important in short-term experiments
(Mennen et al. 1996). Habitual high-fat diets seem to
increase both FVIIc and FVII antigen. Increased postprandial responses of FVIIc are seen after high-fat test meals
regardless of the type of fat. It seems that the change in
fasting FVIIc is part of a general change in concentrations of
vitamin K-dependent proteins, while changes in non-fasting
FVIIc activities are primarily mediated by activation of the
factor VII zymogen (Bladbjerg et al. 1995). The activation
of factor VII has been suggested to be related to free fatty
acid production during lipolysis of triacylglycerol-rich lipoproteins (Silveira et al. 1994).
There are only a few reports about the effects of diet on
AT-III. Early studies indicate that n-6 polyunsaturates might
increase plasma AT-III, while long-chain n-3 have either no
effect or may increase it. A recent study comparing ALA
with EPA+DHA indicated that ALA might have a beneficial
effect on plasma AT-III levels (Freese & Mutanen, 1997).
Supplementation for 16 weeks with long-chain n-3 fatty
acids of patients with chronic atherosclerotic disease
induced a significant increase in plasma levels of tissue
factor pathway inhibitor, indicating down-regulation of the
extrinsic pathway of blood coagulation (Berrettini et al.
1996). In an earlier study, a shorter supplementation period
did not produce such an effect (Hansen et al. 1994). In the
study of Berrettini et al. (1996) a significant reduction of
F1Гѕ2 plasma levels was found also. A slight but significant
decrease in F1Гѕ2 has been reported after a high-stearic-acid
diet when compared with a high-myristic and -lauric acid
diet (Bladbjerg et al. 1995). Circulating amounts of F1Гѕ2
were not different between low-fat and high-monoene diets
(Lopez-Seguara et al. 1996).
In a long-term study with a low-fat (26 % energy) highfibre diet, tPA activity increased significantly in healthy
subjects, while no change in tPA antigen was found
(Marckmann et al. 1993). However, the reduction of the
Cardiovascular system
total dietary fat content alone (from 39 to 31 % energy) had
no effect. Several studies have found no effect on plasma
tPA activity of dietary long-chain n-3 fatty acids (Eritsland
et al. 1994), olive oil (Lopez-Segura et al. 1996), maize oil
(Hellsten et al. 1993) or stearic acid or trans fatty acids
(Mutanen & Aro, 1997).
Effects of the dietary composition on PAI-1, either antigen or activity, are not consistent (Hornstra, 1992; Hellsten
et al. 1993). Long-chain n-3 fatty acid supplementation
mainly increases PAI-1 antigen and either increases, or
has no effect on PAI-1 activity. In a recent study, PAI-1
activity increased similarly with either ALA or EPA+DHA
supplementation (Freese & Mutanen, 1997). There are only
a few studies addressing the effects of other dietary fatty
acids on PAI-1. A high-oleic-acid diet (fat 38 % energy,
monoenes 24 % energy) decreased both PAI-1 activity and
antigen when compared with a high-carbohydrate diet (fat
27 % energy, monoenes 13 % energy). The decrease was
accompanied by a parallel decrease in plasma insulin levels
(Lopez-Segura et al. 1996). Maize oil supplementation
resulted in decreased PAI-1 activity (Hellsten et al. 1993),
but in another study olive oil did not have an effect
(Oosthuizen et al. 1994). Changes in total fat and fibre
intake did not affect PAI-1 either (Marckmann et al. 1993).
No changes in D-dimer concentrations have been
detected in some recent studies with long-chain n-3 fatty
acids (Eritsland et al. 1994, 1995), trans fatty acids (Almendingen et al. 1996; Mutanen, 1997) or stearic acid (Mutanen
& Aro, 1997). A decrease in D-dimer level was found in the
study of Mutanen & Aro (1997) when the subjects changed
from their habitual diet (polyunsaturated : saturated fatty acid
ratio (P : S) 0.36) to more saturated type of diet (P : S 0.24).
Data concerning the effects of other dietary factors on
coagulation and fibrinolysis are scarce. The results from a
low-fat, high-fibre experiment by Marckman et al. (1993),
however, indicate that some components of dietary fibre
may affect coagulation and fibrinolysis. Two other studies
support this assumption (Nilsson et al. 1990; Sundell &
Ranby, 1993). Recently, in a large Finnish cohort of middleaged men an inverse association was observed between the
intake of dietary fibre and the risk of CHD. Adjustment for
serum cholesterol did not change the results, indicating that
in the mechanism lipoprotein metabolism is not involved
(Pietinen et al. 1996).
Platelets are important contributors to both coagulation
and fibrinolysis. Although tissue factor present in monocytes and the blood vessel wall, in combination with
activated factor VII (FVIIa), is the main initiator of coagulation, activated platelets, by exposing phosphatidyl serine
at their surface, provide the preferred surface on which
coagulation occurs. This platelet procoagulant activity, also
called platelet factor 3, is closely related to platelet aggregation. AT-III can rapidly inhibit FVIIa that is bound to
tissue factor, thus inhibiting the start of coagulation
(Rapaport & Rao, 1995). How platelet membrane fatty
acid composition affects the exposure of phosphatidyl
serine, or how tightly the fatty acid composition of phosphatidyl serine is regulated is not known at present. The
functional association between fatty acids and tissue factor
presentation in tissue-factor-containing cells is not known
There is some evidence that long-chain saturated fatty
acids might provide a contact surface for activation of
clotting factors XII and IX (Mitropoulos, 1994). Activation
of these factors can cause the activation of factor VII and
thus increase FVIIc.
Fibrinolysis also occurs at the platelet surface after direct
binding of plasminogen, tPA and plasmin. Once bound, tPA
manifests enhanced catalytic activity to convert plasminogen to plasmin, thereby enhancing thrombolysis. Formed
plasmin also binds to the platelet surface and, at low
concentrations, reduces fibrinogen binding which results
in reduced platelet aggregation. At high concentrations,
however, plasmin activates platelets (Loscalzo et al.
1995). How fatty acids would regulate either the production
of plasminogen or tPA in the endothelium or their activation
on the surface of platelet membranes is not clear as yet. In
endothelial cells, both tPA and PAI-1 productions seem to
be mediated by protein kinase C activation (Rydholm et al.
1995) and thus may be influenced by fatty acids.
There are two recent reviews on lipoprotein metabolism
and thrombosis (Mitropoulos, 1994; Miller, 1995). The
current opinion is that fatty acid composition of lipoprotein
particles may be important for the activation of the contact
system of coagulation. Furthermore, high blood lipid levels
may change platelet function by influencing platelet membrane composition and fluidity.
4. Immune-mediated processes underlying CHD
Maintaining the vascular integrity and defending the circulatory system against pathogenic processes require regulatory interactions among blood cells and between blood cells
and the vessel wall. The interacting cells are leucocytes
(monocytes and T-lymphocytes) and platelets in the circulation, and endothelial cells and SMC in the vessel wall
(Ross, 1995). These processes are controlled through activation of adhesion receptors already present on resting
blood cells and endothelium, or through the expression of
new receptors on the cell surface (Frenette & Wagner,
Cell activation, production of chemoattractants and cell
growth factors are key components in these events, which
are involved in repair and defence systems, but also, under
certain conditions, in tissue injury and disruption in the
cardiovascular compartment. Long-term processes also trigger the participation of key components in cellular immunity, such as T-lymphocytes and macrophages (Lodish et al.
1995). These cells are recognized to play a role in inflammatory and immune-mediated processes in atherosclerosis,
since T-lymphocytes are present in the arterial plaque
(Jonasson et al. 1986), and antibody responses to plaque
constituents have been detected (Palinski et al. 1989).
4.1. Immunocompetent cells involved in the
atherosclerotic lesion
4.1.1. Endothelial cells. The endothelium, which lines
vessel walls and acts as a permeability barrier controlling
the exchange of nutrients and fluids, is a dynamic
component of the artery. It provides a non-adherent surface
for leucocytes and platelets, maintains the vascular tone
G. Hornstra et al.
by releasing vasoactive molecules such as NO, PGI2 ,
endothelin and angiotensin II, and produces and secretes
growth factors and cytokines. The endothelium also forms
and maintains the connective tissue matrix, has the capacity
to modify plasma lipoproteins, and provides anti- and
procoagulant activities. When these functions are altered, in
the initiation of the atherosclerotic lesions, leucocytes
adhere to the vessel wall, following the formation of cell
adhesion proteins (ICAM-1, VCAM-1 etc.) (Springer, 1990;
Poston et al. 1992). There is formation of oxidativelymodified particles and an accumulation of lipoproteins in
the subendothelial space (Simionescu et al. 1986). Associated modifications take place, such as altered vascular
tone, the inability to regenerate wound sites and to prevent
platelet adhesion, thrombosis and coagulation. In addition,
growth factors and cytokines are released after cell
4.1.2. Smooth-muscle cells. The second major type of
cell in the arterial wall is the SMC. During the formation of
arterial lesions, SMC, monocyte-derived macrophages and
T-lymphocytes accumulate in the lesion, and this process is
associated with deposition of connective tissue matrix and
lipid. SMC, which are activated to migrate from the media
into the intima and to proliferate there, produce a variety of
growth factors, and the genes for these molecules and for
cytokines (e.g. interleukin-1, tumour necrosis factor-a) are
induced by various agents (Stemme & Hansson, 1994).
SMC are present in two phenotypic states: the contractile
and the synthetic. In the contractile state, they respond to
vasoactive agents, whereas in the synthetic state they
express genes for growth factors and cytokines and also
produce various forms of connective tissue matrix.
4.1.3. Immunocompetent leucocytes. In addition to the
constitutive cells of the vessel wall, all forms of lesions
contain elements of specialized chronic inflammation, e.g.
monocyte-derived macrophages and T-lymphocytes. The
macrophages, in addition to acting as scavengers and as
antigen-presenting cells, produce growth-regulatory proteins and could contribute to lipoxygenase-mediated generation of oxLDL. Macrophages are the main source of
foam cells, since they take up oxLDL through scavenger
receptors and a putative oxLDL receptor (Stemme &
Hansson, 1994). They can also produce growth factors
and chemotactic molecules for other monocytes, endothelial
cells and SMC.
T-lymphocytes represent the second type of cells derived
from the circulation and found in common atherosclerotic
lesions (Jonasson et al. 1986). These cells appear to be in a
low degree of activation, and have a low proliferation rate
(Gordon et al. 1990). Large numbers of T-lymphocytes are
generally found in lesions associated with risk factors, e.g.
hyperlipidaemia, diabetes, and hypertension.
4.1.4. Mechanisms for the recruitment of blood cells in
arterial lesions. Recruitment of lymphocytes and monocytes, their binding to the endothelium, and adhesion of
activated platelets to monocytes and endothelium, all these
processes are mediated by cell–cell adhesion molecules.
Movement of leucocytes from the blood into tissues,
contributing to tissue oedema and necrosis following
ischaemia, involves additional adhesion molecules, such
as ICAM-1 and VCAM-1.
Growth factors and cytokines participate in cell
interactions and in the development of the arterial lesions.
They have been detected in atherosclerotic plaques in vivo,
by in situ detection methods. Evidence for the activation of
cell–cell interactions in atherosclerotic disease is now
obtained from the assessment of plasma levels of cell
adhesion molecules in atherosclerotic patients (Blann &
McCollum, 1994). These levels were found to differ
depending on the type of dyslipidaemia (Hackman et al.
4.2. The immune system response modulates
atherosclerosis progression
One of the products of activated T-cells, interferon-g
inhibits SMC proliferation in vitro and in vivo. Therefore,
reduced plaque growth would be expected following
increased interferon-g production by cells in the plaque. It
has been found, in fact, that T-cell depletion leads to
increased lesion size after experimental arterial injury
(Hansson et al. 1991), and that cyclosporin A, an inhibitor
of T-cell functions, accelerates atherosclerosis in hypercholesterolaemic mice (Roselaar et al. 1995). Interferon-g
also down-regulates the expression of the scavenger receptor by human macrophages, inhibiting foam-cell formation
in vitro (Geng & Hansson, 1992). The following additional
observations indicate protective effects of the immune
system with respect to the progression of atherosclerosis:
in LDL-receptor-deficient rabbits hyperimmunized with
homologous oxLDL, there is a substantial reduction in the
progression of the lesions (Palinski et al. 1995); elimination
of T-lymphocytes with monoclonal antibodies results in
larger proliferative lesions in balloon-catheterized rat
aortas (Hansson et al. 1991), and mice lacking cytotoxic
T-cells develop much larger lesions in the aorta (Fyfe et al.
1994). On the other side, early studies have shown that
immunization of rabbits with HSP/65 induces an inflammatory type of lesion (Xu et al. 1992).
It is too early, at this point, to conclude what would be the
net effect on atherogenesis of a local immune response in
the plaque. The same holds for the effect of systemic
immune responses, although the systemic antibody response
to the plaque autoantigens against oxLDL tends to correlate
with aggravation of the disease.
4.3. Nutrition and the immunological aspects of
An array of major and minor components of the diet is able
to modulate some functional factor of various types of cells,
including those that participate in the formation of the
arterial plaque and involve the immune system in atherosclerotic disease.
Among the major components of the diet, polyunsaturated fatty acids play an important role in atherogenesis.
Among the minor components of the diet, antioxidants have
been reported to affect the atherosclerotic process. This
heterogeneous class of compounds includes antioxidant
vitamins and a large number of molecules, e.g. flavonoids
and polyphenols, present in several foods. While some of
these effects may be attributed to typical antioxidant
Cardiovascular system
activities, such as reduced production of reactive O species
and of the compounds generated by them (e.g. oxLDL),
other effects appear to be mediated by effects on cellular
functions. We will consider only the activities of antioxidants on cell-mediated processes. Those concerning the
direct effects on the production of reactive O species in
biological systems are discussed by Diplock et al. (1998).
4.3.1. Effects of n-3 fatty acids on cellular immune
response and inflammatory events in atherogenesis. As
recently reviewed by Calder (1996), polyunsaturated fatty
acids such as arachidonic acid (20 : 4n-6), EPA (20 : 5n-3)
and DHA (22 : 6n-3) affect functional variables in various
types of cells, including those involved in inflammation and
immunity. The compounds of the n-3 series have been
shown to be particularly potent and, therefore, their effects
will be more specifically discussed.
In vitro effects. ALA (18 : 3n-3), EPA and DHA have
been shown to reduce the proliferation of human lymphocytes (Kelly & Parker, 1979; Santoli et al. 1990). They also
inhibit the response to antigens and the ability of antigenpresenting cells to present antigen (Fujikawa et al. 1992),
and suppress the production of interleukin-2 (Calder &
Newsholme, 1992), a major stimulator of the proliferation
of lymphocytes and regulator of cytotoxic T-lymphocytes,
natural killer cells and B-cells. This type of action suggests
that n-3 polyunsaturated fatty acids may play a role in
controlling cellular immune processes in atherogenesis.
EPA appears to be more active, but ALA has also been
shown to exert some of the effects mentioned. However, the
experimental conditions in these studies are often far from
physiological and, consequently, the relevance of these
studies is questioned.
Ex vivo effects. Feeding fish oils, often in large
amounts, to animals suppresses the response of spleen,
thymus, lymph node and peripheral blood lymphocytes to
mitogenic stimuli (Kelley et al. 1988) and reduces the
proportion of spleen lymphocytes bearing the interleukin-2
receptor (Yaqoob & Calder, 1993). Results concerning the
effects on the phagocytic activity of macrophages are
conflicting, but reduction of macrophage function has
consistently been reported in various animal models and
in human subjects. Finally, chemotaxis of blood neutrophils
and monocytes towards a variety of chemoattractants is
reduced after n-3 administration (Sperling et al. 1993).
In feeding studies, n-3 fatty acids appeared to directly
affect cellular adhesion processes, as shown by the reduction in the expression of adhesion molecules in T-lymphocytes (Sanderson et al. 1995). These effects have been
further investigated in in vitro systems, where n-3 fatty
acids appeared to reduce the expression of various adhesion
molecules, as well as the binding between monocytes and
endothelial cells (De Caterina et al. 1995a).
A reduced production of various cytokines by peripheral
blood monocytes after n-3 fatty acid supplementation has
been reported in human subjects (Endres et al. 1993,
1995b), but later studies by others have not always given
similar results. Studies with respect to lymphocyte-derived
cytokines are limited and the results are somewhat contradictory too.
Clinical aspects of immunomodulation by n-3 fatty
acids. Clinical studies aiming to assess the effects on
functional variables of the immune system in human
subjects have shown that diets rich in n-3 long-chain
polyunsaturates v. diets rich in n-6 fatty acids (mainly
linoleic acid) decrease the ex vivo synthesis of the cytokines
interleukin-1b and tumour necrosis factor-a and reduce
T-cell proliferation (Meydani et al. 1993). Suppression of ex
vivo interleukin-2 production and of mononuclear cell
proliferation was observed in a cohort of the same study
(Endres et al. 1993). These effects on cytokine synthesis
may take place at the level of transcription, as is suggested
from the observed reduction of mRNA (Kaminski et al.
1993). The potential benefits of n-3 fatty acids in controlling
cellular events in atherogenesis are indirectly supported by
in vitro studies showing reduction of the expression of
cytokine-induced pro-atherogenic and pro-inflammatory
proteins in human endothelial cells by DHA (De Caterina
et al. 1994). These studies concerning the effects of n-3 fatty
acids on the function of cells involved in the remodelling of
vessel walls and in the atherosclerotic process under
pathogenic conditions suggest that these fatty acids exert a
protective action with respect to the arterial wall. It should
be kept in mind, however, that the in vitro results should be
carefully evaluated in the context of the experimental
conditions in order to make comparisons with the in vivo
situation. Moreover, the in vivo or ex vivo effects have been
obtained in general with high doses of the n-3 fatty acids, for
relatively short periods of treatment, whereas few studies
have been made mimicking the dietary situation (relatively
low intakes of long duration).
Mechanisms of action. Long-chain polyunsaturates of
the n-3 family may act at the cellular level through various
mechanisms which can be summarized as follows.
(a) Modulation of eicosanoid production by cells of the
immune system, especially reduction of proinflammatory PGE2 and leukotriene B4 .
(b) Modulation of membrane fluidity.
(c) Modulation of signal transduction pathways, especially
those involving lipid mediators, protein kinase C and
Ca2Гѕ mobilization.
(d) Modulation of the expression of genes involved in
cytokine production or in peroxisomal proliferation,
fatty acid oxidation and lipoprotein assembly.
Safety. Although the issue of safety of n-3 fatty acids
has not been specifically addressed, there are some
reports of alterations of liver function in rodents, at high
levels of intake. On the other hand, there is no evidence of
negative effects even at relatively high levels of intake, in
population groups. It is recommended however, on the
basis of some reports of enhanced susceptibility of LDL
enriched in n-3 fatty acids to oxidation in vitro (a marker
which has a somewhat disputable significance), to increase
the intake of antioxidants, such as vitamin E, as a preventive
4.3.2. Antioxidants. Uncontrolled oxidative stress in
the cardiovascular system is considered to promote the
progression of arterial wall lesions through various
mechanisms, the major ones being the enhanced oxidation
of lipoproteins (resulting in greater atherogenicity and
enhanced accumulation in the cells of the vascular wall) and
the activation of cells involved in the pathogenesis of
G. Hornstra et al.
atherosclerosis (monocytes, endothelium, SMC, platelets),
as a consequence of enhanced formation of activators
(oxLDL, cytokines, eicosanoids, etc.).
In various types of cells, reactive O species may act at the
transcriptional level through the activation by cytokines of
the transcription factor nuclear factor-kB (Schreck et al.
1992). The major antioxidants in the diet, found also
in plasma, are tocopherols, mainly a, but also b and g,
b-carotene and other carotenoids, ubiquinone (coenzyme
Q10 ), flavonoids, and other plant polyphenols (all lipidsoluble), and vitamin C, which is water-soluble.
In vitro and ex vivo studies have shown effects of natural
antioxidants on immune competent cells (Middleton &
Kandaswami, 1992; Faruqi et al. 1994) and cells in the
cardiovascular system. In addition, potent synthetic antioxidants have been demonstrated to inhibit the expression of
genes coding for cytokines (DeForge et al. 1992) and to
reduce VCAM-1 gene expression in human vascular
endothelial cells (Marui et al. 1993).
5. Diet, hypertension and heart function
CHD is strongly related to both systolic and diastolic blood
pressure in a graded fashion (MacMahon et al. 1990) and
treatment of hypertension results in a reduction in coronary
disease-related events (Collins et al. 1990). Hypertension
due to known factors or diseases (i.e. secondary forms of
hypertension) is distinguished from primary hypertension
where no known clinical cause for the persistent elevated
blood pressure can be identified (essential hypertension). In
approximately 90–95 % of hypertensives the causes are
5.1. Aetiology of hypertension
5.1.1. Membrane function. The role of ion transport
across cell membranes in the development of hypertension
has been studied extensively. Some cellular activities, such
as Naþ –Liþ counter transport, ouabain binding sites (Naþ ,
Kþ -ATPase) or activity, Liþ –Kþ co-transport, or Liþ
leakage, are considered to reflect ion channel function and
there is a strong genetic influence on the association
between these markers of ion channel activity and
hypertension. Depending on the specific marker, the genetic
make-up can �explain’ 20–60 % of the occurrence of
hypertension (Williams et al. 1991). In contrast, environmental influences (including dietary factors) are much less
important and explain between 0 and 16 %. This is
important when considering the effect of dietary fatty
acids that may change cellular membrane fatty acid
composition (see section 5.3). The alterations in ionic
channel activity are assumed to lead to increased intracellular Ca2Гѕ content and activity, contraction of the arterial
smooth muscles and, ultimately, to vasoconstriction. The
gene effect related to decreased ouabain-binding sites which
are associated with increased intracellular NaГѕ levels (and
presumably with increased intracellular Ca2Гѕ activity due to
Naþ –Ca2þ exchange) may occur in 8 % of hypertensives.
Other ion-channel-related gene effects mentioned are less
common: 2–3 %. In contrast, some �ion channels’ are
associated with obesity and the combined gene effect occurs
in 10 % of the hypertensive population.
5.1.2. Role of humoral mediators. A number of
vasoactive substances (angiotensin, endothelin) have been
implicated in the development of hypertension. Endothelin1 is a potent vasoconstrictor produced by endothelial cells,
but most endothelin is not secreted luminally and hence
plasma levels may not adequately reflect local production.
Inhibitors of endothelin synthesis or blockade of receptors
can reduce blood pressure in genetic hypertensive animal
models. Interestingly, noxious stimuli (including oxLDL
and cytokines) cause endothelial cells to synthesize
endothelin-1 (Boulanger et al. 1992). Thus oxLDL could
lead to exaggerated endothelin-1 production in atherosclerotic vessels.
The renin–angiotensin system plays an important role in
the homeostasis of salt and water. The conversion of
angiotensinogen to angiotensin I by renin is rate limiting.
Angiotensin II is produced by an angiotensin converting
enzyme (EC Angiotensinogen levels have been
related to hypertension and a gene resulting in higher
angiotensinogen levels has been described. This complex
system with positive and negative feedback mechanisms has
many aspects that may be important in the aetiology of
hypertension. Obesity, excess energy intake and increased
angiotensinogen levels have also been linked. A deletion
polymorphism of the angiotensin converting enzyme gene
has been thought to be connected with the development of
CHD (Cambien et al. 1992) and increased pressor responsiveness to angiotensin I in normotensive men (Ueda et al.
1995). However, no linkage with essential hypertension was
observed (Jeunemaitre et al. 1992) and apparent linkage
with CHD is now strongly contested.
5.1.3. Insulin resistance. Insulin resistance is closely
related to hypertension and hyperlipoproteinaemia. Much
interest is focused on the metabolic syndrome X, not to be
confused with the cardiac syndrome X (chest pain with
normal coronary arteries on angiography). The metabolic
syndrome is associated with central obesity (although not
always), hyperinsulinaemia, hypertriacylglycerolaemia,
maturity onset diabetes and hypertension and will be
discussed in more detail in section 6. Hyperinsulinaemia
has been shown to be related to the development of
hypertension in prospective studies (Skarfors et al. 1991;
Lissner et al. 1992). Since insulin also affects ion transport
and acts as a growth factor, it is thought that these
mechanisms may lead to hypertension (Stout, 1990).
5.1.4. Environmental factors. Environmental factors
also play a role. Dietary intake of Na will increase blood
pressure although some human subjects are more saltsensitive than others. Yet there is a range of responses and it
would be an oversimplification to dichotomize the population into those who are either salt-sensitive or not
(Weinberger, 1990). On the other hand an inverse association between dietary KГѕ intake and blood pressure is
recognized. It may be by this mechanism that fruit and
vegetable consumption (rich in KГѕ ) helps to prevent
hypertension, although the lower blood pressure in women
consuming a fruit- and vegetable-rich diet and in vegetarians may be independent of K (Ascherio et al. 1996a; Beilin
& Burke, 1995). In addition, fruit and vegetables may help
to lower the dietary fat intake and the development of
obesity. Smoking also increases blood pressure acutely for
Cardiovascular system
up to 30 min and when it is considered that many smokers
may smoke twenty cigarettes per day or more, blood
pressure would be raised for long periods of time (Groppelli
et al. 1992). However, earlier studies in which blood
pressures were compared between smokers and nonsmokers before smoking failed to document this (Groppelli
et al. 1992). The diet of smokers differs from that of nonsmokers and in the UK smokers consume more salt and
fewer essential fatty acids, in particular linoleic acid (Fulton
et al. 1988).
Epidemiological studies have generally found positive
associations between alcohol consumption and blood pressure (World Hypertension League, 1991). Alcohol in large
quantities will contribute significantly to energy intake and
may lead to obesity and hypertriacylglycerolaemia. Alcohol
intake is associated with other unfavourable lifestyle factors
such as smoking and low physical activity.
5.2. Strategies to reduce CHD by lowering blood pressure
5.2.1. Intervention trials. Many of the early clinical trials
had insufficient statistical power to evaluate the benefits of
blood pressure reduction in hypertensives in terms of CHD
(and stroke). Recently, despite a wide range of treatments
with drugs, inclusion criteria, etc. these trials have been
subjected to meta analysis. CHD was reduced by 10–14 %
by hypertensive treatment. (The reduction in haemorrhagic
stroke was 40 %.) The reduction in CHD events is less than
was predicted from observational studies and this has led to
concern about possible adverse side-effects of drugs
(MacMahon et al. 1990). b-Blockers adversely affect lipid
and glucose metabolism, whilst high doses of diuretics
share some of these metabolic effects. Neither Ca nor Mg
supplements reduce blood pressure in subjects that are not
deficient in these minerals. Algorithms have been developed
for the treatment of essential hypertension. After it has been
established that blood pressure is really elevated (several
readings after reasonable rest on several occasions) a nonpharmacological approach to lower blood pressure is commenced (see section 5.2.2). Only thereafter is the need for
pharmacological treatment considered as part of a multiplerisk-factor approach to management. For reasons given earlier,
traditional therapy with diuretics and b-blockers is often
replaced by angiotensin converting enzyme inhibitors, Caantagonists or a1-adrenoceptor blockers.
5.2.2. Individual v. population approach. There are
more hypertensive subjects in populations with a high
median blood pressure level. Most CHD events are seen in
the many patients with mildly elevated blood pressure,
although their risk is less than that of the small group of
severe hypertensive subjects. These arguments form the
basis for the population strategy to prevent hypertension and
CHD by non-pharmacological measures: reduction in salt
and high alcohol intake, avoidance of obesity and increasing
the dietary KГѕ : NaГѕ ratio. It is estimated that the average
systolic blood pressure would be lowered by some 8 mmHg.
The magnitude of this blood pressure lowering effect would
be difficult to discern in an individual. Nevertheless, at the
population level such a reduction in systolic blood pressure
in middle-aged men has the potential to reduce CHD and
stroke mortality by 16 and 23 % respectively.
5.3. Dietary fatty acid composition and hypertension
There are some epidemiological observations that suggest
that dietary polyunsaturated fatty acids, whether n-3 or n-6,
may reduce blood pressure. However, dietary intervention
studies are contradictory. Diets supplemented with n-6
polyunsaturates (mainly linoleic acid) do not consistently
reduce blood pressure. There have been many supplementation studies with mixtures of the fish-oil-derived n-3 longchain polyenes EPA (20 : 5n-3) and DHA (22 : 6n-3). Many
of these studies, in normotensive, hypertensive, hypercholesterolaemic and CHD patients were primarily designed to
examine the effect of n-3 long-chain polyenes on plasma
lipids and generally the number of subjects in the study was
too low. The two largest studies, examining the effect in 350
healthy or in 156 hypertensive men and women are still very
small in comparison with current cardiovascular trials
(BГёnaa et al. 1990; Trials of Hypertension Collaborative
Research Group, 1992). Nevertheless, they documented no
benefit in healthy subjects and a reduction in hypertensives.
A recent meta analysis has been carried out. There are
significant differences between the studies in terms of blood
pressure recording (single or repeated, automatic device or
random zero sphygmomanometer, blinding of patients and
observers, choice of olive or n-6 polyunsaturated oil as a
placebo, etc.). The results suggest that n-3 long-chain
polyenes (mainly EPA) reduce blood pressure in a dosedependent manner in hypertensives, but have little or no
effect in healthy volunteers (Morris et al. 1993). The
mechanism is not clear, but is assumed to be by a reduction
in the production of the vasoconstrictor TxA2 . It is widely
held that linoleic acid and n-3 long-chain polyenes could
affect blood pressure because they can change membrane
fatty acid composition and/or membrane fluidity and
thereby alter ion-channel activity and prostaglandin synthesis. The fatty acid composition of membrane phospholipids
is hardly changed by diets rich in linoleic acid. Long-chain
polyenes of the n-3 family, on the other hand, markedly
reduce the level of arachidonic acid in phospholipids. A
shift from TxA2 to TxA3 , devoid of powerful vasoconstricting properties, is now accepted. Fish oil may not reduce
arachidonic acid in phosphatidyl inositol, a phospholipid
central to a1-adrenoceptor-mediated inositol pathway
signal transduction (MacLeod et al. 1994). However, little
is known about the effect of fish oil on the fatty acid
composition of small arterial resistance vessels (MacLeod
et al. 1994). In view of these uncertainties it is clear that a
meta analysis is no substitute for a properly conducted trial.
What is needed is a large double-blind controlled trial in
hypertensive patients, in whom TxA2 production or a1adrenergic mechanisms have been implicated in their
5.4. Heart function
The maintenance of blood pressure and perfusion of organs
is the main function of the heart. The energy for this is
derived from oxidative phosphorylation in mitochondria. As
myocardial energy reserves last but a few seconds, there is a
constant need of O2 supply. Cardiac output can vary
enormously, due to large changes in the emotional and
G. Hornstra et al.
environmental influences, and changes in O2 consumption
will follow in its tract. The function of the heart is controlled
by the autonomic nervous system. Increasing O2 requirements are met by increased O2 extraction and blood
flow (vasodilatation). The lumen of coronary vessels is
controlled by a tonic vasoconstriction mediated by a1adrenergic receptors balanced by a vasodilatation under
the influence of adenosine, prostaglandins and endothelialderived relaxing factor (NO). The heart uses a variety of
substrates (non-esterified fatty acids, glucose, lactate, etc.)
depending on the nutritional state. Non-esterified fatty acids
form the main substrate, but the importance of glucose
increases during the fed state.
5.4.1. Effect of diet. The contractile function of the
heart is under autonomic control and there is no evidence
that myocardial function is sensitive to dietary factors under
conditions of adequate perfusion and O2 supply. However,
when O2 supply is limited due to atherosclerotic lesions in
coronary vessels or microvessel disease, the function may
become compromised. Oxidative metabolism of glucose
requires less O2 than that of non-esterified fatty acids to
produce ATP and may help to improve myocardial function
under those conditions. Relatively few experimental studies
have examined whether myocardial function is sensitive to
changes in the dietary fatty acid composition. Early studies
in rats have limited value as control diets were deficient in
essential fatty acids (Semafuko et al. 1984; Wince et al.
1984). However, a linoleic acid-rich diet may increase
contractile force in isolated papillary muscles and improve
the relationship between left ventricular work and filling
pressure in the isolated perfused rat heart (de Deckere & ten
Hoor, 1979). It should be noted that these preparations may
have a limited O2 supply and some of these effects may not
apply to the well-oxygenated heart. A reduction in basal and
a1-adrenoceptor-mediated peak left ventricular pressure by
a large intake of fish oil has been reported, but no effect was
observed in pigs. Yet, endothelium-derived relaxing factor,
which was reduced in some studies by feeding fish oil, can
reduce cardiac contractility (Mohan et al. 1994). Thus the
potential of n-3 and perhaps also of n-6 fatty acids to
influence cardiac contractility under conditions of limited
O2 supply or at high work loads can be envisaged. However,
without a clear mechanism and understanding of conflicting
reports it is too early to speculate.
5.5. Function of the ischaemic heart
Acute myocardial ischaemia occurs when coronary flow can
no longer meet the O2 requirements of the heart. Ischaemia
may be precipitated by an increased O2 demand due to severe
exercise or stress and/or acute vasoconstriction or thrombus in
the coronary vessel. Prolonged ischaemia results in acute MI
and necrosis of the muscle that is inadequately or not perfused.
The occurrence of serious ventricular arrhythmias very early
is the principal cause of sudden cardiac death. In a patient who
has survived this critical period, the loss of ventricular mass
may result in heart failure. Early restoration of blood flow
(dissolution of the thrombus) may prevent necrosis; however,
reperfusion may also stimulate the production of free radicals,
which may reduce the function and induce serious ventricular
arrhythmias (see Diplock et al. 1998).
5.5.1. Dietary fatty acids and arrhythmia. Diets rich in
linoleic acid protect against experimental ischaemiainduced arrhythmias (LepraВґn et al. 1981; McLennan et al.
1985; Riemersma et al. 1988). There is no consensus about
the mechanism that underlies the anti-arrhythmic effect of
such diets (Riemersma et al. 1988; Charnock, 1994). It is
assumed to be mediated by prostaglandins as a result of
changing phospholipid fatty acid composition. However,
the protective effect may not be abolished by non-steroidal
anti-inflammatory agents (LepraВґn et al. 1981; Sargent,
1990). Some, but not all, studies have also shown a
protection against the so-called ischaemic reperfusioninduced arrhythmias (in contrast to ischaemia-induced
arrhythmias discussed earlier) for reasons that are not
clear. It is important to point out that diets rich in linoleic
acid are low in saturated fatty acids. Thus, it is possible that
saturated fatty acids are pro-arrhythmic (Riemersma et al.
1988). However, fewer arrhythmias were also observed
when the control diet was rich in monoenoic fatty acids
(McLennan, 1993).
Large amounts of fish oil, in quantities that would be
difficult to consume in the context of a Western diet, protect
against the development of serious ventricular arrhythmias
in animal studies (McLennan et al. 1989, 1990; Sargent &
Riemersma, 1990). Conflicting reports have appeared as to
whether this protection extends itself to reperfusion-induced
arrhythmias. The underlying mechanism is assumed to be
due to increased PGI3 and reduced TxA2 production.
Alternatively, it may be due to a direct inhibition of the
voltage-sensitive NaГѕ channel by non-esterified EPA (Weylandt et al. 1996). It is worth emphasizing again that diets
rich in polyunsaturated fatty acids usually reduce the intake
of saturated fat.
Saturated fat intake is not reduced when animals receive
small fish-oil supplements (0.4 % energy). Long-term supplementation with small amounts of fish oil was not antiarrhythmic, yet the well-documented biochemical effects
(reduction of phospholipid arachidonate content and an
increase in the amounts of EPA and DHA) were observed
(Riemersma, 1995). Epidemiological and clinical data suggest that small amounts of fish oil may prevent CHD
mortality, reduce the risk of lethal events following MI
(Burr et al. 1989), and lower the chance of primary cardiac
arrest (related to serious ventricular arrhythmias) in the
community. It has been suggested that ALA as part of a
Mediterranean diet may reduce events after an acute MI (De
Lorgeril et al. 1994). This is an intriguing possibility
(McLennan & Dallimore, 1995), but requires confirmation
in a single-factor study. The convergence between experimental and human studies suggests that dietary fatty acid
composition may indeed reduce the risk of atherosclerosis
(via haemostatic and/or immunological effects) and lethal
coronary events by prevention of serious ventricular
6. Insulin resistance, obesity and
non-insulin-dependent diabetes mellitus
Both experimental data and epidemiological studies suggest
that abnormalities in lipid- and lipoprotein metabolism, as
well as the presence of hypertension, are associated with an
Cardiovascular system
increased cardiovascular risk. In addition, several other risk
indicators, for example insulin resistance, obesity and
NIDDM interfere with lipid metabolism and hypertension.
The relationship between insulin resistance and subsequent CHD morbidity and mortality is evident from several
epidemiological and a few prospective studies. In the
Helsinki Police Officers study (PyoВЁraВЁlaВЁ, 1979), for instance,
982 men with an age range of 30–59 years were followed
over a period of 10 years. In this study, high 1 and 2 h postglucose insulin levels were independent predictors of CHD
end-points, where fasting insulin was not an independent
contributor. The Paris Prospective Study (Fontbonne et al.
1991) examined the incidence of fatal and non-fatal CHD in
7038 males aged 43–54 years for a period of 15 years.
Analysis at 15 years showed that the 2 h post-load insulin
level was a significant, independent predictor of death from
6.1. Insulin resistance, cardiovascular disease and
cardiovascular risk factors
Insulin resistance may be defined as a diminution of the
biological response to a given concentration of insulin. It is
a heterogeneous syndrome with both genetic and environmental factors playing a determinant role in the development. Several factors proposed in this respect have been
reviewed by Despre`s & Marette (1994). These include
genetic factors, such as an excessive accumulation of
visceral fat, and circulating factors, such as free fatty
acids, sex-steroid hormones, tumour necrosis factor-a,
hyperinsulinaemia and hyperglycaemia, with main target
tissues muscle, adipose tissue and liver. In addition, the
morphology of skeletal muscle may itself contribute to the
insulin resistance syndrome, since it has been demonstrated
that the proportion of oxidative fibres (type I) and capillary
density are positively correlated with insulin action in vivo,
as determined by the hyperinsulinaemic euglycaemic clamp
technique (Lillioja et al. 1987). Environmental factors
associated with the insulin resistance syndrome include
lack of physical activity and intake of dietary fat (Despre`s
& Marette, 1994).
Large-scale, prospective epidemiological studies
revealed a clear association between insulin resistance and
CHD (PyoВЁraВЁlaВЁ, 1979; Fontbonne et al. 1991). Factors which
may contribute to this association are certainly the abnormalities in lipid metabolism and hypertension.
6.1.1. Lipid abnormalities and insulin resistance. There
is now abundant evidence for a relation between hyperinsulinaemia and/or insulin resistance and various lipid
abnormalities which are known risk factors for CHD and
other macrovascular complications. Results of the Helsinki
Heart Study (Manninen et al. 1992) and of the PROCAM
study (Assman & Schulte, 1992) both demonstrate that the
characteristic dyslipidaemia associated with an insulinresistant hyperinsulinaemic state is associated with a marked
increase in CHD risk. The alterations in lipid metabolism
commonly associated with insulin resistance have been
reviewed by Frayn (1993). The insulin resistant state is
characterized by elevated plasma triacylglycerol concentrations (particularly elevation of VLDL-triacylglycerol and
VLDL-apoB), a decreased plasma HDL-cholesterol
concentration (especially HDL2 -cholesterol) and the presence of small, dense LDL particles. The presence of this
dense LDL phenotype may result from an overproduction of
apoB, induced by an increased availability of free fatty acids
(Sniderman et al. 1992). Although cause and effect have
never been properly elucidated, it seems that hyperinsulinaemia causes dyslipidaemia, because correction of the
dyslipidaemic state leaves the insulin resistance unchanged,
whereas correction of the insulin resistance by weight
reduction, increased physical activity, and a low-fat
diet is immediately followed by the normalization of
In most studies concerning the relationship between
lipoprotein metabolism and the risk of CHD, only fasting
blood lipids and lipoproteins were considered. However, as
proposed by Zilversmit (1979) almost 20 years ago and
more recently by Patsch (1987), postprandial hyperlipaemia
may be particularly atherogenic, especially since a major
part of lifetime is spent in the period between food ingestion
and 6–8 h thereafter. The magnitude of postprandial lipaemia differs substantially among individuals, including those
considered to be normolipidaemic on the basis of fasting
blood lipid values (Patsch, 1987). Factors which have been
reported to influence postprandial lipaemia include basal
triacylglycerol and HDL-cholesterol concentrations
(O’Meara et al. 1992), obesity (Lewis et al. 1990; Potts et
al. 1995), diabetes mellitus (Stinson et al. 1993) and insulin
resistance (Frayn, 1993).
Lewis et al. (1990) demonstrated that even normolipidaemic obese subjects have greater postprandial lipaemia and
triacylglycerol enrichment of HDL after ingestion of a highfat meal. Potts et al. (1995) concluded that a disturbed
triacylglycerol clearance in subcutaneous adipose tissue is
related to elevated plasma triacylglycerol concentrations
and reduced HDL-cholesterol levels. Roust & Jensen
(1993) investigated postprandial free fatty acid kinetics in
obese persons and concluded that impaired suppression of
adipose tissue lipolysis is a potentially important abnormality present in upper body obesity. Since obesity, and
particularly abdominal obesity, is frequently associated
with insulin resistance, it could be expected that insulin
resistance also might interfere with the magnitude of postprandial lipaemia.
The effects of insulin on lipid metabolism occur both in
adipose tissue and the liver. Insulin mediates the activation
of lipoprotein lipase in adipose tissue; the consequence of a
disruption at this level will result directly in an impaired
postprandial triacylglycerol clearance (Frayn, 1993). Other
actions of insulin comprise the suppression of non-esterified
fatty acid release in adipose tissue by inactivation of
the hormone-sensitive lipase (EC and increased
re-esterification, and the suppression of hepatic secretion
of VLDL-triacylglycerol in the liver. The latter may lead to
inappropriate postprandial VLDL-triacylglycerol secretion
and the presence of large triacylglycerol-enriched VLDL in
the postprandial period. As a consequence, neutral lipid
exchange with LDL may lead to small, dense LDL particle
formation (Frayn, 1993).
These observations show a clear relationship between
insulin resistance and certain disturbances in lipoprotein
metabolism which contribute to the development of
G. Hornstra et al.
cardiovascular diseases. However, to ascertain a causal
relationship, future research has to concentrate on intervention studies which may elucidate the sequence of events.
6.1.2. Hypertension and insulin resistance. Arterial
hypertension is an established risk factor for CHD. Several
large studies have already reported on the relationship
between insulin resistance and hypertension. Some authors
described a positive relationship between insulin concentrations and blood pressure (Welborn et al. 1966; Lucas et al.
1985; Modan et al. 1985) but others were unable to
demonstrate such a relationship (Cambien, 1987; Asch et al.
1991). The reason for this discrepancy may be found in
ethnicity (Saad et al. 1991) and in the presence of obesity
(Cambien, 1987). On the basis of published reports, at least
half of the patients with hypertension can be considered to
have insulin resistance and hyperinsulinaemia (Reaven et al.
The possible mechanisms underlying a relationship
between insulin and blood pressure are complex and can
be divided into direct and indirect effects. Hypertension
may result directly from insulin resistance through the
stimulatory effect of high insulin concentrations on vascular
smooth muscle proliferation (Banskota et al. 1989). Insulin
also enhances renal Na retention directly via its effects on
the proximal tubuli (DeFronzo, 1981) and indirectly through
stimulation of the sympathetic nervous system and augmentation of angiotensin II-induced aldosterone secretion (Rocchini et al. 1990). Stimulation of the sympathetic nervous
system by insulin may also have a direct hypertensive effect
(Landsberg & Krieger, 1989).
Hypertension frequently occurs in combination with
other metabolic alterations such as disturbances in lipid
metabolism, obesity and NIDDM. Since insulin resistance
seems to be the common link between these factors, the nonpharmacological treatment approach should focus on the
increase of insulin sensitivity. Effective tools are weight
reduction, increased physical activity, low-fat diet and
perhaps consumption of foods that reduce the insulinaemic
response. This strategy probably results in a lowering of the
blood pressure as well.
6.2. Nutritional aspects
Although insulin resistance also occurs in persons with
normal body weight, it is a common feature in obese
patients with or without impaired glucose tolerance or
NIDDM. In this specific patient group, diet and exercise
are two common, non-pharmacological approaches for
treatment. Considering the aim of the present review, only
relevant dietary factors will be discussed.
In obese, insulin-resistant patients, several studies have
examined the effect of overall weight loss, by diet or a
diet Гѕ exercise combination, on cardiovascular risk factors
such as lipid and lipoprotein abnormalities and hypertension. From the results, it is clear that weight loss is
accompanied by improved insulin sensitivity and a subsequent better metabolic profile (Colman et al. 1995).
Whether specific dietary components may influence the
status of insulin resistance in obese and non-obese persons
is not fully understood.
The relationship between dietary factors and physical
activity with hyperinsulinaemia was examined in 389 nondiabetic men, 70–89 years of age, who participated in the
Zutphen Elderly Study (Feskens et al. 1994). A significant,
negative association was observed between insulin levels
(during an oral glucose tolerance test) and the intake of
dietary fibre and polyunsaturated fatty acids, which could
not be accounted for by energy intake, BMI, physical
activity, prescribed diets or the presence of CHD. In contrast, insulin levels increased with the increasing intake of
saturated fatty acids and alcohol. Apart from overweight,
physical activity and dietary factors such as the intake of
fatty acids, fibre, carbohydrates and alcohol, were independently associated with hyperinsulinaemia and insulin
In a study with 544 non-diabetic women (aged 30–84
years), the habitual intake of total dietary fat was positively
related to fasting insulin concentrations, particularly among
sedentary women. The positive relation of dietary fat content with the percentage of body fat accounted for a
substantial proportion (П®30 %) of the association of dietary
fat with insulin concentrations (Mayer et al. 1993).
In a group of male Swedish elite athletes, diet modification during 1 year resulted in decreased insulin levels in
conjunction with a decreased relative fat energy content.
Insulin levels returned to baseline amounts when the relative
fat energy content increased again (Tegelman et al. 1996).
Information about the effect of specific fatty acids on
insulin metabolism is scarce. The incorporation of n-3 fatty
acids, and of DHA (22 : 6n-3) in particular, into phospholipids, prevents the expected insulin resistance in rats fed on
a high-fat diet (Storlien et al. 1991). In human subjects,
decreased insulin sensitivity is associated with decreased
concentrations of certain long-chain polyunsaturated fatty
acids (20 : 4n-6, 22 :4n-6, 22 : 5n-6, and 22 : 5n-3) in skeletal
muscle phospholipids (Borkman et al. 1993). Specifically,
decreases in C20–C22 long-chain polyunsaturated fatty
acids were associated with increased insulin resistance.
This raises the possibility that changes in the fatty acid
composition of muscle modulate the action of insulin. The
results of the study of Borkman et al. (1993) demonstrate
that in patients with coronary artery disease, linoleic acid
(18 : 2n-6) correlated directly with hyperinsulinaemia, but
this was not the case in normal controls. Since insulin has an
effect on D6 -desaturation, the conversion of linoleic acid to
g-linolenic acid (18 : 3n-6) may be impaired. In addition,
Pan et al. (1995) demonstrated that an impaired insulin
action and obesity are independently associated with
reduced D5 -desaturase activity. In these circumstances,
the direct supply of g-linolenic acid (impairment of D6 desaturase) or arachidonic acid (impairment of D5 -desaturase) may be of value (Horrobin, 1993). Obesity was also
found to be associated with reduced elongase activity and
higher D9 -desaturase activity (Pan et al. 1995). Trans fatty
acids interfere with desaturation and elongation of 18 : 2n-6
and 18 : 3n-3 (ALA), thereby further contributing to
decreases in C20–C22 long-chain polyunsaturated fatty
acids. A decrease in C20–C22 polyunsaturates leads to
increased fatty acid synthesis, lipogenesis, insulin resistance
and hyperinsulinaemia, with the subsequent development
of obesity, hypertension, NIDDM and CHD (OstlundLindqvist et al. 1985; Simopoulos, 1994).
Cardiovascular system
Further investigations are needed to evaluate if the
essential fatty acids linoleic acid and ALA influence insulin
resistance and, if so, whether this effect requires desaturation and elongation of these fatty acids.
As was already demonstrated in a few studies, postprandial triacylglycerol concentrations correlate with the degree
of hyperinsulinaemia and/or insulin resistance, at least in
obese persons. The influence of dietary factors on postprandial lipaemia was investigated by Jeppesen et al.
(1995). The acute effects of varying amounts of fat and
fructose were studied in eleven healthy, non-diabetic subjects with a wide range of plasma triacylglycerol concentrations. Increasing the dietary intake of fat from 5 to 40 to 80 g
led to a significant increase in postprandial concentrations of
both triacylglycerol and retinyl palmitate. Furthermore,
adding 50 g fructose to 5 g fat also led to a significant
increase in postprandial concentrations of triacylglycerol
and retinyl palmitate. Chronic intake of fish oil (64 mg n-3
fatty acids/kg body weight per d) reduced postprandial
lipaemia in eight normolipidaemic volunteers. This effect
was not due to increased chylomicron clearance but more
probably to reduced chylomicron production or secretion
(Harris & Muzio, 1993). Further research is needed to
elucidate whether these effects are reflected in changes in
insulin sensitivity in these persons. The results suggest that
it may be possible to modulate insulin sensitivity and
subsequent cardiovascular risk factors by diet. However,
further research on mechanisms is unavoidable to determine
the real functional component in the diet.
7. Hyperhomocysteinaemia and cardiovascular risk
7.1. Causes of hyperhomocysteinaemia
Although there is some evidence that increased plasma
homocysteine levels are caused by a massive export of
homocysteine from tissues into plasma (Guttormsen et al.
1996), it is usually thought to result from impaired elimination of plasma homocysteine, either by a defective methylation to methionine or by a reduced trans-sulfuration to
cystathionine and cystathione. Betaine (an oxidation product of choline) and folic acid (in the form of 5-methyltetrahydrofolate) are the methyl donors for the
transmethylation of homocysteine. 5-Methyltetrahydrofolate is obtained from the reduction of 5,10-methylenetetrahydrofolate which is catalysed by the enzyme 5,10methylenetetrahydrofolate reductase (EC;
MTHFR). In this reaction, methylcobalamin, derived from
vitamin B12 , serves as a cofactor. A deficiency or reduced
activity of MTHFR results in increased plasma homocysteine levels. Such a reduced MTHFR activity has been
shown to result from a series of mutations in the gene
coding for this enzyme (Frosst et al. 1995; Goyette et al.
Transfer of a methyl group from betaine to homocysteine
requires the active enzyme betaine : homocysteine methyltransferase (EC, whereas pyridoxal-50 -phosphate, a
form of vitamin B6 , is required as a cofactor. A reduction in
this pathway of homocysteine methylation has not been
reported so far (Dudman et al. 1996).
Because folic acid is an important methyl donor for the
methylation of homocysteine, methylcobalamin (derived
from vitamin B12 ) is the necessary coenzyme in this reaction
and B6 -derived pyridoxal-50 -phosphate is required for
homocysteine removal by trans-sulfuration, folate deficiency and/or a poor status of the vitamins B6 or B12 may
be a nutritional reason for hyperhomocysteinaemia.
7.2. Athero-thrombotic mechanisms of
7.2.1. Interaction with lipoproteins. In the chemical
pathology of atherosclerosis, homocysteine is thought to
play an important role, because the free amino groups of
LDL can be thiolated by homocysteine thiolactone, causing
aggregation and increased uptake of LDL by macrophages,
explaining lipid deposition in atheromas. Homocysteine
thiolactone, released from homocysteinylated LDL within
the vascular wall, promotes intimal injury, oxidation of
cholesterol and unsaturated lipids, platelet aggregation,
thrombogenic factors, myointimal hyperplasia, deposition
of sulfated glycosaminoglycans, fibrosis and calcification of
atherosclerotic plaques (McCully, 1993). It has also been
suggested (Harpel & Borth, 1992) that homocysteine
increases the atherogenic and antifibrinolytic potential of
7.2.2. Smooth-muscle cell proliferation. Homocysteine
has been shown to stimulate vascular SMC proliferation, a
hallmark of arteriosclerosis, possibly by increasing the
transcription rate of cyclin A (Tsai et al. 1996).
7.2.3. Endothelial functions. In cell culture studies, it
was demonstrated that homocysteine significantly lowers
endothelial cell growth (Tsai et al. 1994). Moreover, as
demonstrated by Dudman et al. (1991) in in-vitro studies,
homocysteine causes endothelial detachment, but since
fibronectin greatly diminished this process, it was considered of limited relevance to atherogenesis in hyperhomocysteinaemia.
It has been suggested that high plasma homocysteine
levels cause endothelial injury, largely as a consequence of
facilitating the generation of H2 O2 from O2 (Stamler &
Loscalzo, 1992; Jones et al. 1994; Hultberg et al. 1995),
although the evidence is not unanimous (Clarke et al. 1992).
H2 O2 , in turn, is presumed to induce dysfunction and
damage to the endothelial cell resulting in platelet activation, coagulation and reduced fibrinolysis.
Further studies by Stamler et al. (1993) indicate that
normal endothelium modulates the potential adverse effects
of homocysteine by releasing NO and forming the adduct
S-NO-homocysteine. The adverse vascular properties of
homocysteine may result from an inability to sustain
S-NO-homocysteine formation owing to an imbalance
between the production of NO by progressively dysfunctional endothelial cells and the levels of homocysteine.
There is no evidence that homocysteine inhibits the
formation of endothelial prostacyclin (Wang et al. 1993).
7.2.4. Functions of blood platelets. As summarized by
Stamler & Slivka (1996), the effect of homocysteine on
platelet function and survival is controversial. Thus, the
shortened survival as measured by Harker et al. (1974) in
homocysteinuria patients could not be reproduced by others
(Uhleman et al. 1976; Hill-Zobel et al. 1982).
G. Hornstra et al.
Homocysteine has been shown to inhibit the ecto-ADPase
activity of human umbilical vein endothelial cells. Because
ADP is a potent platelet aggregatory agent, this action of
homocysteine may enhance platelet aggregability (Harpel
et al. 1996).
7.2.5. Coagulation and natural anticoagulants. Hyperhomocysteinaemia has been associated with a consumption
coagulopathy, resulting in reduced amounts of clotting
factor VIIc and AT-III. However, there is some evidence
that deficient synthesis of these substances is involved,
which is normalized on treatment with pyridoxine plus
folate (Schienle et al. 1994).
In male CHD patients, homocysteine levels were significantly correlated with fibrinogen content and plasma viscosity (von Eckardstein et al. 1994). Within the patient group of
this study, both fibrinogen and homocysteine contents significantly increased in parallel with the number of stenosed
coronary vessels.
Homocysteine was shown to induce tissue factor procoagulant activity in cultured human endothelial cells in a
time- and concentration-dependent manner by tissue factor
gene transcription (Fryer et al. 1993).
In monkeys, Lentz et al. (1996) demonstrated that dietinduced hyperhomocysteinaemia is associated with significantly decreased thrombomodulin anticoagulant activity. In
contrast, van den Berg et al. (1995) noted increased plasma
levels of thrombomodulin in young patients with peripheral
arterial occlusive disease and hyperhomocysteinaemia after
methionine loading. Since the thrombomodulin levels
decreased on treatment with pyridoxine plus folic acid, in
this patient group hyperhomocysteinaemia appears to be
associated with enhanced thrombomodulin levels. Together
with the increased levels of vWf, this is considered by the
authors to be a marker of endothelial dysfunction.
Homocysteine inhibits the expression and activity of
endothelial cell surface thrombomodulin, the thrombin
cofactor responsible for activation of a natural anticoagulant, protein C (Harpel et al. 1996). Although Rodgers
& Conn (1990) demonstrated homocysteine to reduce protein C activation by endothelial cells in vivo, this finding
could not be confirmed by others (Bienvenu et al. 1993;
Aronson et al. 1994). Homocysteine also lowers expression
of the natural anticoagulant heparan sulfate proteoglycan on
the surface of porcine aortic endothelial cells in culture, as
reflected by the reduced binding of another natural anticoagulant, AT-III (Nishinaga et al. 1993).
Increased coagulation and fibrinolysis in hyperhomocysteinaemia in vivo has recently been substantiated by
increased concentrations of thrombin–antithrombin complexes and D-dimers (Hamano et al. 1996).
7.2.6. Fibrinolysis. In stroke patients, plasma homocysteine levels appeared significantly related to the
concentrations of tPA, but not to PAI-1 (Lindgren et al.
1996). Similar results had also been found by Bienvenue
et al. (1993) in fifty patients with arterial and venous
thrombosis. These authors failed to demonstrate a significant relationship between plasma homocysteine and plasminogen levels. Van den Berg et al. (1995) found normal
tPA levels in young patients with peripheral arterial
occlusive disease and hyperhomocysteinaemia after methionine loading. Since homocysteine has been shown to inhibit
the binding of tPA to endothelial cells in culture (Hajjar,
1993), it may interfere with the fibrinolytic properties of the
endothelial surface.
7.2.7. Altered gene expression. By using a modified
non-radioactive differential display analysis to evaluate
gene expression in cultured human umbilical vein endothelial cells, Kokame et al. (1996) demonstrated that
homocysteine can alter the expressivity of multiple genes,
including a stress protein, which may contribute to
7.3. Hyperhomocysteinaemia or B-vitamin status of
primary importance in cardiovascular risk?
In many studies relating hyperhomocysteinaemia to cardiovascular risk, increased plasma levels of homocysteine are
associated with reduced amounts of folic acid, vitamin B12
(cobalamin), vitamin B6 and/or pyridoxal-50 -phosphate
(Ubbink et al. 1993; Jacobsen et al. 1994; Pancharuniti
et al. 1994; Dalery et al. 1995; Robinson et al. 1995, 1996;
Chasan-Taber et al. 1996; Petri, 1996). In addition, cobalamin-deficient patients usually have increased plasma levels
of homocysteine (Stabler et al. 1990). This implies that a
relative deficiency of these B-complex vitamins rather than
the high homocysteine plasma levels may be the actual
cardiovascular risk factor (Chasan-Taber et al. 1996). Findings by Schmitz et al. (1996) that homozygosity for the
C677T mutation, associated with increased plasma homocysteine level, is not associated with increased risk of MI,
irrespective of folate intake, support this contention. Comparable results were obtained by Ma et al. (1996), who
suggest that a gene–environment interaction might increase
the risk by further elevating plasma homocysteine, especially when folate intake is low. Further evidence for a
primary role of folate deficiency in cardiovascular risk
comes from studies by Selhub and co-workers (Selhub
et al. 1995; Selhub, 1996) who demonstrated that plasma
concentrations of folate and pyridoxal-50 -phosphate as well
as folate intake were inversely related to extracranial carotid
stenosis after adjustment for other known risk factors. In a
group of 367 elderly patients undergoing coronary angiography, Herzlich et al. (1996) observed no significant trend
in change in homocysteine as the extent of coronary artery
disease increased. However, a low vitamin B12 status was
shown to be associated with a lower left ventricular ejection
fraction, suggesting a primary role for the cobalamin status
in determining left ventricular function. Verhoef et al.
(1996) also demonstrated that plasma levels of vitamin B6
and folate (but not of vitamin B12 ) were inversely associated
with the risk of MI, independently of other potential risk
factors. From their studies, Robinson et al. (1995) conclude that low pyridoxal-50 -phosphate confers an independent risk for coronary artery disease and Ellis &
McCully (1995) observed that the treatment of patients
with carpal tunnel syndrome and related disorders with
vitamin B6 was associated with only 27 % of the risk of
developing cardiac chest pain or MI compared with
patients who had not taken vitamin B6 . Dalery et al.
(1995), however, did not observe differences for folate,
vitamin B12 or total vitamin B6 between CHD patients and
Cardiovascular system
7.4. Dietary B-vitamins lower plasma homocysteine
In numerous studies, increased plasma homocysteine levels
appear to be associated with reduced plasma folate concentrations (Verhoef et al. 1996) and since folate is the main
methyl donor in the conversion of homocysteine to methionine, folate supplementation may be the preferred way to
lower homocysteine-mediated cardiovascular risk. In their
meta-analysis Boushey et al. (1995) calculated that an
additional intake of 200 mg folate/d would reduce the
plasma homocysteine content by about 4 mmol/l and that
by increasing dietary folate in the USA 13 500–50 000 CHD
deaths per year could be avoided. As suggested by Jacques
et al. (1996), individuals carrying a gene mutation resulting
in expression of a sub-normal activity of the homocysteine
transmethylation enzyme MTHFR may have a higher folate
requirement. Consequently, this population may certainly
require folate supplementation to prevent hyperhomocysteinaemia.
Van den Berg et al. (1995) demonstrated, in young
patients suffering from peripheral arterial occlusive disease
and hyperhomocysteinaemia after methionine loading, that
treatment with pyridoxine plus folic acid resulted in normalization of homocysteine metabolism and ameliorated
endothelial dysfunction as reflected by a change towards
normal circulating levels of vWf and thrombomodulin.
Schienle et al. (1994) reported a case study, demonstrating
that in a patient with homocystinuria due to cystathionine-bsynthase deficiency and thromboembolic disease, treatment
with pyridoxine plus folate not only led to normalization of
amino acids in urine and plasma and of plasma levels of
plasma coagulation and anti-coagulation factors, but also
prevented further thromboembolic episodes.
8. Critical assessment of the science base
8.1. Identification of criteria
In this section, the science base presented previously will be
critically evaluated, using the following criteria of decreasing importance (except criterion 5).
Criterion 1. Plausible and validated evidence does exist for
the involvement of the various variables investigated as
(anti)risk factors or -indicators in the aetiology of CHD. For
risk factors a causal involvement in CHD aetiology should
have been proven; for risk indicators such a causality is not
Criterion 2. Well-designed human intervention studies
have been published, demonstrating that higher intakes (or
body levels) of the food items considered lower the levels of
the identified risk factors or indicators.
Criterion 3. Prospective, statistically validated epidemiological evidence is available that links higher intakes (or
body levels) of these food items to reduced levels of the risk
factors or indicators.
Criterion 4. Retrospective epidemiological data are present
which demonstrate a statistically validated association
between intake or body levels of the food items investigated
and the levels of the risk factors or indicators identified
under criterion 1.
Criterion 5. Clear evidence exists for the safety of the food
items considered.
8.2. Evaluation of the present knowledge base with respect
to food functionality
8.2.1. Plasma lipoproteins. Results from well-designed
intervention trials clearly demonstrate that the plasma
concentration of LDL-cholesterol is a causal risk factor
for CHD. Most probably, plasma HDL-cholesterol concentration is an anti-risk factor, but confirmation still depends
on results of intervention studies showing that an isolated
increase in the plasma HDL-cholesterol concentration
significantly lowers the risk of CHD. Evidence for plasma
VLDL or triacylglycerol levels being associated with the
risk of CHD is mainly based on epidemiological studies.
Therefore, the plasma VLDL or triacylglycerol concentration can only be considered a risk marker for CHD. The
same holds for the plasma concentration of Lp(a), which has
been demonstrated to be a powerful risk marker in
epidemiological studies.
It should be mentioned that so far the lipoprotein profile
has mainly been investigated in blood sampled under fasting
conditions, whereas man is usually in a state of postprandial
hyperlipidaemia for at least 8 of every 24 h. Since the
importance of the postprandial lipoprotein profile for determining the risk of CHD has only been superficially investigated, it will not be emphasized here.
Taking these considerations into account, dietary saturated fatty acids can be classified as CHD-risk-promoting
nutrients because, as compared with carbohydrates, they
increase plasma LDL-cholesterol concentrations more
strongly than plasma HDL-cholesterol levels (chain
lengths up to sixteen C atoms) or reduce the plasma HDLcholesterol concentration (stearic acid, 18 : 0), even if they
seem to lower slightly the plasma Lp(a) concentration.
Dietary trans-monounsaturated fatty acids increase LDLand reduce HDL-cholesterol levels in plasma; moreover
they increase the plasma Lp(a) concentration. Foods low in
saturated and trans fatty acids and high in linoleic acid and
ALA can, therefore, be classified as functional with respect
to lowering the lipoprotein-associated risk of CHD.
The cis-unsaturated fatty acids oleic acid, linoleic acid
and ALA reduce the plasma concentration of LDL-cholesterol, whereas they hardly affect plasma HDL-cholesterol
and Lp(a) concentrations. Therefore, foods enriched in these
unsaturated fatty acids can be classified as functional in
reducing CHD risk. Oils rich in the highly unsaturated fatty
acids EPA and DHA have consistently been shown to lower
plasma VLDL concentrations and may, therefore, reduce
CHD risk. However, in certain population groups they
increase the plasma LDL-cholesterol concentration. So,
with respect to lipoprotein effects, foods enriched in EPA
and/or DHA cannot be classified as functional in reducing
CHD risk by virtue of their effect on the plasma lipoprotein
Dietary soluble fibre and certain phytosterols can be
classified as functional in lowering CHD risk, because
they improve the plasma lipoprotein profile. Although
ethanol and a number of fat replacers have similar effects,
their side-effects may hamper their use as functional foods.
Insufficient evidence is available with respect to soyabean
protein preparations, garlic, inulin and oligofructose.
Finally, the available evidence does not support the
G. Hornstra et al.
classification of mono- and disaccharides, resistant starch,
fermented milk products, tocopherols and tocotrienols as
functional food components.
8.2.2. Arterial thrombosis. In Western societies with
ageing populations the modulation of thrombosis tendency
is likely to become an important approach to the prevention
of CHD. The main problem today, however, is the lack of
reliable variables to measure the prothrombotic state in
human subjects. In addition, there is a considerable lack of
indicators reliably reflecting thrombotic risk in man. So far
it has not been shown that changes found in platelet function
measured in vitro significantly predict changes in thrombosis tendency in vivo. The plasma levels of factors involved
in coagulation and fibrinolysis do not necessarily reflect the
degree to which these phenomena really occur. Similarly,
the predictive value of endothelial cell function for CHD has
been insufficiently evaluated.
Diet, especially dietary fatty acids, has been shown to
affect many of the previously mentioned variables, but the
mechanisms involved are largely unknown. Consequently,
increasing mechanistic knowledge about the influence of
dietary factors on platelet, leucocyte and endothelial functions and on coagulation and fibrinolysis in vivo, is required
for improving dietary strategies to control the prothrombotic
state. According to current knowledge, long-chain n-3 and
n-6 fatty acids are particularly able to modulate both
endothelial cell and platelet functions. However, the optimal
n-6 : n-3 fatty acid ratio and the effect of these fatty acids on
the antioxidant status of the body is not clear. The same
holds for the mechanisms by which platelet and/or leucocyte
fatty acid composition affect coagulation and fibrinolysis in
man. Also the role of dietary factors as regulators of the
interaction between different cell types involved in thrombogenesis has been insufficiently studied so far. Because of
all these uncertainties, there is no solid evidence for any
food item to be considered �functional’ with respect to
lowering platelet and endothelial functions, coagulation
and fibrinolysis.
8.2.3. Immunological interactions. The immune system
responses in the cardiovascular system cannot be considered
risk factors with respect to the atherosclerotic process,
because of a lack of evidence for the causal involvement of
these responses in atherogenesis. Therefore, the term risk
indicators should be used.
Although various studies have shown that a high intake of
n-3 fatty acid-rich foods (fish), or of n-3-rich preparations
(fish oils) may exert antiatherosclerotic activities, there is no
direct evidence that these effects are mediated by modifications of immune responses participating in the atherogenic
process. Some indirect evidence may be provided by the
results of some, but not all, studies showing favourable
effects of n-3 intake on the rate of re-stenosis of dilated
coronary arteries (for reviews, see Gapinski et al. 1993 and
Cairns et al. 1996), a process which appears to involve cells
of the immune system and the proliferation of cells of the
arterial walls (Westerband et al. 1997). Additional studies
are required to substantiate the effects of n-3 fatty acids on
re-stenosis following angioplasty. However, these results
may not disclose the mechanism(s) of n-3 activities.
Diets rich in antioxidants have been shown to exert
protective effects with respect to the atherogenic process.
They have also been shown to affect the activities of
immune competent cells and to inhibit the expression of
genes coding for cell–cell adhesion molecules, which play a
role in the development of the arterial lesions. As for the n-3
long-chain polyenes, however, there are no statistically
validated epidemiological, prospective, or intervention
data indicating that these effects may be mediated by
modulation of immune system responses.
8.2.4. Hypertension. CHD is strongly related to both
systolic and diastolic blood pressure in a graded fashion and
treatment of hypertension results in a reduction in coronary
disease-related events. Therefore, hypertension is a risk
factor for coronary artery disease.
Reports on the blood pressure-reducing effect of linoleic
acid are inconsistent. With respect to n-3 long-chain polyenes meta-analyses suggest that these fatty acids may reduce
blood pressure in hypertensive, but not in normotensive,
subjects. Consequently, n-3 long-chain polyenes may be
considered �functional’ with respect to reducing increased
blood pressure. Since it is not known whether these fatty
acids will prevent normotensive people from becoming
hypertensive, blood pressure-related functionality of these
fatty acids is restricted to hypertensive subjects. A diet rich
in fruit and vegetables also helps to lower blood pressure;
however, the mechanism involved has not yet been
The potential of n-3 and perhaps also of n-6 fatty acids to
influence cardiac contractility under conditions of limited
O2 supply or at high work loads can be envisaged, but
evidence is available from in vitro and animal studies only,
results are not consistent and mechanisms involved remain
controversial. The same holds for the reported preventive or
reducing effects of n-6 and n-3 fatty acids on arrhythmia:
the data are largely based on animal studies and underlying
mechanisms are hardly known. Therefore, insufficient evidence is available for the classification of unsaturated fatty
acids as functional with respect to cardiac contractility and
prevention of arrhythmia.
8.2.5. Insulin resistance. Although several excellent
studies are available, demonstrating a link between insulin
resistance, obesity, NIDDM, metabolic abnormalities and
coronary artery disease, cause-and-effect relationships have
not been proven by statistical means. Consequently, these
conditions can only be regarded as risk indicators, not risk
From epidemiological studies it is suggested that the
intake of dietary fibre (positively) and the intake of dietary
fat (negatively) affect insulin sensitivity. However, welldesigned intervention trials of sufficient size and duration
concerning the effect of either of these dietary components
on insulin sensitivity have not yet been performed. The
relatively short-term and mostly small studies that have
been reported were largely carried out in obese subjects in
which many physiological variables (e.g. general food
habits, body weight, insulin sensitivity, blood pressure)
are different from normal-weight subjects.
There are some data from intervention studies about
the effect of specific fatty acids on insulin metabolism.
However, mechanisms underlying these associations
have not yet been elucidated. Further intervention studies
will be important in determining the sequence of events.
Cardiovascular system
In these studies, the use of stable isotopes will be
Taken together, there is insufficient evidence to classify
any of the food items �functional’ with respect to insulin
resistance and related conditions.
8.2.6. Hyperhomocysteinaemia. The view that the
level of homocysteine is a risk factor for cardiovascular
disease is exclusively based on epidemiological investigations, most of which were case–control studies. In-vitro
studies with, mainly, endothelial cell cultures clearly
demonstrate an endothelium-activating effect of homocysteine, possibly resulting in thrombogenic conditions.
However, in vivo data to confirm this thrombogenic
potential of plasma homocysteine are not available as yet.
Because of the rather consistent inverse relationship
between plasma levels of homocysteine and of folate,
vitamin B12 and/or vitamin B6 , no final answer can be given
to the question of whether hyperhomocysteinaemia or a
reduced vitamin status is ultimately associated with an
increased cardiovascular risk. Since no well-designed
intervention studies have been reported showing that
reducing hyperhomocysteinaemia or increasing the folate
and/or B-vitamin status causes a reduction in cardiovascular
risk, plasma homocysteine, folate, vitamin B6 and vitamin
B12 levels can be considered (anti)risk indicators at best.
Increasing the consumption of folate and/or vitamins B12
or B6 lowers plasma homocysteine quite consistently, but
whether this will result in a reduced cardiovascular risk
remains to be proven.
In principle, improvement of the folic acid status by
dietary folate supplementation may mask or even precipitate
clinical manifestations related to vitamin B12 deficiency.
However, extensive studies in more than 700 elderly participants in the Framingham Heart Study revealed that the
benefit of folate fortification through projected decreases in
homocysteine level and heart disease risk greatly outweigh
this risk (Tucker et al. 1996). Moreover, concerns about
masking cobalamin deficiency by folic acid supplementation could be lessened by adding cobalamin to folic acid
supplements (Boushey et al. 1995).
9. Conclusions and recommendations for further
9.1. Plasma lipoproteins
Dietary lipids are able to affect lipoprotein metabolism in a
significant way, thereby modifying the risk of cardiovascular disease. Although effects of the individual dietary fatty
acids and dietary cholesterol on fasting serum lipids and
lipoproteins have been studied extensively, possible interactions among fatty acids or with dietary cholesterol, as well
as postprandial effects, are only poorly understood. This
should be investigated more thoroughly in well-controlled
dietary trials, using recently developed techniques. For
example, stable-isotope methodology should be used to
measure apoprotein metabolism, or to measure in mononuclear cells mRNA levels of the LDL receptor and of
hydroxymethylglutaryl CoA reductase. Also, effects on
other lipid variables like, for example, cholesterol ester
transfer protein-activity and lipoprotein particle sizes,
should then be taken into account so as to increase our
understanding of the dietary effects on lipoprotein metabolism. In addition, special attention should be paid to
(potential) gene–diet interactions. These remarks, of
course, also apply to other dietary components that interfere
with cholesterol absorption.
9.2. Arterial thrombosis
Platelet function may possibly affect cardiovascular risk and
the relatively low platelet content of n-3 polyunsaturated
fatty acids may present a risk for platelet hyperactivity.
Insufficient evidence is available to reliably link endothelial
cell function to cardiovascular risk. Increased blood coagulability and reduced fibrinolytic activity are associated with
increased risk for cardiovascular disease, but causality has
not been proven and, consequently, �functional foods’
cannot be identified.
Further research is needed in the following areas.
(1) Prospective validation studies should be performed to
find out to what extent the presently available putative
indicators of arterial thrombosis tendency (i.e. platelet
aggregation in vitro, urinary excretion of thromboxaneand prostacyclin metabolites and of specific platelet
proteins, plasma concentrations of soluble forms of cell
adhesion molecules, activation fragments of clotting
factors, and fibrin degradation products) reflect the
risk for arterial thrombosis.
(2) Depending on the results, it may be necessary to
develop and validate new methods to measure in vivo
arterial thrombosis tendency in human subjects and to
search for and prospectively validate more specific in
vivo activation markers for platelets, endothelial cells,
leucocytes, clotting factors and the fibrinolytic process.
(3) Well-designed intervention studies should be initiated
to investigate the effect of selected dietary components
(e.g. the various n-3 and n-6 fatty acids and their
combination, antioxidants, fibre) on the processes participating in arterial thrombus formation. These studies
should not only measure effects, but should also try and
unravel the mechanisms involved.
9.3. Immunological interactions
Long-chain polyenes of the n-3 family and antioxidants are
examples of food components endowed with various biological activities, which can be assessed in in vitro and in
ex vivo experiments. These activities include modification
of immune system responses of cells participating in
atherogenesis, which may thus be considered markers of
an active state of this process. Certain foods are rich in n-3
fatty acids (e.g. fish rich in the n-3 long-chain polyenes and
some vegetable oils, such as soyabean and low-erucic acid
rapeseed, rich in ALA). Other foods (e.g. vegetables and
vegetable oils, fruits) are rich in various types of antioxidants (vitamins, flavonoids, polyphenols, etc.). Diets
based on high intakes of these foods are, therefore, expected
to exert beneficial health effects on the atherosclerotic
process, as shown by various studies. However, the variable
contents, from both a quantitative and qualitative point of
G. Hornstra et al.
view, of these bioactive components in these foods make it
difficult to define them as �functional foods’. In addition,
although beneficial effects, for instance on the cardiovascular system, have been shown in human studies, there is
little evidence, from statistically validated epidemiological,
prospective, or intervention studies, that these effects may
be mediated by modulation of immune system responses.
As to the safety of high intakes of foods rich in n-3 longchain polyenes, the absence of detrimental effects, except
for possible minor intestinal dysfunctions, in the reported
studies, indicates that they should be considered safe. The
same holds for foods rich in antioxidants, since there is no
evidence that a high consumption of these foods results in
detrimental effects.
We are at an early stage of examining the role of immune
function on the development of atherosclerotic plaques and
there is a great need to develop strategies for studying the
effects of macro- and micronutrients on the function of the
immune system. These should take place at different levels
of complexity and biological organization, and using dietary
investigations that are relevant to the diets consumed in the
Western world.
Strictly standardized in vitro experiments are required to
obtain new information on the role of the cells involved in
the onset of the arterial lesions, and main research areas are
functional activities, and their controlling factors, of the
main cell types participating in the formation of atherosclerotic plaques. These activities need to be tested either
alone or during cell–cell interactions, and should involve
the assessment of the factors responsible for these events
(expression of cell-adhesion molecules, cytokines and
growth factors). As to the underlying mechanisms, both
short-term effects, mediated by fast cell-signalling pathways, and long-term processes, generally mediated by gene
activation and transcription, need to be studied in detail. In
this context, special attention should be paid to the interplay
between functionally specialized cells in the vessel wall,
e.g. endothelial cells and SMC, and inflammatory and
immune cells. Recruitment of these latter cells from the
circulation into the vessel wall is a major factor in controlling locally the progression of the lesions, whereas systemic
immune responses may differently modulate the process.
Clearly, studies of the in vivo effects of nutrients on these
steps represent the first approach in the identification of
active components and they will also shed some light on
potential and most promising mechanisms of action.
While animal studies allow the assessment of pathological events at the organ and tissue level and of the effects of
treatments on these processes, this can obviously not be
done in human subjects. The most important aspects of
research on cell-mediated processes in atherogenesis, i.e.
human studies, and on the effects of drugs and nutrients, are
therefore also the most difficult ones and completely rely on
specific markers of the disease state. Therefore, a most
important area of research is the assessment of clear
relationships between different stages and forms of the
disease and selected markers of cellular and immune activation, to be detected in the circulation and possibly in urine.
More specifically, measurements of soluble adhesion
molecules in plasma and, possibly, of cleavage products
in urine, may improve the diagnosis and the evaluation of
prognosis of the disease. In addition this may help to
establish and follow the impact of nutrient supplementation.
9.4. Hypertension
There are many different reasons for hypertension in individuals. In the aetiology of hypertension, the genetic component is definitely stronger than environmental factors,
including diet. Future work should consider the multiple
reasons that may lead to hypertension. The effect of dietary
fatty acids on blood pressure should be examined in patients
in whom TxA2 production and/or a1-adrenergic mechanisms are implicated in hypertension. The effect of individual
fatty acids, such as ALA, EPA and DHA, on the development of atherosclerosis (via haemostatic or immunological
effects) and lethal coronary events should be examined in
large well-designed trials in man.
9.5. Insulin resistance
Several studies indicate an existing relationship between
insulin resistance and cardiovascular disease. Factors which
may contribute are fasting and postprandial lipoprotein
levels in plasma, as well as hypertension. Environmental
factors include the lack of physical activity and the intake of
dietary fat. It may be possible to modulate insulin sensitivity
and subsequent cardiovascular risk factors by diet, more
specifically by decreasing the total amount of dietary fat and
increasing the proportion of polyunsaturated fatty acids.
However, additional studies on the mechanisms involved
are required to understand the real function of these dietary
Further research should also focus on intervention
studies, not only to test the efficacy of specific fatty acids,
dietary fibre, low-energy diets, etc., but also to try and
explain the mechanisms underlying the observed changes.
Moreover, these studies will be helpful in determining the
sequence of events.
Further investigations are also needed to evaluate
whether the essential fatty acids linoleic acid and ALA
ameliorate insulin resistance and, if so, whether this effect
requires desaturation and elongation of these fatty acids. For
these studies, the use of stable isotopes is instrumental.
9.6. Hyperhomocysteinaemia
Compelling evidence is now available for the association
between the plasma level of homocysteine and the risk of
cardiovascular disease, although further studies are needed
to substantiate the causality of this relationship. In addition,
well-designed intervention trials are required to prove the
beneficial role of dietary supplements containing folate and
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# Nutrition Society 1998
British Journal of Nutrition (1998), 80, Suppl. 1, S147–S171
Functional food science and gastrointestinal physiology and function
S. Salminen1 , C. Bouley2 , M.-C. Boutron-Ruault3 , J. H. Cummings4В¬ , A. Franck5 , G. R. Gibson6 ,
E. Isolauri7 , M.-C. Moreau8 , M. Roberfroid9 and I. Rowland10
Department of Biochemistry and Food Chemistry, University of Turku, SF-20500 Turku, Finland
Groupe Danone, 15, Av. GalileВґe, F-92350 Le Plessis-Robinson, France
U290 INSERM, Hoˆpital St Lazare, 107, rue du Faubourg Saint-Denis, F-75010 Paris, France
Dunn Clinical Nutrition Centre, Hills Road, Cambridge CB2 2DH, UK
Raffinerie Tirlemontoise – ORAFTI, Aandorenstraat 1, B-3300 Tienen, Belgium
Institute of Food Research, Reading Laboratory, Earley Gate, Reading RG6 6BZ, UK
University of Tampere Medical School, PO Box 607, SF-33101 Tampere, Finland
INRA – Unite´ d’Ecologie et de Physiologie du Syste`me Digestif, Baˆtiment 440 R-2,
Domaine de Vilvert, F-78352 Jouy-en-Josas Cedex, France
UCL, Ecole de Pharmacie, Tour Van Helmont, Avenue E. Mounier, 73, B-1200 Brussels, Belgium
University of Ulster, Coleraine BT52 1SA, UK
1. Introduction
2. Intestinal microflora: physiology and functions
2.1. The normal flora
2.2. Fermentation and short-chain fatty acids
2.2.1. Physiology and health
2.2.2. Acetate
2.2.3. Propionate
2.2.4. Butyrate
2.3. Interactions between the intestinal microflora and
epithelial cells
2.4. The concept of healthy microflora
3. The gastrointestinal immune system
3.1. Gut-associated lymphoid tissue (GALT)
3.2. The structure of GALT and cell distribution
3.3. Immunophysiological regulation
3.4. Regulation of antigen transfer
3.5. Interactions between the intestinal microflora and
the GALT
4. Mucosal cell proliferation and differentiation
4.1. Cell proliferation
4.2. Differentiation
4.3. Apoptosis
4.4. Mucosal enzymes
5. Gastrointestinal function and disease
5.1. Gastrointestinal infections
5.2. Normal bowel habit
5.3. Constipation
5.4. Irritable bowel syndrome (IBS)
5.5. Inflammatory bowel disease
5.5.1. Crohn’s disease
5.5.2. Ulcerative colitis
5.6. Food allergy
5.7. Colorectal cancer
6. Methodology
6.1. Human intestinal microflora
6.2. Functional analysis of the gut microflora
6.2.1. Bacterial enzymes
6.2.2. Bacterial metabolites in faeces
6.2.3. Assessment of cytotoxicity, genotoxicity and
mutagenicity of faeces
6.2.4. Susceptibility of functional markers to
dietary change
6.3. Digestibility and bioavailability of foods
6.4. Large-bowel function
6.5. Gut-associated lymphoid tissue
6.6. Epithelial cell proliferation and colon
6.6.1. Biological markers for colorectal
6.6.2. Cell proliferation
6.6.3. Differentiation
6.6.4. Apoptosis
6.6.5. Products used in experimental
6.6.6. Types of lesion
6.6.7. Transgenic mouse models for colon cancer
6.6.8. Limits of experimental models
7. Human studies on the effects of food and food
7.1. Prebiotics
7.2. Probiotics
7.2.1. Alleviation of lactose intolerance symptoms
7.2.2. Immune enhancement
Abbreviations: GALT, gut-associated lymphoid tissue; IBS, irritable bowel syndrome; Ig, immunoglobulin; ILSI, International Life Sciences Institute; IQ, 2amino-3-methyl-7H-imidazo[4,5-f ]quinoline; MTT, 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide; 7-OHIQ, 7-hydroxy-2-amino-3,6dihydro-3-methyl-7H-imidazo[4,5-f ]quinoline-7-one; rRNA, ribosomal RNA; SCFA, short-chain fatty acids.
*Corresponding author: Dr J. H. Cummings, fax Гѕ44 (0)1223 413763, email [email protected]
S. Salminen et al.
7.2.3. Acute gastroenteritis
7.2.4. Faecal mutagenicity and enzymes
7.3. Diet and colon cancer
7.3.1. Dietary protective factors
8. Safety issues
8.1. Prebiotics
8.2. Probiotics
9. Critical evaluation of present knowledge
9.1. Intestinal microflora
9.2. Mucosal function
9.3. Gastrointestinal physiology
9.4. Methodology
Human studies on health benefits
9.5.1. Prebiotics
9.5.2. Probiotics
9.5.3. Diet and colon cancer
9.6. Safety
10. Recommendations for future research priorities
10.1. Intestinal microflora
10.2. Short-chain fatty acids and intestinal microflora
10.3. Diet and cancer
10.4. Immune system
10.5. Gut mucosa
The gut is an obvious target for the development of functional foods, acting as it does as the
interface between diet and the metabolic events which sustain life. The key processes in digestive
physiology which can be regulated by modifying diet are satiety, the rate and extent of
macronutrient breakdown and absorption from the small bowel, sterol metabolism, the colonic
microflora, fermentation, mucosal function and bowel habit, and the gut immune system. The
intestinal microflora is the main focus of many current functional foods. Probiotics are foods
which contain live bacteria which are beneficial to health whilst prebiotics, such as certain nondigestible oligosaccharides which selectively stimulate the growth of bifidobacteria in the colon,
are already on the market. Their claimed benefits are to alleviate lactose maldigestion, increase
resistance to invasion by pathogenic species of bacteria in the gut, stimulate the immune system
and possibly protect against cancer. There are very few reports of well-designed human
intervention studies with prebiotics as yet. Certain probiotic species have been shown to shorten
the duration of rotavirus diarrhoea in children but much more work is needed on the mechanism
of immunomodulation and of competitive exclusion and microflora modification. The development of functional foods for the gut is in its infancy and will be successful only if more
fundamental research is done on digestive physiology, the gut microflora, immune system and
mucosal function.
Gastrointestinal function: Microflora: Immune system
1. Introduction
One of the most promising areas for the development of
functional foods lies in modification of the activity of the
gastrointestinal tract by use of probiotics, prebiotics and
synbiotics. To understand the potential value of these
functional foods and to be able to develop new approaches
it is necessary to study the normal human intestinal flora,
fermentation, the gut immune system, mucosal function and
the principal gut-related diseases.
2. Intestinal microflora: physiology and functions
2.1. The normal flora (Gibson & Macfarlane, 1995)
Bacterial numbers and composition vary considerably along
the human gastrointestinal tract. The total bacterial count in
gastric contents is usually below 103 /g, with numbers being
kept low due to the acid lumen pH. In the small intestine,
numbers range from approximately 104 /ml contents to about
106 –107 /ml at the ileocaecal region. The main factors
limiting growth in the small bowel are the rapid transit of
contents and secretion of bile and pancreatic juice.
The human large intestine is an intensely populated microbial ecosystem. Several hundred species of bacteria are
usually present, with typical numbers of about 1011 –1012 /g.
The majority of these bacteria are strict anaerobes.
Table 1 lists bacteria commonly isolated from the human
colon. Bacterial counts of individual species range over
several orders of magnitude, and the nutrition and metabolic
products of different bacterial groups vary considerably.
Most bacteria growing in the colon are non-sporing anaerobes
and include members of the genera Bacteroides, Bifidobacterium and Eubacterium among many others. Clostridia
are also represented, although they are outnumbered by the
non-sporing anaerobes, as are facultative anaerobes such as
streptococci and enterobacteria. Quantitatively, the most
important genera of intestinal bacteria in animals and man
are the bacteroides and bifidobacteria, which can account
for 30 % and 25 % of the total anaerobic counts respectively.
The Gram-negative Bacteroides group (e.g. Bacteroides
ovatus, Bacteroides fragilis, Bacteroides thetaiotaomicron)
are thought to be numerically predominant. The genus
contains both proteolytic and saccharolytic species.
Amongst the Gram-positive, non-sporing rods, several
genera are numerically significant. Obligate anaerobes
include eubacteria and bifidobacteria, such as Bifidobacterium bifidum and Bifidobacterium infantis, which are
prominent in the faeces of breast-fed infants. The genus
Lactobacillus contains many species that occur in the gut of
most warm-blooded animals. Although numerically important in the alimentary tract, their ecological significance has
not been conclusively determined.
Gastrointestinal physiology and function
Table 1. Bacteria, their substrates and products in the human large intestine (From Macfarlane et al. 1995)
(log10 /g dry wt faeces)
Saccharolytic, some amino acid fermenting
Saccharolytic and amino acid fermenting
As for the clostridia
Amino acid fermenters
Saccharolytic, lactate fermenting
Carbohydrate and amino acid fermenting
Amino acid fermentation, carbohydrate
also assimilated
As for streptococci
GВ№ rods
GГѕ rods
GГѕ rods
GГѕ rods
GВ№ rods
cocco bacilli
A, P, S
A, B, L
A, L, f, e
A, P, B, L, e
A, L
A, B, L
A, P
A, L, S
L, A
B, A, L
Mixed acids
GГѕ, Gram-positive; GВ№, Gram-negative; A, acetate; P, propionate; B, butyrate; L, lactate; S, succinate; f, formate; e, ethanol.
Several types of spore-forming rods and cocci are also
inhabitants of the gut. The genus Clostridium is probably the
most common: C. perfringens, C. bifermentans and C. tetani
are regularly isolated, albeit in relatively low numbers,
and are of significance in human and veterinary medicine.
Facultative and obligately anaerobic Gram-positive cocci
are also numerically important. The strict anaerobes include
Peptostreptococcus, Ruminoccus, Megasphaera elsdenii
and Sarcina ventriculi. The facultatively anaerobic streptococci are well represented by many species from Lancefield
group D, including S. faecalis, S. bovis and S. equinus, and
some from group K, such as S. salivarius, which is usually
associated with the mouth. Gram-negative anaerobic cocci
include Veillonella and Acidaminococcus.
Although not numerous, the Gram-negative facultative
anaerobic rods include a number of important pathogens. For example, members of the Enterobacteriaceae,
particularly Escherichia coli, are usually thought of as
characteristic intestinal bacteria.
The large-gut microflora is acquired at birth. Initially,
facultatively anaerobic strains dominate. Thereafter, differences exist in the species composition that develops and this is
largely controlled by the type of diet. The faecal flora of
breast-fed infants is dominated by bifidobacteria. In contrast,
formula-fed infants have a more complex microbiota with
bifidobacteria, bacteroides, clostridia and streptococci all
being prevalent. After weaning, a pattern that resembles the
adult flora becomes established (Ducluzeau, 1993).
The principal role of the intestinal microflora is to salvage
energy from carbohydrates not digested in the upper gut,
through fermentation. The major substrates for fermentation
are dietary carbohydrates that have escaped digestion in the
upper gastrointestinal tract. These include starch that enters
the colon (resistant starch), as well as NSP, e.g. cellulose,
hemicelluloses, pectins and gums. Other carbohydrate sources
available for fermentation are non-digestible oligosaccharides, various sugars and sugar alcohols (Cummings et al.
1997). In addition, proteins and amino acids can be effective as
growth substrates for colonic bacteria. These include elastin,
collagen and albumin, as well as bacterial protein released
following cell lysis. Pancreatic enzymes represent a source of
N. Bacterial secretions, lysis products, sloughed epithelial cells
and mucins may also make a contribution as fermentation
substrates. Total substrate availability in the human adult colon
is 20–60 g carbohydrate and 5–20 g protein/d (Cummings &
Englyst, 1987; Cummings et al. 1989).
Significant regional differences occur in bacterial activity
in the colon. The right (proximal) colon is characterized by a
high substrate availability (due to dietary input), low pH
(from acids produced in fermentation) and rapid transit. The
left, or distal, colon has a lower concentration of available
substrate, the pH is approximately neutral and bacteria grow
more slowly. The proximal region tends to be a more
saccharolytic environment than the distal gut, the latter
having higher bacterial proteolysis.
In addition to its role in fermentation the large-intestinal
microflora contributes towards health in a number of other
ways. The development of the intestinal microflora provides
the basis for a barrier that prevents pathogenic bacteria from
invading the gastrointestinal tract. The composition of the
intestinal microflora together with the gut immune system
allows resident bacteria to exert a protective function. In
addition gut bacteria are involved in vitamin synthesis (especially vitamins B and K) and in the metabolism of xenobiotics.
Thus, modification of the flora by dietary means offers one of
the most effective opportunities for development of functional
2.2. Fermentation and short-chain fatty acids
(Binder et al. 1994; Cummings, 1995;
Cummings et al. 1995)
Through fermentation, bacterial growth is stimulated (biomass), and short-chain fatty acids (SCFA) and the gases H2 ,
CO2 and CH4 are produced.
S. Salminen et al.
SCFA are the major end-products of bacterial fermentative reactions in the colon and are the principal anions in
the hindgut of man and all other mammals. The SCFA are
acetate, propionate and butyrate but other significant endproducts of carbohydrate fermentation include lactate,
ethanol, succinate, formate, valerate and caproate (Table
1). Branched-chain fatty acids such as isobutyrate, 2-methylbutyrate and isovalerate may be formed from the fermentation of amino acids that originate in proteolysis. The other
end-products from bacterial metabolism of proteins include
NH3 , phenols, indoles and amines, some of which have toxic
properties (Macfarlane & Macfarlane, 1995).
The amount of SCFA, which is usually in excess of
100 mmol/kg contents, and the molar ratios of the three
principal acids produced by fermentation, vary substantially, depending on the substrate. This has been studied
extensively in vitro using single-chamber chemostat models
of the gut inoculated with intestinal micro-organisms.
Yields vary from 40–60 % (g SCFA/100 g substrate utilized), with molar ratios of acetate from 60–80, propionate
14–22 and butyrate 8–23 (Cummings, 1995). Whilst acetate
is produced in all fermentation systems in vitro, it is the
major product of pectin breakdown. Similarly, the highest
molar ratios of propionate are seen characteristically with
arabinogalactan and guar gum as substrate. Amounts of
butyrate vary perhaps more than any other according to
substrate but the polysaccharide that is associated with the
highest relative amounts is starch. In animal studies, wheat
bran seems to give rise to high concentrations of SCFA in
the gut, despite the fact that it is relatively poorly fermented,
especially in human subjects (Cheng et al. 1987; McIntyre
et al. 1991). Studies in human subjects to determine
amounts of SCFA in the gut are difficult, but evidence
suggests that caecal concentrations of SCFA are approximately double those in the recto-sigmoid area (Cummings et
al. 1987).
The amount of SCFA produced in human subjects is very
difficult to determine. Studies of arterio–venous differences
across the gut indicate that 300–500 mmol are produced
each day, whilst in individual cases this may reach 1–2 mol.
Few dynamic studies have been carried out in man because
of problems accessing the portal vein and differential
metabolism of SCFA by individual tissues. The situation
is complicated by endogenous production of acetate by
the liver. Future stable-isotope studies may give more
information in this area.
SCFA production in the large intestine can be observed
qualitatively by measuring levels in blood. However, only
acetate appears in significant amounts in peripheral blood,
although this responds in both time and amount to substrate fermentation in the large intestine (Pomare et al.
1985; Lifschitz et al. 1995).
2.2.1. Physiology and health. All SCFA are rapidly
absorbed from the hindgut and stimulate salt and water
absorption. They are then metabolized principally by the gut
epithelium, liver and muscle, with virtually none appearing
in urine and only small amounts in faeces.
One of the most important properties of SCFA is their
trophic effect on the intestinal epithelium. All three major
SCFA are trophic when infused into the large intestine,
although butyrate seems to be the most effective and
propionate the least. What is perhaps more interesting is
that infusion of SCFA into the hindgut leads to trophic
effects in the small intestine (Sakata, 1987; Frankel et al.
1994) although the mechanisms for this are not fully
determined. These trophic properties of SCFA have important implications, particularly for patients receiving enteral
or parenteral nutrition, and in maintaining the mucosal
defence barrier against invading organisms.
2.2.2. Acetate. Acetate is the principal SCFA in the gut.
It is taken up by the epithelium, appears in portal blood and
eventually passes through the liver to peripheral tissues
where it is metabolized by muscle. In animal studies, the
liver secretes free acetate when levels in portal blood fall
below a critical level. Uptake and utilization of acetate by
many tissues has been shown and is the principal route
whereby the body obtains energy from carbohydrates not
digested and absorbed in the small intestine. Current
evidence suggests that the energy value of fermented
carbohydrate is 6·3–8·4 kJ/g (1·5–2 kcal/g) (Livesey,
1990; Roberfroid et al. 1993).
2.2.3. Propionate. In ruminant species, propionate is a
major glucose precursor but this is not an important role in
hindgut fermenting species such as man. Propionate is
largely cleared by the liver and has not been shown
consistently to have significant effects on carbohydrate
metabolism in human subjects. In vitro, propionate inhibits
uptake of acetate into the cholesterol synthesis pathway, and
in both rats and pigs propionate supplementation of the diet
reduces cholesterol levels in blood. In human feeding
studies of propionate only one out of three currently
reported shows any change in blood cholesterol levels
(Venter et al. 1989; Todesco et al. 1991; Stephen, 1994).
2.2.4. Butyrate. Butyrate is the most interesting of the
SCFA, since in addition to its trophic effect on the mucosa it
is an important energy source for the colonic epithelium
and regulates cell growth and differentiation. Butyrate is
almost entirely cleared by the colonic epithelium and is the
principal energy source for the epithelial cells (Bugaut &
Bentejac, 1993; Cummings, 1995). A defect in butyrate
metabolism has been identified in ulcerative colitis patients
and may be induced by S compounds generated in the largebowel lumen (Roediger et al. 1993; Pitcher & Cummings,
The effect of butyrate on cell growth and differentiation is
of great importance and has been the subject of a number of
studies (Boffa et al. 1992; McIntyre et al. 1993). Butyrate
brings about a concentration-dependent slowing of the rate
of transformed cell growth and promotes expression of
differentiation markers in vitro, thus leading to reversion
of cells from a neoplastic to a non-neoplastic phenotype
(Kim et al. 1980, 1994; Whitehead et al. 1986; Gibson et al.
1992). In vitro studies with colonocytes suggest an interaction between long-chain fatty acids, which result in
decreased viability and differentiation of the cells, and
butyrate, which has the opposite effect (Awad et al.
1991). In carcinogen-induced animal models of largebowel cancer, however, butyrate, either from fermentable
carbohydrate sources such as resistant starch or purified
NSP such as pectin, leads to increased cell turnover and in
some studies increased tumour formation (Sakamoto et al.
1996; Young et al. 1996). The proliferative effects of
Gastrointestinal physiology and function
butyrate are probably not of pathological significance.
Butyrate increases the proliferative index at the bottom of
the crypt and thereby has a trophic effect on the mucosa. It
does not, however, increase the proliferative index of the
surface of the crypt (type II abnormality), which is closely
connected with risk of colorectal cancer. Moreover, the
relevance of these models to human carcinogenesis is
doubtful for a number of reasons (see section 6.6). The
expression of several genes is affected by butyrate and
butyrate response factors have been identified in the
upstream element of certain genes (Kim et al. 1994).
2.3. Interactions between the intestinal microflora and
epithelial cells
Although attachment to the epithelium is thought to be an
important factor whereby bacteria colonize the gut, the
mechanisms that allow certain species to maintain themselves in specific locations in the intestinal tract are
largely unknown. An interesting new observation is that
the intestinal microflora can influence expression of epithelial glycoconjugates, which may serve as receptors for
attachment of (pathogenic) micro-organisms. Recent papers
by Bry et al. (1996) and Umesaki et al. (1995, 1997) report
that host epithelial cells in the small intestine express
fucosylated glycoconjugates in response to the presence of
specific, strictly anaerobic bacteria (B. thetaiotaomicron and
a segmented filamentous bacterium SIF13). Attachment of
some pathogenic micro-organisms is decreased by the
mutually beneficial crosstalk between the indigenous
microflora and the host (Umesaki, 1989). The observation
that one species can induce epithelial surface structures
which influence attachment of other bacteria has significance for the use of CaCo-2 cell lines or gnotobiotic animals
as model systems to study adherence or infectious diseases
as well as for strategies to prevent and to treat gastrointestinal diseases. Thus, adhesion of bacteria to mucosal
cell lines is important, but their mucus-adhering and
degrading properties also need to be addressed.
2.4. The concept of healthy microflora
It is a long-held belief, originating probably with Metchnikoff at the turn of the century, that some gut bacteria are
Fig. 1. Generalized scheme of predominant groups of colonic bacteria, indicating how the genera may exhibit potentially harmful and beneficial
functions. GГѕ, Gram positive; cfu, colony-forming units.
S. Salminen et al.
beneficial to health, whilst others may be harmful (Fig. 1).
That bacteria in the gut can be harmful through production of toxins causing diarrhoea, mucosal invasion and
activation of carcinogens is self-evident. Such bacteria are
thought to include the Clostridia, sulfate reducers and amino
acid-fermenting species.
Potentially health-giving bacteria are thought to include
principally the bifidobacteria and lactobacilli. These two
genera do not include any significant pathogenic species and
their dominance in the faeces of breast-fed babies is thought
to provide protection against infection. In adults they may
be the principal species responsible for barrier function
and for stimulating healthy immune function. In addition,
bacteria act in symbiosis with the host through fermentation.
However, the colonic microflora is a complex interactive community of organisms and its functions are a
consequence of the combined activities of the microbial
components. Thus manipulation of the human intestinal
flora offers the potential to improve health through a variety
of mechanisms.
3. The gastrointestinal immune system
3.1. Gut-associated lymphoid tissue (GALT)
(Brandtzaeg et al. 1989)
The first, and in normal individuals only, contact that
ingested bacteria, including probiotics, have with the
immune system is with the GALT. The human intestine
represents the largest mass of lymphoid tissue in the body,
containing over 106 lymphocytes/g tissue. In addition,
about 60 % of the total immunoglobulin (Ig; several
grams) produced daily is secreted into the gastrointestinal
tract. GALT is part of the mucosal immune system (i.e.
gastrointestinal tract, respiratory tract, oral cavity, urogenital tract and mammary glands) and has unique cell
types and mechanisms of immunity. The special nature of
intestinal immunity has evolved under constant exposure
to environmental antigens, whilst requiring an effective
response to an invading pathogen despite the presence of
dietary antigens. The difference between immune responses
to dietary proteins and antigens of colonizing bacteria may
play a role in the prevention of hypersensitivity reactions to
food proteins.
3.2. The structure of GALT and cell distribution
Intestinal immune cells are organized in different compartments: aggregated in follicles and the Peyer’s patches;
distributed within the mucosa as diffuse lymphocyte
populations; and in the epithelium (reviewed by McKay &
Perdue, 1993). The GALT T-lymphocytes are not homogeneous. These are classified as CD4Гѕ helper/inducer
cells and CD8Гѕ suppressor/cytotoxic cells, generating
different cytokine profiles with distinct yet unproven
functions (reviewed by Brandtzaeg et al. 1989; Brandtzaeg,
1995). The majority of the intra-epithelial T-cells have a
suppressor/cytotoxic phenotype, contrasting with the lamina
propria cells, which show mainly a helper/inducer phenotype. The lamina propria is also endowed with lymphocytes belonging to the B-cell lineage. These are mainly
memory cells and plasmocytes, where 70–90 % of them are
IgA-producing cells.
The epithelial layer of the small-intestinal mucosa is
arranged in folds, consisting of villi and crypts, which
increase the absorptive surface area. The epithelium consists
of a single layer of absorptive columnar epithelial cells,
goblet cells and intra-epithelial lymphocytes. The intraepithelial lymphocytes are a heterogeneous population of
cells. In the mouse, the primary intra-epithelial lymphocytes
are CD3Гѕ, CD8Гѕ, T-cells with a g/d-T-cell receptor (T-cell
receptor 1) and in man CD8 T-cells expressing an a/b-cell
receptor (T-cell receptor 2). The proportion of T-cell
receptor 1 cells in the epithelium is greater than in peripheral
blood. The g/d-T-cell receptor cells are thought to mature in
the epithelium rather than in the thymus, thus their
development might be more susceptible to environmental
exposures. Intra-epithelial lymphocytes are known to
non-major-histocompatibility-complexrestricted and major-histocompatibility-complex-restricted
cytotoxicity, and regulate neighbouring immune and
epithelial cells by secreting cytokines.
The epithelium is surrounded by the lamina propria,
which comprises lymphoid organs such as reticular tissue
and which contains plasma cells, T-helper cells, granulocytes and mast cells. The lamina propria is surrounded by
smooth-muscle tissue. Along the small intestine are Peyer’s
patches, which are organized lymphoid follicle aggregates.
The Peyer’s patches are more accessible to micro-organisms
than other epithelial surfaces of the gut, because they have
reduced numbers of the mucus-secreting goblet cells. In
addition, the epithelial layer of the Peyer’s patches contains specialized transport cells called M-cells, which lack
microvilli and are able to phagocytose both soluble antigens
and micro-organisms.
3.3. Immunophysiological regulation (Brandtzaeg, 1995)
Different components of the mucosal immune system act
to focus a specific response against offending antigens. The
first line in this defence, immune exclusion involving IgA
antibodies, is non-inflammatory (Brandtzaeg, 1995). The
best-characterized component of the mucosal immune
defence is the secretory IgA system (Brandtzaeg, 1995).
IgA antibody production is abundant at mucosal surfaces.
IgG-, IgM- and IgE-secreting cells function also, but at a
significantly lower frequency in GALT. In contrast to IgA in
serum, secretory IgA is present in dimeric or polymeric
form in the gut. The predominance of IgA in the mucosal
immune system results from IgA-selective T-cell regulation in GALT, particularly in the Peyer’s patches, where
specific immune responses are generated (Biewenga et al.
1993). After being synthesized by IgA precursor cells,
polymeric IgA is transported to the mucosal surface by
epithelial transcytosis mediated by the polymeric immunoglobulin receptor, the secretory component. Secretory IgA
is resistant to intraluminal proteolysis, and does not activate
complement or inflammatory responses, which makes IgA
ideal for protecting the mucosal surfaces. Hence, the main
function of secretory antibodies is, in cooperation with nonimmunological defence mechanisms (Sanderson & Walker,
1993), to mediate immune exclusion of foreign antigens by
Gastrointestinal physiology and function
preventing epithelial adherence and penetration of invasive
pathogenic micro-organisms, neutralizing toxins and viral
Although GALT is mainly involved in specific immune
protection of the gut, there is evidence for a �common
mucosal immune system’: an immune response initiated in
GALT can affect immune responses at other mucosal
surfaces (Brandtzaeg, 1995). The lymphocytes activated
within Peyer’s patches disseminate via mesenteric lymph
nodes, thoracic duct and the bloodstream back to the
lamina propria, and traffic between other secretory tissues,
including the respiratory tract and the lachrymal, salivary
and mammary glands.
There are differences between the upper and lower parts
of the human GALT in isotype distribution of immunoglobulin-producing cells. Two IgA subclasses are available
(IgA1 and IgA2 ). IgA1 immunocytes predominate in the
small-intestinal mucosa, while IgA2 are most frequent in
normal colonic mucosa. IgA2 in the colon is resistant to
most bacterial proteases that cleave IgA1 (Brandtzaeg,
Immune elimination is directed towards removal of
foreign antigens that have penetrated the mucosa. This
second line of defence involves antibodies such as IgG
and a large number of mediators such as inflammatory
cytokines, which are considered to be responsible for
the pathophysiology associated with local inflammation
(Brandtzaeg, 1995). Immune regulation pertains to the
state of specific hyporesponsiveness induced by prior oral
administration of antigens, inducing oral tolerance. Consequently, hyporesponsiveness to ubiquitous antigens such as
dietary antigens is a hallmark of the intestinal immune
system (Weiner et al. 1994). This has been taken to be a
combined effect of immune exclusion and suppression of
the systemic immune response, but it is still a matter of
3.4. Regulation of antigen transfer
(Isolauri et al. 1993a, b)
Antigens are proteins foreign to the host. Factors that
influence antigenicity include molecular complexity, solubility and concentration. Most antigens are macromolecules
in the molecular mass range 10 000–70 000 Da.
Apart from the barrier function, the intestinal mucosa is
efficient in assimilating antigens. For this purpose, there
are specialized antigen transport mechanisms in the villous
epithelium and particularly in the Peyer’s patches (Heyman
et al. 1982). The manner in which an antigen is transported
across the mucosa determines the subsequent immune
response (Heyman et al. 1982; Heyman & Desjeux, 1992;
Isolauri et al. 1993a, b; Sanderson & Walker, 1993).
Most antigens are excluded by a well-functioning
mucosal barrier but an immunologically important fraction
of antigen does bypass it (Heyman et al. 1982; Isolauri et al.
1993a, b). Antigens are absorbed across the epithelial layer
by transcytosis, and the main degradative pathway entails
lysosomal processing of the antigen. A minor pathway
allows the transport of unprocessed antigens. The Peyer’s
patches are covered by a unique epithelium and antigen
transport across this is characterized by rapid uptake and
reduced degradation of antigens. In health, paracellular
leakage of macromolecules is prevented because intact
intercellular tight junctions maintain the barrier to macromolecules. Consequently, in healthy subjects antigen transfer is well controlled and aberrant antigen absorption does
not occur.
There is evidence that during the process of absorption
across the intestinal mucosa, dietary antigens are altered
into a tolerogenic form (Weiner et al. 1994). By interfering
with this process intestinal inflammation is an important
risk factor for the development of hypersensitive disorders
(Fargeas et al. 1995).
3.5. Interactions between the intestinal microflora
and the GALT (Moreau & Coste, 1993)
After birth, the intestine is rapidly colonized by bacteria,
which probably act as a source of antigens and non-specific
immunomodulators. The dual role of the digestive flora on
the immune system should be emphasized. Bacteria can be
considered as antigens able to elicit specific systemic and
local immune responses. Furthermore, they exert a considerable influence on the number and distribution of the
GALT cell populations and play an important role in the
regulation of immune responses. These data have emerged
mainly from animal studies using germ-free and gnotobiotic
animal models (see section 6.5). As direct evidence from
human subjects is scarce, we can only extrapolate from
experimental results obtained in mice. Such studies are
important to determine the exact role played by different
bacteria present in the digestive flora, with the aim of
improving the bacterial equilibrium and allowing the best
immune modulation by functional foods. The cellular and
molecular events by which the digestive flora modulates the
immune system are still poorly understood.
The digestive flora is the major antigenic stimulus
responsible for the migratory pathway and maturation of
precursor lymphoid cells present in the Peyer’s patches.
Consequently, it acts on the development and maturation of
the IgA plasmocytes. In germ-free mice, IgA-plasmocyte
number is decreased tenfold as compared with controls.
It has been shown that the sequential establishment of
the digestive flora from birth to weaning is responsible
for the progressive increase in IgA plasmocyte numbers in
the lamina propria of the small intestine in the growing
normal mouse. In addition, Gram-negative bacteria such as
Escherichia coli and Bacteroides play an important role in
this immunologically non-specific effect.
The digestive flora also modulates the specific immune
responses at local and systemic levels. It allows the persistence of the systemic unresponsiveness to an antigen,
induced by a previous feeding with the same antigen
(oral tolerance) (Moreau & Gaboriau-Routhiau, 1996)
and shortens the abrogation of oral tolerance mediated
by cholera toxin or E. coli toxin (Gaboriau-Routhiau &
Moreau, 1996), which seems to be a property of Gramnegative bacteria (M. C. Moreau and V. Gaboriau, unpublished results). In another study the presence of the gut flora
modulated the intestinal antibody IgA response to rotavirus.
Recently, the development of an experimental model of
adult germ-free mice infected with a heterologous strain of
S. Salminen et al.
rotavirus allowed investigation of the immunomodulating
properties of a strain of Bifidobacterium on the enhancement
of the intestinal anti-rotavirus IgA antibody response at
cellular and faecal levels (Moreau et al. 1998). At the
systemic level, in gnotobiotic mice harbouring a human
strain of Bifidobacterium in the intestine or two bacterial
strains from yoghurt, Lactobacillus bulgaricus and Streptococcus thermophilus, increases of the specific antibody
response in serum and in the phagocytic activity of peritoneal phagocytes were observed respectively (Moreau et
al. 1994).
4. Mucosal cell proliferation and differentiation
(Wright & Alison, 1984)
The intestinal mucosa of rodents and other mammals is
renewed every 2–3 d (Wright & Alison, 1984). Maintenance of the architecture of the colonic mucosa, in
particular of mucosal crypts, is a consequence of the balance
amongst a number of factors. Proliferation of stem cells
occurs near the base of the crypt. As enterocytes migrate up
the crypt they differentiate, mature and become functional
in terms of absorption and mucin secretion.
4.1. Cell proliferation
This has been well described and a wide variety of methods
are available for its estimation in vitro and in vivo in both
animals and man (Goodlad & Wright, 1982; Goodlad,
4.2. Differentiation
Light and electron microscopic examination of human
colonic tissue has revealed that stem cells differentiate
into a number of cell types, including mucus-secreting
cells, columnar cells (thought to have an absorptive and a
secretory function) and intestinal endocrine cells. Histochemical studies have shown alterations in secreted glycoproteins between differentiated and undifferentiated regions
of the small and large intestine, indicating that a modification of carbohydrate structures accompanies goblet cell
differentiation in rat and man (Boland et al. 1992). Furthermore, the mucin of normal colonic mucosa differs markedly
from that in cancerous tissue and �transitional tissue’ in the
early stages of neoplastic development (Boland et al. 1992).
A characteristic of tumours is the presence of poorly
differentiated cells: consequently, a dietary treatment that
encourages differentiation is potentially beneficial.
4.3. Apoptosis
Apoptosis (genetically programmed, autonomous cell death)
associated with the removal of damaged cells is considered
to be a protective event.
4.4. Mucosal enzymes (Szarka et al. 1995)
Phase I, cytochrome P450 enzymes and phase II drugmetabolizing enzymes such as glutathione S-transferase
(EC and UDP-glucuronosyl transferase (EC are widely distributed in the intestinal mucosa.
These enzymes are involved in the biotransformation of
mutagens, procarcinogens, steroids and other compounds of
exogenous and endogenous origin. Modulation by dietary
compounds may result in protection against toxic and
carcinogenic damage to tissues (Wattenberg, 1983). In
terms of deactivation, the enzyme glutathione S-transferase
is of particular importance. It is present in many tissues
in a variety of forms (p, m and a) and plays a critical
role in protecting tissues from xenobiotics and carcinogens. Glutathione S-transferase activity has been found to be
lower in individuals at high risk from colon cancer when
compared with controls (Szarka et al. 1995).
5. Gastrointestinal function and disease
5.1. Gastrointestinal infections
(Gracey, 1993; Savarino & Bourgeois, 1993)
Acute infections of the gut are usually self-limiting, characterized by diarrhoea and often vomiting. The principal
pathogens are viruses and bacteria such as Escherichia
coli, Campylobacter spp, Vibrio cholerae, Staphylococcus
aureus, Bacillus cereus, Clostridium perfringens, Salmonella spp, Shigella spp, Yersinia spp and a number
of protozoa, especially Giardia lamblia, Entamoeba
histolytica and Cryptosporidium parvum.
Bacteria causing infection are usually classified according to whether they secrete an enterotoxin (toxigenic) or
invade the bowel wall (invasive). Toxigenic diarrhoeas
include cholera, and both enteropathogenic and enterotoxigenic E. coli, whilst the classic invasive organisms are
Shigella (dysentery), Salmonella (typhoid) and enteroinvasive E. coli. Rotaviruses are most commonly found in
diarrhoea of children and invade the small-intestinal epithelium. Acute diarrhoea is responsible for 3–4 million deaths
annually worldwide, many of which are children, in which it
accounts for 20–30 % of all mortality.
Rotavirus is the most common cause of acute childhood
diarrhoea. It is primarily seen in infants and young children,
with a peak incidence between 6 months and 2 years of
age. Rotaviruses invade the highly differentiated absorptive
columnar cells of the small-intestinal epithelium, where
they replicate. This results in partial disruption of the
intestinal mucosa with loss of microvilli and decreased
villus : crypt ratio. Rotavirus infection is associated with
increased intestinal permeability. Jalonen et al. (1991)
found increased lactulose : mannitol urinary recovery
ratios in patients with acute diarrhoea compared with nondiarrhoeal patients. Concomitantly, the levels of immune
complexes containing dietary b-lactoglobulin in sera were
significantly higher in patients with rotavirus diarrhoea
than in non-diarrhoeal patients. Enhanced macromolecular absorption in rotavirus gastroenteritis has been
shown in several studies (Heyman et al. 1987; Isolauri
et al. 1993a, b). A local immunoinflammatory reaction
impairs the intestine’s barrier function. Impaired barrier
function and defective handling of intraluminal antigens
in the epithelial cells may be an important pathogenic
mechanism in acute and chronic gastrointestinal disorders. It may abrogate tolerance to ubiquitous antigens,
Gastrointestinal physiology and function
including bacteria residing in the intestine (Duchmann et al.
Chronic infection of the gut is much rarer and seen only in
persons who have anatomical abnormalities of the gut such
as blind loops, strictures or fistulas. Chronic infection with
Tropheryma whippelli causes Whipple’s disease and intestinal bacteria are responsible for tropical sprue. Tuberculosis affects the gut, especially the ileo-caecal region,
and chronic carrier states occur with amoebas.
The main indigenous bacteria of the large intestine
resist invasion by pathogenic species and this is part of
the human host defence against diarrhoeal illness. This
barrier function provided by the gut flora may be impaired
during antibiotic use, where diarrhoea is common. Antibiotic-associated diarrhoea is usually due to invasion with
toxin-producing species such as Clostridium difficile or
Clostridium septicum.
5.2. Normal bowel habit (Cummings, 1993, 1994)
Bowel habit is defined by the amount of stool passed,
frequency of defecation and consistency of stool. It varies
very widely throughout the world with daily stool weights in
the range 100–400 g/d and stool frequency of three times
per day to three times per week. In European countries
and North America, daily stool weight is of the order of
100–150 g/d (Cummings et al. 1992). Bowel habit is controlled principally by two factors, first diet, and second gut
motor activity (transit time). The foods that affect bowel
habit are those which reach the large intestine, i.e. are nondigestible. The dietary components falling into this category
are lactose (in lactase-deficient individuals), sugar alcohols,
non-digestible oligosaccharides, resistant starch and NSP.
Dietary fat and protein have little effect on bowel habit
unless they are rendered non-absorbable by some technique
(e.g. sucrose polyester).
The mechanism by which non-digestible foods affect
bowel habit depends on their fermentability. Foods that
are not fermented appear in faeces and cause bulking
depending on their inherent mass or water-holding capacity
(i.e. bran and other intact cell-wall material). Most foods
that reach the large intestine are fermented, yielding SCFA,
which are absorbed and do not contribute to faecal bulk, and
H2 and CH4 , which can expand faecal bulk but not mass.
Fermentation also stimulates bacterial growth to produce
biomass, which is the principal mechanism of increasing
stool mass. A final mechanism that needs to be borne in
mind is the interrelationship between intestinal bulk and
motor activity. As bulk in the large intestine increases, so
motor activity is stimulated and, in general, the greater the
bulk the more rapid the transit. Motor activity expressed as
transit time may also modulate stool output independently
of dietary bulk (Cummings, 1993, 1994).
5.3. Constipation
Constipation is a disorder of motor activity of the large
bowel traditionally defined in terms of bowel frequency.
The main symptom in constipation is straining at defecation,
and discomfort, distension and incomplete rectal emptying
are all considered part of the condition. Total gut transit time
is generally prolonged in constipated subjects. There are many
causes of constipation, with diet one of the common reasons,
particularly low-NSP diets, gluten-free diets, �low-residue’
diets and enteral feeds. Treatment of simple constipation is
usually in the first instance by dietary means. The principle is
to increase fermentable carbohydrates in the diet, especially
NSP from whole-grain cereals. Thus, diet has a major role to
play in controlling bowel habit.
5.4. Irritable bowel syndrome (IBS) (Thompson & Heaton,
1980; Thompson & Gomborone, 1993)
IBS is one of the commonest disorders seen in the hospital
gastroenterology clinic, but it is poorly understood. IBS (or
irritable colon, mucus colitis, spastic colon) is a disorder of
motor activity of the whole bowel, although colonic symptoms usually predominate. It occurs very widely throughout
the world and is commoner in women. IBS has two main
presenting features, abdominal pain and altered bowel habit
(Thompson & Heaton, 1980).
The cause of IBS is unknown but it occurs in many
patients following dysentery or antibiotic use. In addition,
patients often volunteer that specific foods upset them (food
intolerance) and stress is clearly contributory. Wheat bran
and other bulk laxatives are frequently given, but results
have been very variable. They may aggravate symptoms
through gas production, although in patients who are
predominantly constipated they are of benefit. Because of
a postulated disturbance in the colonic microflora in IBS a
number of groups are currently trying the use of probiotics
to �normalize’ the flora.
5.5. Inflammatory bowel disease (Podolsky, 1991;
MacDonald, 1993; Tytgat et al. 1995)
Two major disorders, Crohn’s disease and ulcerative colitis,
are conventionally grouped together under the heading
inflammatory bowel disease because both are characterized
by chronic inflammation in the gut. However, it is best to
consider them as separate conditions because they have
characteristically different pathology, clinical courses,
complications and management. The aetiology of neither
is known.
5.5.1. Crohn’s disease. Crohn’s disease may affect
any part of the gut from mouth to anus. Characteristically
it occurs in the ileocaecal region and colon, and the
inflammation is patchy or discontinuous. It frequently recurs
after surgical resection of the affected areas of gut at or near
the point of anastomosis of the bowel. The involved
intestine is thickened, with ulceration of the mucosa,
stricturing and fistula formation. Mouth ulcers and perianal
abscesses are characteristic. Histologically there is transmural inflammation, with mononuclear cells, lymphoid
aggregates and granulomata.
Crohn’s disease occurs worldwide although it is uncommon in Central and South America, Africa and Asia. It is
seen less frequently than ulcerative colitis, although rates
have increased fivefold since 1950. It is predominantly a
disease of the young with peak occurrence between the ages
of 20 and 30 years with a second peak between 70 and
80 years. The cause is unknown but genetic factors are
S. Salminen et al.
important with 10 % of patients having a close relative
with the disease. Diet has been implicated, especially
sugar, but a multicentre trial in which a diet excluding
sugars and rich in NSP was given did not result in any
conclusive benefit in patient management (Ritchie et al.
1987). More likely aetiological factors include bacteria and
other micro-organisms.
Diet has an important role to play in the management of
Crohn’s disease. For patients who do not respond to conventional treatment in Crohn’s disease, various enteral and
parenteral regimens providing bowel rest have been used.
The rationale for such treatments is that the absence of
food antigens in the bowel lumen reduces inflammatory
immune reactions, motor and digestive activity, and gives
the mucosa a chance to heal itself.
Most patients relapse soon after introduction of normal
food following bowel rest regimens. It has been suggested
that dietary modification to exclude foods likely to cause
symptoms (predominantly cereals, dairy products and meat)
can lead to extended periods of remission.
5.5.2. Ulcerative colitis. Ulcerative colitis is a chronic
inflammatory condition of the mucosa of the large bowel
that causes bloody diarrhoea. It is one of the diseases of
modern civilization, being first described with certainty in
1909 and predominantly affecting industrialized populations. It has an overall prevalence of 40–120 cases per
100 000 of the population in Western countries and is
uncommon, although beginning to emerge, in Africa and
India. It affects the sexes equally and usually presents
between the ages of 20 and 40 years.
Its cause is unknown but there is probably a genetic
component, with a 15-fold increased risk amongst close
relatives of patients. Animal models point to an involvement
of the immune system, but also to the necessary presence of
bacteria in the colon to produce colitis. The observation
that healthy colonocytes use butyrate for their metabolism and that this is defective in ulcerative colitis has
raised the possibility of dietary factors in its aetiology.
However, no convincing evidence for a dietary factor has
emerged, although the inhibition of butyrate oxidation by
S-compounds leaves the possibility of diet combining with
the colonic microflora as a possible initiating factor of the
inflammation (Pitcher & Cummings, 1996).
5.6. Food allergy (Isolauri, 1995)
Food allergy is defined as an immunologically mediated
adverse reaction against dietary antigens. Food allergy can
affect several organ systems, the symptoms commonly
arising from the gut, skin and respiratory tract. Despite the
wide spectrum of clinical manifestations, there are at least
two prerequisites for the development of food allergy:
dietary antigens must penetrate the intestine’s mucosal
barrier, and the absorbed antigens must cause harmful
immune responses. The immaturity of the immune system
and the gastrointestinal barrier may explain the peak prevalence of food allergies in infancy. In food allergy,
intestinal inflammation and disturbances in intestinal permeability and antigen transfer occur when an allergen
comes into contact with the intestinal mucosa. During
dietary elimination of the antigen, the barrier and transfer
functions of the mucosa are normal. It has, therefore, been
concluded that impairment of the intestine’s function is
secondary to an abnormal intestinal immune response to the
offending antigens.
5.7. Colorectal cancer (Faivre et al. 1985)
Colorectal cancer is unequally spread throughout the world
(Burkitt, 1971). It is amongst the three most common
malignancies in most industrialized countries including
Western Europe, and its survival rate has improved little
during the past decades, being of the order of 40 % at 5
years. The most common locations in high-risk countries are
the left colon and the rectum, whereas right-colon cancers
are proportionally more common in low-risk areas such as
Japan. About 5 % of colorectal cancers are truly genetic
diseases (hereditary nonpolyposis colorectal cancer and
familial polyposis coli), transmitted as autosomal dominant,
but the majority of colorectal cancers are sporadic, and are
mostly influenced by environmental factors, in particular
diet, with a potential interaction between a genetic background and diet (Boutron et al. 1996). From 70 to 80 % of
left colon and rectal cancers in Western countries follow the
so-called adenoma–carcinoma pathway, with possibly less
in the case of right-colon cancers (Bedenne et al. 1992).
This is of major importance as it provides the opportunity of
studying precancerous lesions both in aetiological studies
such as case–control and cohort studies, and for intervention studies, where studying adenoma recurrence or
growth is easier and brings results more rapidly (Boutron
& Faivre, 1993).
6. Methodology
6.1. Human intestinal microflora (Collins & Gibson, 1998)
The identification of factors controlling or influencing the
composition of the human intestinal microflora, including
prebiotics and probiotics, may be compromised by the
precision of current methodologies for determining bacterial
composition which are based almost entirely on phenotypic
approaches. Whilst these have met with some success,
when done properly, they are time-consuming, laborious
and lack the resolving power necessary to analyse the
complex microbiota at the species or subspecies level.
Traditional gut microbiological methodologies are
usually based on morphological and biochemical properties
of the organisms (Table 2). Whilst such an approach is costeffective and allows the processing of replicate samples, the
procedures used may be unreliable and may lack resolution.
For example, the metabolic plasticity of organisms is
problematic and the test used may not be reproducible.
Phenotypic characterization does not allow a high degree of
fidelity and is most useful for genus level identification. In
some cases this situation is eased if the test organism
exhibits a specific metabolic trait. For example, bifidobacteria may be detected, on a qualitative basis, by the
production of fructose-6-phosphate phosphoketolase activity. An additional problem is that traditional cultivationbased methods may result in underestimation of microbial
diversity, due to the presence of organisms that cannot be
Gastrointestinal physiology and function
Table 2. Methods for study of the human gut microbiota
Morphological and biochemical
Straightforward to carry out. Can run in
parallel a large number of replicates.
Relatively inexpensive
Specific biomarkers, e.g. certain
cell-wall antigens, cellular fatty
acids, plasmid profiles,
serotyping, resistance to
Ribotyping (RNA polymorphisms)
Cultural procedures may not be required
16 S ribosomal RNA typing
Reliable. Very high discriminatory power
High fidelity. Reliable. Cumulative database
allows placement of unknown species.
Applicable to culturable and non-culturable
forms. Allows probe development
cultivated, which therefore elude isolation. This has led
to the development of alternative strategies for assessing
microflora changes.
The detection of biomarkers that may be attributed to
certain components of the flora offers some potential.
However, to be wholly effective there would be a need for
all the major bacterial components of interest to be separable by individual biomarkers, e.g. changes in cellular fatty
acids. This may be feasible but would be difficult to prove in
a reliable manner.
An attractive solution to the problem of determining
microflora changes accurately, lies in the application of
modern high-resolution molecular genetic techniques.
Recent advances in the field of molecular biology are
revolutionizing the characterization and identification of
micro-organisms (Pace, 1996). For example, molecular
sequence analysis, particularly of ribosomal RNA (rRNA),
provides a powerful tool for determining the genetic interrelationships of micro-organisms, and allows systematic
monitoring of the gut flora response to dietary intervention.
By utilizing diagnostic sequences within the rRNA, it is
possible to design gene probes that facilitate precise
identification. The use of polymerase chain reaction
technologies may also allow access to non-culturable
micro-organisms. Over the next few years, 16 S rRNA
sequence analysis is expected to rapidly advance our
knowledge of the true genetic diversity of the gut microbiota, including organisms that evade traditional
identification, due either to a lack of taxonomic resolution
and/or non-culturability.
Molecular approaches have already been used to determine changes in the composition of the microbial gut flora
(Langendijk et al. 1995; Kok et al. 1996; McCartney et al.
1996; Wilson & Blitchington, 1996). Clearly, however,
before such techniques are routinely used in gut microbiological applications, the fidelity and efficacy of such
methods need to be rigorously evaluated. A comparative
phylogenetic framework of gut micro-organisms, based on
genetic material such as 16 S rRNA, would allow highly
discriminatory and dependable diagnostic probes to be
developed. Preferably, the probes should be validated
using different procedures, such as in situ and dot blot
Involves operator subjectively to recognize different
colonial and cellular morphologies. Lack of
discriminatory power and subject to metabolic plasticity
of the organisms. Applicable only to culturable bacteria
Cannot assign the position of hitherto unknown species.
Relies on all test organisms having unique biomarker.
Stability of the biomarker may be questionable
Applicable only to culturable forms. Cannot assign the
taxonomic position of any unknown species
Costly for both reagents and large-scale equipment, e.g.
automated sequencers. Recommended for partial use
Another approach to analyse the genetic diversity of
complex microbial populations is denaturing gradient gel
electrophoresis or temperature gradient gel electrophoresis.
The technique is based on the separation of polymerase
chain reaction-amplified fragments of genes coding for
16 S rRNA, all of the same length (Muyzer et al. 1993).
This results in unique separation patterns for different
microbial populations, and will contribute to the description of changes or differences in microflora composition of
uncharacterized microbial populations.
The potential benefits of such technologies in gut microbiology, especially dietary modulation for improved health,
are large, particularly when used in conjunction with traditional phenotypic procedures. The use of molecular genetic
approaches for qualitative and quantitative monitoring of the
human intestinal microbiota will constitute an essential step
forward for determining the validity of the functional food
concept, when directed towards the role of the gut flora.
6.2. Functional analysis of the gut microflora
(Rowland, 1995)
The complexity of the gut microflora, coupled with timeconsuming procedures necessary to identify and enumerate
the anaerobic components, makes their characterization
by conventional methodology difficult and expensive. In
particular, such methods are not suited to studies involving
large numbers of subjects or treatment regimens. Less
comprehensive studies (e.g. identification of major
groups) are open to the criticism that any induced changes
occurring in genera or species other than those being
enumerated will be missed. Furthermore, although bacteriological investigations are useful in describing the basic
ecology of the gut, they are of less value in studies of
metabolism, nutrition and cancer.
An alternative approach is to use biochemical assays that
measure the functional activity of the flora as a whole and
thus permit deductions to be made regarding the role of the
flora in the metabolism of dietary components. In addition,
by selecting microbial enzyme activities or metabolic endpoints resulting in compounds with potentially toxic or
beneficial effects, probable health consequences for the
host can be assessed.
S. Salminen et al.
6.2.1. Bacterial enzymes. The bacterial enzymes commonly assayed include b-glucuronidase (EC, bglucosidase (EC, azoreductase, nitroreductase,
nitrate reductase (EC, the conversion of precarcinogen 2-amino-3-methyl-7H-imidazo[4,5-f ]quinoline
(IQ) to 7-hydroxy-2-amino-3,6-dihydro-3-methyl-7Himidazo[4,5-f ]quinoline-7-one (7-OHIQ). The substrates
of these enzymes and the functional and health implications
of their products have been extensively reviewed (Rowland,
1995). For example, bacterial b-glucuronidase in the colon
is able to release carcinogens from hepatically derived
glucuronic acid conjugates and is a critical factor in the
enterohepatic circulation of drugs and other foreign
compounds. b-Glucosidase hydrolyses plant glycosides to
release the aglycones, many of which are mutagenic,
although some exert anti-carcinogenic activity (Rowland,
1995). Azo- and nitroreductases reduce their substrates to
amines, which are usually more toxic than the parent
compound, and nitrate reductase generates the highly
reactive and toxic anion nitrite. Bacterial conversion to
7-OHIQ is one pathway of activation of the pre-carcinogen
IQ (formed during cooking of meat and fish) to a genotoxic
6.2.2. Bacterial metabolites in faeces. Faecal metabolites that are indicators of bacterial activity relevant to
colonic health include NH3 , a toxic product of bacterial
breakdown of protein and urea (Clinton, 1992), and phenols
and cresols, which are derived from amino acid catabolism
by gut bacteria (Macfarlane & Macfarlane, 1995). The
production of NH3 is closely related to bacterial activity and
is associated with certain toxic events in the gastrointestinal
tract. NH3 is considered to be a potential tumour promoter in
the colon, and has been postulated to enhance neoplastic
transformation in the gut.
Other gut bacterial products with possible adverse effects
on the colonic mucosa include N-nitroso compounds, which
are potentially carcinogenic substances formed by bacterial
catalysis of the reaction of nitrite and nitrogenous compounds in the colon (Rowland, 1995), diacylglycerol, a
putative tumour promoter derived from lipid breakdown,
and secondary bile acids, deoxycholic and lithocholic acids,
also putative tumour promoters.
6.2.3. Assessment of cytotoxicity, genotoxicity and
mutagenicity of faeces. An alternative approach to assaying enzymes or metabolites in faeces is to assess
toxicological activity of fractions using short-term tests
for toxicity, genotoxicity and mutagenicity. This provides a
direct estimate of the potential of the faecal sample to
damage the colonic mucosa and has been used to provide
insights into possible processes involved in colon cancer.
Usually, the aqueous phase of human faeces (faecal water)
is used (Rafter et al. 1987). Cellular toxicity can be
assessed using rapid colorimetric assays in multiwell
plates. For example the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983)
involves the pH-dependent conversion of water-soluble
MTT to water-insoluble formazan and provides a reliable
estimate of viable cell number.
Recently, using the Comet assay, it has been shown
that the genotoxicity of faecal water varies markedly
between individuals, with at least some of the DNA
damage occurring via an oxidative mechanism (Venturi
et al. 1997).
6.2.4. Susceptibility of functional markers to dietary
change. In animal models, major changes in activities of
bacterial enzymes and in levels of bacterial metabolic
products have been seen after a wide range of dietary
changes. These include the type and level of dietary fat and
protein, supplementation with dietary fibre and resistant
starch, and addition of oligosaccharides (Rowland, 1991). In
many cases changes were seen in the absence of alteration in
composition of the gut flora.
In contrast, modification by diet of bacterial metabolism
in human subjects has proved more difficult. However,
changes in enzyme activities and concentrations of NH3 ,
phenol and cresol have been detected in volunteers consuming lactulose (Terada et al. 1992) and lactobacilli
(Goldin & Gorbach, 1984).
A low level of NH3 production in the gut is associated
with low-protein, high-fibre diets, which appear to be
protective against cancer of the colon. NH3 levels have
been shown to be elevated in rats consuming a diet containing high-risk factors for colon cancer (Hambly et al.
The cytotoxicity of faecal water strongly correlates with
bile acid concentration in faeces and is increased in individuals on high-fat diets (Rafter et al. 1987) and decreased
in subjects on high-resistant-starch diets (van Munster et al.
6.3. Digestibility and bioavailability of foods
The methodology for studying non-digestibility of foods is
an important area for understanding the effects of food
and food components on intestinal microflora and
physiology. A compilation of current methodology is
given in Table 3. It is important to identify the nature of
molecules, their chemical bonds and molecular size to
understand digestibility. In vitro digestion studies and
markers for absorption and excretion are of value. Animal
models offer a means of simulating different digestion
extremes and human volunteer studies enhance the understanding of in vivo digestibility of foods and bioavailability
of nutrients.
6.4. Large-bowel function
Study of large-bowel function is extremely difficult, mainly
because of its inaccessibility. However, the large bowel has
unique aspects of metabolism; the principal events in the
lumen are anaerobic and end-products such as H2 and SCFA
are not produced by other biochemical reactions in the body.
These products are absorbed and appear in blood and breath,
and can be used to study intraluminal events. Many investigators have used faeces and their composition as a guide to
intracolonic events. Such studies have often been criticized
because of the lack of representativeness of faeces, but for
some aspects of colonic metabolism, such as the gut microflora of the lumen, they are probably acceptable. The study
of the large intestine has spawned a large number of in vitro
models, particularly of fermentation. A short summary of
methods is given in Table 4.
Gastrointestinal physiology and function
Table 3. Methods to study the digestibility and bioavailability of foods
1. Chemistry
Identifies nature of molecules, chemical bonds,
molecular size, etc. (e.g. most b-glucans are not
Studies with pancreatic and other enzymes
2. In vitro model
3. Blood appearance
4. Breath
5. Ileostomy model
6. Intestinal intubation:
jejunum, ileum, colon,
perfusion/aspiration, single
or multilumen tubes
7. Faecal analysis
8. In vitro fermentation
9. Animal models:
Requires dedicated carbohydrate chemistry
Glucose tolerance, chylomicrons. Applicable to a large
number of samples
H2 or CH4 , 13 CO2 . Simple non-invasive for human
Probably the gold standard for study of digestion in
stomach and small bowel
Valuable for dynamic studies of flow rate, site of digestion
in small bowel, nutrient concentrations and physical
form, and identification of intermediate products of
Relatively straightforward and gold standard for overall
digestion and fermentation
Various batch and continuous culture methods available.
Good for modelling physiology
Gives greater access to tissues. Germ-free, human
associated and conventional flora
Care needed to mimic conditions in gut and hence
rate and extent of digestion
Requires human subjects and can be affected by
factors other than those determining digestion
Difficult to quantify. Prolonged studies (16–24 h)
needed for fermentation. Large subject variability
Requires access to patient population. May
underestimate losses to caecum because of
microbial colonization of ileum and fermentation
in bag
Very invasive human studies. Need X-ray control.
Presence of tube may alter normal physiology.
Difficult to make quantitative
Requires accurate faecal collections with balance
markers and good methods validated for faeces
Needs well-founded microbiology laboratory
Animal facilities needed. Extrapolation to man
Table 4. Methods for studying large-bowel function
6.5. Gut-associated lymphoid tissue
Focus of study
Bowel habit
Motor function
Diary record
Motility recording
Recto-anal manometry and pelvic floor
Transit time:
whole gut – non-absorbable markers
Partial transit time:
small bowel – isotope-labelled meal and
gamma scanning
– lactose breath H2
large bowel – X-ray following marker ingestion
Microflora, and enzymic activities
Fat, N, biomass, carbohydrate, DM, bile acids,
sterols, pH
Faecal water: osmolarity, pH
bile acids
Occult blood
Transport physiology
Batch cultures, single chemostats,
multi-chamber chemostats
Short-chain fatty acids, branched-chain fatty
acids, bile acids
H2 , CH4 , 13 CO2
General histology and immuno-staining
DNA adducts
Mutational analysis (e.g. Kras)
Proliferation markers
Composition and structure
Barium studies
Plain film of abdomen
Sigmoidoscopy (including flexible)
Faecal analysis
In vitro models
Blood (including
portal blood)
Mucosal biopsies
Exfoliated cells
The presence of the digestive flora has a considerable
influence on the immune system of the host (see section
3.5) and the principal methodology to study this is the use of
germ-free animals, also termed axenic animals. By comparing germ-free and conventional animals, it is possible to
highlight the role of the digestive flora in immune function.
Moreover, the role played by bacteria isolated from the
digestive flora, or used as probiotics, can be analysed by
inoculating the gut of germ-free animals with these bacteria.
These are called gnotobiotic animals. Recently, germ-free
mice associated with human flora have been developed
allowing in vivo studies of functional properties of
probiotics and prebiotics used in human nutrition.
The advantages of gnotobiotic animal studies are to
determine which kind of immune response a given bacteria
established in the gut is able to exert, e.g. non-specific
and/or specific immune response; inductive or suppressive
immune response. Many methodologies are available. They
are summarized in Table 5 and marked with (H) when they
are applicable to human studies. The disadvantages of
gnotobiotic models are the expensive animal facilities
they need and the limits of the animal species studied.
Moreover, it is not certain that a modulating bacterial
effect observed in gnotobiotic conditions will be expressed
in conventional conditions. Thus, other studies using conventional animals are needed. Current methods utilize in
vitro cultures of systemic or intestinal lymphoid cells (De
Simone et al. 1993), cellular assays with colonic cell lines
(Schiffrin et al. 1995) and in vivo assays with conventional
animal models (Perdigon et al. 1996). Two types of specific
immune response can be assessed at the intestinal level.
These include the suppression of humoral and cellular
immune responses to chronically administered antigens at
the systemic level (immune regulation) and the induction
of a protective IgA antibody response at the mucosal level
S. Salminen et al.
Table 5. Selected methods to study intestinal immune function
Immune response
Proliferative assays
In vitro: measurement of the
proliferation of systemic or
intestinal lymphoid cells after
stimulation with mitogens or
cell components
Easy for systemic assays: from
blood samples (H)
Cytokine production
In vitro: after proliferative assays
Phagocytic activity
In vitro: peritoneal cells,
circulating cells
Development of new
methodologies (H)
Biomarkers (H)
Easy for systemic assays: from
blood samples (H)
Modulation of molecular
expression in intestinal cell
In vitro: HT-29, CaCo-2 cell line
Facs analysis, histochemical
Measured by ELISA or ELISPOT
at several levels:
(1) In serum: soluble IgA
(2) In blood: circulating IgAproducing cells
(3) In faeces
IgA antibody response
(4) In saliva
(5) Whole gut lavage fluid
Oral tolerance to dietary antigens
Ex vivo: proliferative assays of
lymphoid blood cells with
specific antigen
Inflammatory cytokine production
by lymphoid blood cells
In vivo: intestinal permeability
In vitro: Ussing chamber
Cell lines originated from human
Specialized line cells
Not easy for intestinal assays in
human subjects: need biopsy
Technical difficulties
Need correlation with in vivo
assays to give a
biological significance
Need correlation with in vivo
assays to give a biological
Correlation with specific immune
response poorly understood
Technical difficulties for intestinal
Adenocarcinoma lines
Absence of correlation with
intestinal cellular environment
Easy in human subjects (H)
Reflects intestinal response (H)
Easy, allows kinetic studies (H)
Easy (H)
Invasive, but avoids intestinal
biopsy (H)
Direct measure of the
unresponsiveness state (H)
Biomarkers: TNF-a (H)
Increase in food
hypersensitivities (H)
Direct correlation with antigen
Does not reflect the intestinal
Does not reflect transepithelial
transport into gut lumen
Individual and daily variations,
proteolytic activity, reflects only
colonic response
Individual and daily variations
Needs hospitalization
Cause or consequence of oral
tolerance breakdown?
Not specific to immunological
Invasive and difficult technology
H, applicable to human studies; IgA, immunoglobulin A; TNF-a, tumour necrosis factor-a; Facs, fluorescence-activated cell-sorting; ELISPOT, enzyme-linked
immunospot assay.
(immune exclusion). The definition of biomarkers is still
incomplete. Several methodologies have been developed for
IgA response measurements. Sampling needs blood collection or intestinal biopsies or stool and saliva collection.
Problems in faeces and saliva handling and daily variations in
expression of IgA activities have not been solved. In the case
of oral tolerance, the biomarkers available in human subjects
are defined as the absence of proliferation of cultured blood
cells with antigen or down-regulation of inflammatory cytokines involved with food allergies (Benlounes et al. 1996;
SuВЁtas et al. 1996a, b). Other methodologies must be developed according to further knowledge about oral tolerance
mechanisms (Weiner et al. 1994).
6.6. Epithelial cell proliferation and colon carcinogenesis
6.6.1. Biological markers for colorectal carcinogenesis.
A relationship between colorectal carcinogenesis and
abnormal cell proliferation has been demonstrated (Lipkin,
1988) in studies of the mucosa in patient groups at high risk of
cancer, e.g. ulcerative colitis and familial adenomatous
polyposis, and in animals exposed to carcinogens that target
the colon. Two changes in colonic cell proliferation have
been described (Risio, 1992): an increase in the total number
of proliferating cells, or hyperproliferation, which is not a
specific marker of cancer risk, and a progressive shift of
proliferating cells to the crypt surface (stage II abnormality)
which is more specific for tumour risk.
6.6.2. Cell proliferation. Several techniques have been
developed to measure cell proliferation in colonic mucosa
and assess the influence of diet. Measurement of crypt-cell
production rate in microdissected crypts is considered to
provide the best assessment of proliferation with the fewest
artifacts. However, since it requires in vivo treatment with
vincristine, it is not suitable for human studies.
Change in the rate of cell proliferation in the normal
mucosa may be less reliable as a biomarker for diet-related
cancer risk (Wasan & Goodlad, 1996) and is just one of the
processes contributing to colonic mucosal crypt architecture. Other events are differentiation, exfoliation and
apoptosis. It is likely that the best predictor of cancer risk
is an overall assessment of these events.
Markers of early epithelial events have been used in
animal models. These include DNA damage, microadenomas and aberrant crypt foci in the mucosa. The microgel
electrophoresis (Comet) assay has been used for assessing
Gastrointestinal physiology and function
DNA damage in the colonic mucosa. Induction of aberrant
crypt foci has been particularly widely used, as it is easy
to observe macroscopically. However, its reliability as a
marker of colorectal tumour risk is a matter of debate. In
man, aberrant crypts and microadenomas, similar to those
described in animals, have been described (Roncucci, 1992)
but their correlation with other well-known markers of risk
has not been established. Other markers of cell proliferation
include PCNA and Mib 1; both are proteins appearing at
specific stages in the cell cycle.
6.6.3. Differentiation. Methods exist to measure the
state of differentiation of the mucosal epithelial cells by
histochemical staining of mucins using binding to specific
lectins. The methods can be applied to tissue sections from
human and animal biopsies after fixation. Such approaches
have been used to study colonic epithelium in rats and
human subjects at various stages of neoplasia. They have
not achieved widespread use for investigating dietary
modification of neoplastic processes.
6.6.4. Apoptosis. Identification of oligonucleotide fragments by in situ end labelling using immunoperoxidase
techniques forms the basis for various methods that can be
applied to sections of colonic tissue from human subjects
and animals and to cell suspensions (Ansari et al. 1993).
Apoptosis in cell cultures from transformed colon tissues
has been determined by measuring cell loss from
monolayers (Hague et al. 1993).
6.6.5. Products used in experimental carcinogenesis
(Martin et al. 1981). Spontaneous colorectal cancer is
exceptional in animal models, but tumours can be created
easily in rats, mice or hamsters using chemicals such as
N-methylnitrosourea and N-methyl-N Р€-nitro-N-nitrosoguanidine (Table 6). These products are direct carcinogens, which explains their local efficacy and their
specificity when administered intra-rectally. Others such
as 1,2-dimethylhydrazine or azoxymethane must be first
metabolized by the liver, then at the level of the target
cell, in order to be carcinogenic.
1,2-Dimethylhydrazine and derived azo and azoxy
alkanes represent the carcinogens most commonly used to
induce intestinal carcinomas in rats, mice and hamsters, by
the oral or subcutaneous route. In rats, precancerous lesions
such as adenomas are exceptional, cancers arising most
commonly without any precursor adenoma, which is the
opposite to what is observed in man. In mice, it is easier to
induce adenomas than carcinomas (Maskens, 1976).
Secondary bile acids are cocarcinogens. They have been
shown to promote colorectal carcinogenesis in animal
models by increasing tumour formation rate induced by
carcinogens, or increasing colonic cell hyperproliferation
through the production of diacylglycerol and stimulation of
protein kinase.
6.6.6. Types of lesion (Weisburger, 1973). The most
studied lesion is adenocarcinoma induced either directly
or, more often, indirectly. When azoxymethane is used by
the subcutaneous or intramuscular route, intestinal carcinomas are formed in 100 % of cases. Small doses induce
tumours in the proximal colon and the caecum, whereas
larger doses produce tumours mainly in the distal colon.
Such tumours arise on flat mucosa and form plaques, thus
mimicking human infiltrating tumours, which arise without any detectable adenomatous tissue and are named de
novo cancers. However, this type of tumour is rare in
human carcinogenesis, particularly in Western countries
where tumours are mainly of the fungating type and arise
in a pre-existing adenoma. Aberrant foci are interesting to
study as they can be easily observed macroscopically.
After a single injection of azoxymethane, aberrant foci
have been described after early slaughter of the animals.
These lesions are considered by some authors to be an
early marker of tumour risk. They are more common in
the rectum (90 % of animals) than the caecum (10 % of
animals), but tend to migrate with time, with a decrease in
rectal lesions and an increase in caecal lesions after 4
Changes in colonic cell proliferation, which will be
described in man elsewhere, are largely used to test the
protective effect of products, e.g. Ca, against secondary bile
acid-induced proliferation.
6.6.7. Transgenic mouse models for colon cancer studies.
A number of inbred mouse models carrying germ-line
mutations at the Apc gene (the murine homologue of APC,
which is mutated in patients with familial adenomatous
polyposis) have been developed. These animals exhibit
spontaneous tumours throughout the intestinal tract, usually
in the first few months of life. They are of use in studies of
the interaction of diet and colon cancer and provide a model
that dispenses with the need for chemical induction of
carcinogenesis. The susceptibility of tumour incidence in
these mice to dietary modulation is under investigation in a
number of laboratories. Table 7 lists four such mouse
models in current use.
Table 6. Current methods for studying colon carcinogenesis in animals
MNNG (N -methyl-N Р€-nitro-N nitrosoguanidine) and MNU
(N -methylnitrosourea)
DMH (1,2-dimethylhydrazine)
Intra-rectal instillations
Azo and azoxy alkane derivatives
Subcutaneous or intramuscular
Secondary bile acids
Intra-rectal or oral
Oral or subcutaneous
Colon adenocarcinomas, squamous
cell anal carcinomas, spleen and
liver haemangiomas
Multiple colorectal adenocarcinomas
and a few adenomas in rats
In mice adenomas, and a few
carcinomas; adenocarcinomas in
plaques (de novo)
Alone: colonic cell hyperproliferation
After DMH: increased number of
S. Salminen et al.
Table 7. Transgenic mouse models currently in use in colon cancer
Min (multiple
neoplasia), usually
used in the heterozygous cross with
C57B16/J mice
Apc 1638T
Apc D716
Tumour yield
per animal
Germline nonsense
mutation at
codon 850 of Apc
About 100
adenomas with
the majority in the
small intestine
Apc disrupted at
codon 1638,
probably leading
to a null allele for
Apc disrupted at
codon 1638
leading to a
Apc disrupted at
codon 716,
leading to a
truncated protein
About five intestinal
No intestinal
tumours at 5
200–500 intestinal
6.6.8. Limits of experimental models (Maskens &
Dujardin Loits, 1981). Experimental carcinogenesis in
animals creates mainly infiltrating de novo carcinomas,
and is an indirect process in most cases, i.e. multiple
metabolic transformations are needed, the last occurring in
the colon itself and involving bacterial enzymes such as bglucuronidase, azoreductase or nitroreductase. In man, in
particular in high-risk countries such as Western Europe,
North America or Australia, most colorectal tumours are
polypoid, fungating, and arise on a pre-existing adenoma. It
has been estimated that, in the distal colon and rectum,
where most tumours arise, over 80 % of cancers arise
through the adenoma–carcinoma pathway. Indirect evidence, such as the type of mutation of the p53 protein,
points to a major role played by secondary bile acids, i.e.
endogenous carcinogenesis, whereas carcinogenesis in
animals is called exogenous carcinogenesis. The role of
the latter may be more important in low-risk countries
such as Japan, but is likely to be of little importance in
high-risk countries. Therefore, it is difficult to extrapolate
from animal models to man, in particular regarding the
importance of bacterial enzymes, apart from the major role
played by the 7 a-dehydroxylase, which converts primary
into secondary bile acids.
7. Human studies on the effects of food
and food components
Among the components likely to be used in functional
foods, prebiotics and probiotics are already used as food
Probiotics have been variously defined (Fuller, 1991,
1992; Havenaar & Huis in’t Veld, 1992) largely on the
basis of their initial use in animal feeds. For the purposes of
human nutrition we suggest that a probiotic is best defined
as �a live microbial food ingredient that is beneficial to
A prebiotic is a �nondigestible food ingredient that
beneficially affects the host by selectively stimulating the
growth and/or activity of one or a limited number of bacteria
in the colon, that have the potential to improve host health’
(Gibson & Roberfroid, 1995).
Another approach is the use of synbiotics. A synbiotic has
been defined as �a mixture of probiotics and prebiotics that
beneficially affects the host by improving the survival and
implantation of live microbial dietary supplements in the
gastrointestinal tract, by selectively stimulating the growth
and/or activating the metabolism of one or a limited
number of health-promoting bacteria, and thus improving
host welfare’ (Gibson & Roberfroid, 1995).
7.1. Prebiotics (Gibson & Roberfroid, 1995)
The key criterion for a food ingredient to be classified as
a prebiotic is selective stimulation in human subjects of
the growth of potentially beneficial bacteria in the gut. A
prebiotic may repress pathogen growth or virulence and
induce systemic effects that can be beneficial to health.
The prebiotics identified today and which have served to
introduce the concept (Gibson & Roberfroid, 1995) are nondigestible oligosaccharides that are fermented in the colon.
They are obtained either by hot-water extraction from
plants, eventually followed by enzymic hydrolysis of the
extracted molecules, or synthesis from a disaccharide using
-osyl transferases. The prebiotics that are available in
Europe at present or that are being developed, belong to
two groups, characterized by the major -osyl monomer they
are composed of; namely, the fructosyl prebiotics (fructooligosaccharides) and the galactosyl prebiotics (galactooligosaccharides). Daily intake of fructosyl prebiotics for
a few weeks leads to a selective stimulation of the growth of
bifidobacteria. Many studies using different dose regimens
and different methods of microbial analysis have shown
selective stimulation of bifidobacteria (Roberfroid et al.
1997). Ito et al. (1993) have reported the same effect for
galactosyl prebiotics. In addition, the experiments reported
by Gibson et al. (1995) demonstrate that the growth of
bifidobacteria is accompanied by a reduction in the number
of other populations, e.g. bacteroides, clostridia and fusobacteria, thus leading to a major modification in the composition of the colonic microflora. Lactitol (4-O-b-d
galactopyranosyl-d-glucitol) has been shown to have
prebiotic properties (Ballongue et al. 1997).
The nutritional properties of prebiotics have been summarized in the report of an International Life Sciences
Institute (ILSI) Europe workshop entitled Colonic Microflora: Nutrition and Health (Roberfroid et al. 1995) and in
the proceedings of the 1st International Conference on
East–West Perspectives in Functional Foods organized by
ILSI (Roberfroid, 1996). In a recent monograph, Cummings
(1997) also reviewed the nutritional properties of prebiotics.
The health-promoting consequences of prebiotic fermentation include increased faecal biomass and consequently
stool weight and/or stool frequency (Roberfroid et al. 1993;
Gibson et al. 1995). Colonic fermentation of prebiotics
produces SCFA mainly acetate, propionate and butyrate
(Roland et al. 1995). Through fermentation and absorption
of SCFA prebiotics have part of their energy salvaged. Their
Gastrointestinal physiology and function
digestible energy is of the order of 4–9 kJ/g (1–2 kcal/g)
(Roberfroid et al. 1993; Molis et al. 1996). This property
has led to their use as bulking ingredients, sugar substitutes
and, for inulin, as a fat replacer (Coussement, 1996).
7.2. Probiotics (Salminen et al. 1996a,b,c)
The bacterial genera most often used as probiotics are
lactobacilli and bifidobacteria. They can be given with
fermented foods such as yoghurt, fermented vegetables or
meats and they may briefly establish in the gut. A number of
health-related effects are documented. Such established
effects are listed in Table 8 and each effect has been
shown in at least two human clinical studies by different
research groups.
Colonic fermentation has been shown to be altered
following probiotic intake either as fermented milks or
freeze-dried cultures. Japanese studies have shown that
oral administration of certain lactic-acid bacteria increases
the numbers of endogenous lactobacilli in faeces. Similarly,
an increase in faecal bifidobacteria has been observed, and
the numbers of clostridia have decreased (Hosoda et al.
1994; Benno et al. 1996). Recent studies by Alander et al.
(1996) have indicated that the potential to colonize human
colonic mucosa differs from that of faecal samples. Thus,
more human studies are clearly needed to understand
changes in both colonic microflora and colonic mucosal
microflora during probiotic intake.
7.2.1. Alleviation of lactose intolerance symptoms. The
majority of the world’s population have low levels of bgalactosidase in their small-bowel mucosa. Most lactasedeficient people are symptom-free if they consume only
limited amounts of milk, except those subjects who are
severely lactose intolerant. A beneficial effect of probiotics
on lactose digestion has been demonstrated (Sanders,
1993). In particular, in studies comparing yoghurt and
milk consumption it was shown that yoghurt consumption
enhances lactose digestion in lactase-deficient subjects
(Kolars et al. 1984; Marteau et al. 1990) and slows
orocaecal transit.
7.2.2. Immune enhancement. An enhancement of the
circulating IgA antibody secreting cell response was
observed in infants supplemented with a strain of Lactobacillus casei, and was correlated with shortened duration
of diarrhoea in the study group when compared with a
placebo group (Kaila et al. 1992). Other studies reported
an enhancement in the non-specific immune phagocytic
activity of granulocyte populations in the blood of human
volunteers after consumption of Lactobacillus acidophilus
and Bifidobacterium bifidum (Schriffin et al. 1995; Marteau
Table 8. Health-related effects of currently available probiotics (for
detailed references, see Marteau & Rambaud, 1993; Lee & Salminen,
1995; Salminen et al. 1996a, b, c)
Alleviation of symptoms of lactose intolerance
Immune enhancement
Shortening the duration of rotavirus diarrhoea
Decreasing faecal mutagenicity
Decreasing faecal bacterial enzyme activity
Prevention of recurrence of superficial bladder cancer
et al. 1997a, b). Since phagocytic activity is involved with
natural immunity and phagocytes are implicated in antibody
immune responses as antigen-presenting cells, it is possible
that stimulation of intestinal IgA antibody responses
induced by lactic-acid bacteria may be explained partly by
an effect on phagocyte cell functions. Ingestion of yoghurt
has been reported to stimulate cytokine production,
including interferon-g in human blood mononuclear cells
(Solis-Pereyra & Lemonnier, 1996).
7.2.3. Acute gastroenteritis. Several studies, by different groups and in different conditions, have shown that
some probiotic lactobacilli significantly shorten the duration
of rotavirus diarrhoea in developed (Guarino et al. 1977)
and developing countries (Raza et al. 1995; Pant et al.
1996). For one strain, a similar clinical effect on the
duration of rotavirus diarrhoea without an increase in
immune response has been reported (Shornikova et al.
1997). Bifidobacterium bifidum (now reclassified as Bifidobacterium animalis) has been reported to prevent rotavirus
diarrhoea (Saavedra et al. 1994).
7.2.4. Faecal mutagenicity and enzymes. Lactic acid
bacteria influence the mutagenicity of intestinal contents
and the levels of faecal microbial enzymes, such as bglucuronidase, b-glucosidase, nitroreductase and urease
(EC Lactobacillus acidophilus has been shown to
decrease faecal and urinary mutagenicity in healthy
volunteers consuming fried minced beef. The same strain
decreased faecal E. coli levels in colon-cancer patients and
reduced faecal b-glucuronidase levels. Similar results have
been reported for many probiotic lactobacilli strains (Goldin
& Gorbach, 1984; Goldin et al. 1992; Ling et al. 1994;
Morotomi, 1996).
The prophylactic effects of oral administration of a
lactobacillus strain on the recurrence of superficial bladder
cancer have been reported in two Japanese studies (Aso &
Akazan, 1992; Aso et al. 1995).
7.3. Diet and colon cancer
The principal risk factors for sporadic colorectal cancer are
high intakes of animal fat and meat, particularly red meat,
excess energy intake, high intakes of refined cereals and of
alcohol, particularly beer for colorectal carcinogenesis, and
lack of physical exercise. A number of dietary factors have
been proposed as potentially protective (Potter et al. 1993).
7.3.1. Dietary protective factors. A high intake of
vegetables and fruit is associated with a decreased risk of
colorectal cancer and adenomas throughout the world
(Trock et al. 1990). Although vegetables such as the
cruciferae may have a specific protective affect via
antioxidant substances such as indoles, all fresh vegetables
seem to be protective, in particular against cancer of the
distal bowel. Components of fruit and vegetables that have
been implicated as protective against colorectal cancer
include NSP (dietary fibre), micronutrients (vitamins C, D
and E, b-carotene, Ca and Se), glucosinolates, phenols,
lignans, flavonoids and isoflavonoids.
One of the most important protective components of
vegetables and unrefined cereals is dietary fibre. The
mechanism may be through a diluting effect of carcinogens,
a reduction in transit time, fermentation and the production
S. Salminen et al.
of SCFA and alterations in N and bile acid metabolism.
Although results of individual case–control studies have
been disappointing, a meta-analysis of thirteen of these
studies (Howe et al. 1992) found a significant inverse
association between fibre intake and risk of colorectal
cancer with a relative risk close to 0В·5 for consumers of
over 27 g/d. When extrapolating these data to the American
population, the authors estimated it to be possible to reduce
the risk of colorectal cancer by 31 % i.e. 50 000 cases per
year by increasing the daily consumption by 13 g. However,
the multiplicity of methods for assessing dietary fibre intake
in these studies makes any quantitative recommendations
Ca and vitamin D are other potentially protective
factors that could easily be used as preventive agents. A
recent American cohort study summarized the relations
between Ca, vitamin D and dairy products (Bostick et al.
1993). Of thirteen studies (nine case–control and four
intervention studies), eight suggested an inverse relationship, which was statistically significant in five. Most studies
that observed a protective effect of Ca came from Nordic or
Anglo-Saxon communities, whereas no study demonstrated
a protective effect of Ca or dairy products in Latin communities. It can, therefore, be suggested that the way Ca is
consumed, and perhaps the general level of Ca consumption, is of importance. It has been suggested that fermented
dairy products could be protective. In a French study
(Boutron et al. 1996), a reduced risk of large adenomas
was associated with a moderate intake of yoghurt.
Other dietary protective factors which have been
investigated in intervention studies of recurrence of adenomas are the so-called antioxidant vitamins. The results of
these studies have so far been disappointing (Greenberg et
al. 1994; MacLennan et al. 1995).
8. Safety issues
(Donohue & Salminen, 1996; Roberfroid et al. 1997)
Safety issues with new prebiotics and probiotics, including
genetically modified bacteria, should be assessed according
to European Union Novel Food Regulations on a case-bycase basis. Safety aspects of currently used prebiotics and
probiotics are discussed in relation to their physiological
8.1. Prebiotics (Van Loo et al. 1995)
Although the European Union regulation on novel foods and
novel food ingredients leaves room for interpretation, it is
very likely that most prebiotics placed into the European
market after this regulation comes into force will fall within
its scope. They will, therefore, be subject to safety and
nutritional evaluation on a case-by-case basis.
Fructosyl-type ingredients, for which prebiotic properties
are claimed, are already in the market. Fructosyl-type
ingredients are natural components of a variety of fruits,
vegetables and cereals, and consequently are consumed
regularly (a few g/d) as part of the current diet (Van Loo
et al. 1995). They are classified as natural food ingredients
and cleared as novel food ingredients. Similarly galactosyl
prebiotics have been cleared as novel food ingredients by
the Health Department of The Netherlands (Staatscourant,
8.2. Probiotics
(Adams & Marteau, 1995; Donohue & Salminen, 1996)
The use of lactic acid-producing bacteria in foods has a
long history and most strains are considered commensal
micro-organisms with little or no pathogenic potential.
Their ubiquitous presence in intestinal epithelium and the
human gastrointestinal tract, and their traditional use in
fermented foods and dairy products attest to their safety.
Members of the genus Lactobacillus are most commonly
given safe or generally recognized as safe (GRAS)
status, whereas members of the genera Streptococcus
and Enterococcus contain many opportunistic pathogens.
Case reports from the literature of lactic acid-producing
bacteria causing clinical infection in human subjects have
recently been analysed in reviews by Gasser (1994) and by
Aguirre & Collins (1993). Both reviews conclude that,
considering their widespread consumption, lactic-acid
bacteria appear to have a very low pathogenic potential.
Two recent Finnish studies confirm that the number of
infections associated with lactic-acid bacteria is extremely
small and no case could be linked to commercial probiotics
(Saxelin et al. 1996a,b).
9. Critical evaluation of present knowledge
Each of the foregoing sections has examined in depth one
aspect of the science of gastrointestinal physiology and
function in relation to probiotics and prebiotics. The following conclusions are derived from the discussions within
each section.
9.1. Intestinal microflora
The intestinal microflora has been studied using traditional
methods. A current problem is the presence of nonculturable species, which require new methodologies to be
developed for their detection and measurement.
The establishment of the normal human intestinal
microflora, its components and metabolic activities, requires
further study. Similarly, knowledge is needed on the composition and activities of the flora in different ethnic groups,
at different ages and in different countries.
Prebiotics and a few probiotics have already been shown
to have the potential to modify significantly the composition
of the intestinal microflora. In particular, the stimulatory
effect of fructosyl prebiotics on the growth of bifidobacteria
is well established. Moreover, prebiotics and probiotics are
interesting tools with which to study the physiological
consequences of changes in metabolic activities following
such modification.
9.2. Mucosal function
There is evidence for a strong interaction between the
intestinal microflora, gut mucosa and GALT and the functions and dysfunctions of the gastrointestinal tract. It has
been shown that probiotics may change the gut mucosal
Gastrointestinal physiology and function
barrier by stabilizing the intestinal mucosa, normalizing
intestinal permeability and improving gut immunology.
However, there are differences in the function and activity
of different probiotics. Another consequence of intake of
prebiotics and probiotics is the prevention of overgrowth
of pathogenic bacteria and viruses. Taken together, these
modifications have been shown to influence the gut barrier
Future human studies should consider intestinal immunity and its modulation by resident probiotic bacteria or
prebiotic components in more detail. Local release of
cytokines induced by inflammatory reactions may amplify
adverse reactions to food components within the intestinal
tract and other parts of the body.
9.3. Gastrointestinal physiology
Gut bacteria play a role in bowel functions like faecal
mass, stool frequency, regulation of colonic pH, production
of SCFA and salvage of energy from non-digestible food
components. Any modification of the microflora is likely to
influence these functions, but most of the results of human
studies with pro- and prebiotics are preliminary and yet to
be confirmed. In particular, the interaction of probiotics and
prebiotics with the endocrine activity of the gut requires
9.4. Methodology
There is clearly a need for new methodology to measure
and characterize the composition of faecal microflora, in
particular in large human nutrition studies.
New biomarkers specifically involved with immune
responses need to be defined and validated for nutritional
studies. Germ-free animals (gnotobiotic mice) offer a model
to clarify the specific effect of a bacterial strain or a food
component on a given immune response.
Development of early markers of carcinogenesis is
urgently required for human intervention studies.
9.5. Human studies on health benefits
9.5.1. Prebiotics (Table 9). There are currently few
reports of well-designed and well-documented human
Table 9. Established and postulated functional effects of currently
available prebiotics in human subjects
Established functional effects
Non-digestibility and low energy
value (< 9 kJ/g)
Stool bulking effect
Modulation of the gut flora,
promoting bifidobacteria and
repressing clostridia
IBS, irritable bowel syndrome.
Postulated areas for future
Prevention of intestinal disorders
(IBS, ulcerative colitis) and
infections, including diarrhoea
Modulation of immune response
Prevention of colon
Reduction in serum levels of
triacylglycerols and
Improved bioavailability of
minerals (Ca, Mg)
Table 10. Postulated clinical effects of probiotics
Areas for future research
Research on mechanisms
Regulation of intestinal motility.
Modulation of intestinal and
systemic immune responses.
Reduction and protection of
radiotherapy-associated intestinal
dysfunction. Prevention of intestinal
Intestinal microflora effects,
immunomodulation effects,
competitive exclusion, cholesterol
intervention studies with prebiotics. Future studies may
indicate differences between the effects of prebiotic
components. Thus, it is important that each compound is
tested separately or in products designed for a particular
9.5.2. Probiotics (Table 10). Alleviation of lactose
maldigestion symptoms is well characterized for some
probiotics. The ability of certain probiotic strains to shorten
the duration of rotavirus diarrhoea has been established in
several studies. Immunomodulation has been demonstrated for some probiotic strains and one strain has been
documented to reduce the recurrence of superficial bladder
cancer in human subjects. Novel methodologies are required
to provide further data on the mechanisms of competitive
exclusion and microflora modification.
For other areas of interest there are still few welldesigned and well-conducted human intervention studies
with probiotics. There are indications of significant differences between probiotic bacterial genera and species, and
between strains of the same species. Thus, it is important
that each strain is tested on its own or in products designed
for a particular function. Further research is needed on the
immune response, intestinal cancer and the mechanisms that
underlie probiotic effects.
9.5.3. Diet and colon cancer. The major challenge at
present is to demonstrate within intervention studies which
specific dietary factors are able to decrease the risk of colon
cancer. Recurrence of large adenomas, the step prior to
cancer, is a promising tool for use in human studies and
could be applied to prebiotics, probiotics and synbiotics
using functional food products as test products. Colon
cancer-related variables need to be carefully characterized
in the diet and among the microflora.
9.6. Safety
The human consumption of prebiotics and probiotics, at
least lactic-acid bacteria, appears to be safe.
10. Recommendations for future research priorities
Evidence has accumulated to support an important role for
gastrointestinal function and the intestinal