Clinical Science (1995) 88, 375-392 (Printed in Great Britain)
375
The influence of trans fatty acids on health: a report
from The Danish Nutrition Council
Steen STENDER, J$rn DYERBERG, Gunhild HqLMER, Lars OVESEN and Brittmarie SANDSTRdM
Danish Nutrition Council, Esplanaden 80, I263 Copenhagen, Denmark
PREFACE
On Saturday 6 March 1993, the Danish News
received a press statement via Reuters’ Bureau
containing the results of a large US study, with the
headlines “Study connects vegetable margarine and
heart disease” and “Replacing butter and other
animal fats by vegetable margarine may cause
rather than prevent heart disease”. This surprising
conclusion was drawn by a research team headed by
Professor Walter Willet of Harvard University in an
article published the same day in the journal Lancet.
The study was based on the food habits and health
of 85 095 American nurses examined in 1980 and
followed up for 8years. Women who daily consumed more than four teaspoonfuls (20g) of margarine had a 50% higher risk of coronary thrombosis. This apparently harmful influence by margarine
could be caused by the trans fatty acids, generated
when vegetable oils are hydrogenated for the production of margarine.
The study and topic received substantial attention
in the Danish media, and, in order to minimize the
confusion regarding different fats and their influence
on health, the Danish Nutrition Council appointed
a task group to evaluate the question in a broader
sense. After more than a year’s work, the report
now available concludes that trans fatty acids may
increase the process of developing arteriosclerosis to
the same, or possibly to a higher, extent than
saturated fats, and possibly have a harmful effect on
the growth of the fetus.
A considerable reduction of the trans fatty acid
content in our diet may prolong the average human
life by up to several months for women and even
more for men. On this basis, the Danish Nutrition
Council recommend a re-adjustment of the industrial process, allowing for a gradual reduction of
trans fatty acids in Danish margarine products
within 2-3years, so that even people with a large
consumption of products containing trans fatty
acids are ensured a low total consumption. The
large amount of trans fatty acids in food is a
phenomenon to which man has not previously been
exposed. There is no reason, therefore, to assume
that a dramatic reduction of the trans fatty acid
content in margarines may have a negative impact
on health. This fact has simplified the Danish
Nutrition Council’s basis of decision.
The Danish Nutrition Council hope that this
report will be considered a weighty document by
the health authorities and the food industry. Compared with the difficult task of prolonging the
average human life in Denmark by affecting lifestyle
elements, an industrial reduction of the trans fatty
acid content in margarine products seems possible
without any considerable inconvenience to the
consumer.
ARNEASTRUP
CHAIRMAN
SUMMARY
Trans fatty acids constitute 0-30% of the fat in
Danish margarines, most in industry and bakery
margarines and usually less in table margarine. The
trans fatty acids make margarines more solid at
room temperature and therefore provide an economical storage advantage. In British and U.S. reports
from 1984-1989, the trans fatty acids were more or
less acquitted of unhealthy effects. During the last
5-6years, however, a series of new studies has been
published regarding both the connection between
the consumption of trans fatty acids and the occurrence of coronary heart disease and the impact on
the lipoprotein level in plasma. Studies suggest that
the consumption of trans fatty acids from margarine
is equally, or perhaps more, responsible for the
development of arteriosclerosis than saturated fatty
acids. In addition, it is now clear that both the fetus
and the breast-fed baby are exposed to trans fatty
acids in relation to the mother’s consumption. A
couple of recent studies suggest a possible restrictive
influence of the trans fatty acids on the weight of
the fetus. The average consumption of trans fatty
acids from margarine in Denmark in 1991 was
approximately 2.5 g/day per person. For about
15OOOO adult Danes, the consumption is assumed to
be more than 5g/day per person. On this basis, the
Danish Nutrition Council recommend that the consumption of trans fatty acids is reduced as much as
possible. This can be done by reducing the fat
content in food and by reducing the trans fatty acid
content in all Danish margarine products to 5% or
less. Thereafter, the group of adult Danes, including
pregnant and breast-feeding women, with a large
consumption of margarine and margarinecontaining products, will on average only consume
2 g of vegetable trans fatty acids/day. This corresponds to the consumption in the low-risk groups in
the above-mentioned epidemiological studies.
376
S. Stender et al
In addition, the Danish Nutrition Council
encourage the producers of margarines to make
products that can be marketed as ‘free of trans fatty
acids’.
TERMS OF REFERENCE
On 8 March 1993, the Danish Nutrition Council
appointed a task group with the following terms of
reference: (i) to evaluate the influence on health of
trans fatty acids in food; (ii) to estimate the Danish
consumption of trans fatty acids and the separate
sources in connection with this; (iii) if relevant, to
develop suggestions as to how the trans fatty acid
content in food can be reduced to an acceptable
level while considering the interests of both the
consumer and the industry.
The task group consisted of Steen Stender (Chairman) MD, Medical Superintendent, Jsrn Dyerberg
MD, Medical Superintendent, Gunhild Hslmer
MSc, Professor, Lars Ovesen MD, Head, and Brittmarie Sandstrom PhD, Research Professor.
Special consultants in the Danish Nutrition
Council have been attached to the task group,
namely Lars Garby MD, Professor and Ole Lander
Svendsen, Registrar.
The section on trans fatty acids and embryogeny
has been worked out in co-operation with Dr
Marianne Hsrby Jsrgensen and Kim Fleischer
Michaelsen MB, Senior Lecturer. The task group
finished its work on 1 March 1994, having held
seven meetings in all.
INTRODUCTION
In the spring of 1993, trans fatty acids demanded
renewed interest, when a US population study [ l ]
showed a connection between a large consumption
of these fatty acids from the diet and an increased
risk of coronary heart disease (CHD).
A large intake of trans fatty acids had previously
been suspected of representing an independent risk
of developing C H D [2]. Several studies have
pointed to trans fatty acids having an impact on the
plasma cholesterol level which is comparable with
the effects of saturated fatty acids [3].
In a report from 1985 [4], a U.S. expert committee concluded: “Human studies indicate that
elaidic acid or partially hydrogenated vegetable oils
are no more, or little more, cholesterolaemic than
oleic acid”. A corresponding report from The British
Nutrition Foundation’s Task Force in 1987 [S]
concluded: “Studies of the effect of diets containing
different amounts of trans fatty acids in man do not
suggest any consistent effect of trans monounsaturated fatty acids on plasma cholesterol levels that is
attributable to the trans geometry per se”. In 1989
[ 6 ] , the US National Research Council concluded:
“Some effects of the trans fatty acid isomers on lipid
or lipoprotein metabolism may remain undetected.
However, most evidence indicates that these
isomers, in the quantities usually consumed in the
US diet, do not influence serum cholesterol concentrations”. In 1991 [7], a UK Department of Health
Report concluded: “The data did not adequately
refute or support the allegations of potential longterm adverse effects on health of isomeric fatty acid
intake in the UK”.
In the main part of the studies that underline the
above conclusions, no direct standards of effect for
C H D have formed the basis of the conclusions, but,
on the contrary, indirect standards of effect have
been used, i.e. total plasma cholesterol level as a
marker for the risk of developing CHD. In the
meantime, new studies [8-121 indicate that the
consumption of trans fatty acids affects the lipoproteins of the blood, i.e. high-density lipoprotein
(HDL), low-density lipoprotein (LDL) and lipoprotein(a) [Lp(a)], in an atherogenic direction without
necessarily being reflected in the total cholesterol
concentration.
It has previously been reported that the feeding of
test animals, over many years and generations, with
large amounts of trans fatty acids has not caused
measurable restraints in growth or changes of the
organs. In the above-mentioned US report [4], it is
concluded: “The low levels of trans isomers found in
tissue lipids of premature infants (0.1-0.9%) indicate
that human placenta is a barrier to the transport of
these isomers”. Within the last couple of years,
however, a few studies of the connection between
the trans fatty acid content in the plasma of mothers
and newborn babies have been concluded. These
studies show that trans fatty acids are conveyed
over the barrier between the placenta and the fetus
and, at the same time, the studies have given
occasion for suspicion about a negative impact from
the trans fatty acids on the weight of the fetus
[13-151.
In December 1993, a review dealing with a part of
all these new findings was published [16]. To the
question of whether the trans fatty acids are neutral
or damaging to health, the authors conclude:
“Although recent evidence appears to favour the
latter, further studies are required”.
In the present report, the newest literature about
the connection between the consumption of trans
fatty acids and CHD, embryogeny and cancer will
be dealt with. In particular, the works which were
inaccessible when the above-mentioned English and
American reports were made will be stressed.
SECTION I. FATTY ACID ISOMERS
1.1 Definition and chemistry
Trans fatty acid is a common designation of a
large number of different fatty acid isomers, formed
during hydrogenation. Fatty acid isomers are fatty
acids with the same gross formula. These can be
divided into positional isomers, where the position
of the double bond in the molecule varies, and
The influence of trans fatty acids on health: a report from The Danish Nutrition
Council
377
rapeseed oil used for nutrition today is regarded as
being free of erucic acid. The fatty acids present in
nature usually have a cis configuration, but trans
fatty acids can be found in both the animal and
vegetable kingdom (see Section 3).
I -2H
1.2 Formation in vitro (industrially) and in vivo
O
I
I
Ni
1
+IH
H/
H/E
H
8
\
;
I
-c
11
H10
NI
9
*
‘10
Fig. I. Principle for the formation of fatty acid isomers during the
process of hydrogenation
Table I. Examples of fatty acid isomers
Positional isomers
Geometrical isomers
cis 181 A9 = (n-9)
Oleic acid
cis 181 A9 = (n-9)
Oleic acid
and
trans 181 A9 = (n-9)
Elaidic acid
cis 181 AI I = (n-7)
cis-Vaccenic acid
cis 1 8 1 A6 = (n- 12)
Petroselinic acid
cis 221 A13 = (n-9)
Erucic acid
and
trans 221 A13 = (n-9)
Brassidic acid
geometrical isomers or cis-trans isomers, where the
configuration around the double bond changes.
Therefore, not all fatty acid isomers are trans fatty
acids. Examples of these are given in Fig. 1 and
Table 1
The numbering of double bonds is made either
from the carboxyl group or from the methyl group.
In the first case the position will be given the prefix
A, in the latter case the position is named ( n - x ) ,
where x is the number of the C-atoms before the
first double bond (previously called a).
Oleic and cis-vaccenic acids occur widely in
nature. Both can be formed in uiuo from acetate
residues, and are found in the animal and vegetable
kingdom. Petroselinic acid, for example, OCCULS in
parsley seeds. Erucic acid is a vegetable fatty acid
found among the Cruciferae and, therefore, originally also in rapeseed. As this fatty acid was
connected with harmful nutritional effects, an
almost erucic-free rape species has been grown. The
Fatty acid isomers are present in partially hydrogenated vegetable oils and marine oils used for the
production of margarine and shortenings. During
the process of partial hydrogenation, developed at
the turn of the century with the purpose of producing cheaper ‘butter-like products’, a more solid and
spreadable fat can be obtained from an oil with a
high content of polyunsaturated fatty acids
(PUFAs). In the hydrogenation process, which takes
place at high temperature and under pressure, the
oil is mixed with a catalyst, usually nickel, and
hydrogen is admitted. Binding of the unsaturated
fatty acids to the surface of the catalyst takes place
and, at the same time, a double bond in the fatty
acid molecule is opened (Fig. 1).
Provided a reaction with the hydrogen takes
place, saturation of the double bond is achieved.
The binding to the catalyst is, however, not stable
and if fatty acids are liberated from the catalyst, the
double bond may be regenerated, either in a cis or a
trans configuration. As the trans configuration is
energetically the most favourable, this structure will
be dominating.
A partial liberation of the fatty acid, which is
anchored at two points, would allow a new bond to
be established at a neighbouring C-atom (no. 8 or
no. 11 in Fig. 1). This means that a positional fatty
acid isomer can be generated. As described above,
either a cis or a trans isomer can be formed.
Provided there are more double bonds in the fatty
acids being hydrogenated, a large number of both
positional and cis-trans isomers may be formed.
The result of the hydrogenation will depend upon
the concentration of the catalyst, hydrogen pressure,
temperature and agitation [17]. Therefore, it is
possible to produce partially hydrogenated oils with
a large variation in amounts of positional, as well as
geometrical, fatty acid isomers from unsaturated
vegetable and marine oils.
By a controlled hydrogenation of soybean oil,
PUFAs may gradually be saturated, first linolenic
acid and then linoleic acid. At the same time, fatty
acid isomers of mainly trans 18:l are generated, but
trans 18:2 isomers are also formed, depending on
the extent of the hydrogenation. The distribution of
the positional fatty acid isomers in the cis as well as
the trans fraction of 18:l from partially hydrogenated soybean oil has been examined by Scholfield
et al. [18], who found that the double bonds were
distributed between positions A6 to A14 for the
trans fraction, with positions A10 and A l l as major
components. For partially hydrogenated marine oils
containing relatively large amounts of Cz0 and C22
370
S. Stender et al.
monoenes, an even larger variation in the distribution of positional fatty acid isomers in the trans and
cis fractions has been found. For eicosapentaenoic
acid (cis 205 A5, 8, 11, 14, 17) and docosahexaenoic
acid (cis 22:6 A4, 7, 10, 13, 16, 19), with the great
number of double bonds, numerous possibilities for
binding to the catalyst and thus for the formation of
reaction products exist. For CzOand Cz2 monounsaturated fatty acids, a distribution from the A7 to
the A17 position has been found for both chain
lengths [19].
During partial hydrogenation of vegetable oils it
is desirable to maintain as high a content of linoleic
acid as possible, In the process, cis,trans and
trans,cis 18:2 are also generated in minor amounts.
However, the content of trans,trans 18:2 is normally
very low.
The amount of long-chain (CzOand Cz2) di- and
tri-unsaturated fatty acids in partially hydrogenated
marine oils varies with the conditions of the hydrogenation, but a total of 2-6% of C20:2and C22:2is
present, whereas the triene content is low. The
isomerism of the fatty acids mentioned is complicated and is not elucidated in detail.
The total content of trans fatty acid isomers in
margarines and shortenings, of which the main part
is trans 18:1, varies widely, but lies in the area of 520% of the fat content.
Another source of formation of fatty acid isomers
is a bacterial hydrogenation in the intestinal tract of
ruminants. The unsaturated fatty acids in the feed,
i.e. in grass and other coarse feed, undergo an
enzyme-catalysed biohydrogenation into saturated
and monounsaturated fatty acids. The latter consist
of both geometrical and positional isomers. Fat in
dairy products and other fat from cows, as well as
meat from sheep and goats, therefore contains small
amounts of fatty acid isomers (3-5% of the fatty
acid content). In butter, it has been shown that the
trans monoene fatty acid isomers have positions of
the double bond varying from A6 to A16, with A9
and All as major components [20], whilst oleic
acid constitutes the dominating fraction (95%)
among the cis monoenes. Trans fatty acids may also
occur as natural components of vegetables; for
example, small amounts of trans 16:l A3 are found
in peas, cabbage and spinach [21].
corresponding fatty acids with unsaturation at even
positions. The melting point of cis 18:l A9 is
approximately 11”C, whereas it is approximately
23°C for both cis 18:l A8 and cis 18:l AlO. The
corresponding values for trans fatty acids are
approximately 45°C for trans 18:l A9 and approximately 52°C for trans 18:l A8 and trans 18:l A10
c221.
Thus, it is not the distribution between the trans
fatty acids alone, but also the cis positional fatty
acid isomers that determine the spreadability of the
edible fat.
A high melting-point for a fat might influence the
absorption from the gut. It has been shown in
human experiments, however, that both trans and
cis 18:l isomers with melting points at about 50°C
were absorbed 90-100% [23].
1.4 Biochemistry and metabolism
With the introduction of a trans double bond in a
fatty acid molecule, the spatial structure will be
changed so that it is straightened out and dimensionally looks like the corresponding saturated fatty
acid (Fig. 2), but with a ‘twist’ in the molecule,
which affects the sectional area and thereby the
space requirements in a membrane. One could
therefore assume that the characteristics of the trans
fatty acids are in between those of the corresponding cis unsaturated fatty acids and saturated fatty
acids.
Regarding the incorporation of fatty acids into
the organism, a number of studies have shown that
the cis as well as the trans C18 monoene isomers
react differently compared with oleic acid and saturated acids. Emken [23] has thus reported that
positional as well as geometrical fatty acid isomers
from foods are incorporated in human plasma triglycerides to a smaller extent than oleic acid, but
the trans fatty acids can be incorporated in the
triglycerides of the depot fat. On the contrary, the
1,33 A
1-1
1.3 Impact of hydrogenation on the melting point of the
fatty acids
As mentioned, the most important reason for
hydrogenation of oils is the demand for an
increased melting point of the fat. All trans fatty
acids have, compared with a corresponding cis fatty
acid, higher melting points; for example, oleic acid
(cis 18:l A9) has a melting point of approximately
11°C while elaidic acid (trans 18:l A9) has a melting
point of approximately 45°C [22].
In general, the C18 monoenes with double bonds
at uneven positions have lower melting points than
-3.01
C’
A-
, y 4
12,521
Fig. 2, Structure of trans (I), cis (11) and saturated fatty acid (111)
with the same number of C-atoms. A cis bond introduces a bend in the
molecule, whereas a trans bond produces a molecular structure with
dimensions much like a saturated fatty acid.
A
The influence of trans fatty acids on health: a report from The Danish Nutrition Council
Fig. 3. Polyunsaturated fatty acid formation through A6 and AS
desaturation of oleic acid, linoleic acid, linolenic acid and elaidic
acid, respectively.
trans isomers are being discriminated in the plasma
cholesterol-ester fraction. For phosphatidylcholine it
was found that the 1-position, which normally contains a large amount of saturated fatty acids, also
had a preference for trans isomers and for certain
positional fatty acid isomers, whereas in the 2position, which normally contains PUFAs, trans
isomers were not present.
These data are in accordance with results from
animal experiments, where feeding with partially
hydrogenated oils clearly showed a preference for
incorporation of trans fatty acids in position-1 of
the membrane phospholipids [24, 251. A preferential
incorporation of the positional isomers also takes
place in the 1-position, resulting in a lower content
of saturated fatty acids in this position [26].
For partially hydrogenated marine oils, containing Czo and C , , fatty acids with trans as well as cis
configuration, a chain shortening to C,, and, to a
lesser extent, to C16 takes place in uiuo before the
incorporation into tissue phospholipids [27]. The
chain shortening is believed to occur in the peroxisomes. In depot fat and organ triglycerides, CZotlis
found, but not Czz:l. The amount of trans fatty
acids present in depot fat is, however, less than in
the food consumed, which implies an increased
metabolism. Studies with humans fed labelled C18
cis and trans monoenes [23] also showed a faster
removal of the trans isomer.
Fatty acid isomers influence the formation of
long-chain PUFAs. These PUFAs are formed from
the essential fatty acids, linoleic acid (cis 18:2 A9,
12) and linolenic acid (cis 18:3 A9, 12, 15), by
introduction of a number of new double bonds
(desaturations) and by elongation of the carbon
chain. The fatty acids synthesized are either normal
components of the cellular membranes of different
tissues or precursors for eicosanoid production. It is
therefore important to understand the influence of
the fatty acid isomers on the above-mentioned
conversion processes, shown in Fig. 3.
Lemarchal and Munsch [28] showed that elaidic
acid could be transformed into cis 5, trans 9 18:2
when rats were fed trielaidin, but not when the diet
contained linoleic acid. The reaction rate of the
enzymes introducing new double bonds in a given
fatty acid can be restrained by a fatty acid competing for the active site of the enzymes. It has been
shown for the rate-limiting enzyme in the reaction,
the A6 desaturase, that the ability to transform a
379
certain fatty acid depends on the level of unsaturation as well as the chain length and the
configuration of the double bonds: 18:3 ( n - 3))18:2
(n-6))cis 18:l A9)trans 18:l A9 [29, 301. With a
sufficient amount of an unsaturated fatty acid
present, the impact of trans monoene fatty acids will
be limited by this model. The conversions have also
been examined for trans isomers of linoleic acid.
With pure fatty acid isomers of 18:2, Privett et al.
[31] showed that only cis 9, trans 12 18:2 was
transformed to the arachidonic acid analogues.
Later studies with mice [23] showed that the conversion rate is identical for cis 9, cis 12 18:2 and cis
9, trans 12 18:2. Despite this, no change was found
in the arachidonic acid content in the membrane
phosphatidylcholine after feeding trans fatty acid
isomers of C18:2to rats, indicating that the limiting
factor is rather the incorporation into the
phospholipids.
Anderson et al. [32] reported that trans,trans 18:2
decreased the conversion of linoleic acid, but as
trans,trans 18:2 is only formed in very small
amounts during hydrogenation, the consumption
through food is insignificant. The impact on the
conversion of linoleic acid is therefore assumed to
be very small.
In experiments with rats, it has been observed
that partially hydrogenated oil, of vegetable as well
as of marine origin, in the food gave a lower
content of arachidonic acid in liver lipids, although
linoleic acid was supplied in appreciable amounts in
the diet [33-351. In incubation experiments with
liver microsomes, it was later shown that partially
hydrogenated vegetable oil containing C18:, isomers
could inhibit the A6 desaturation, whereas the partially hydrogenated marine oil, which apart from
C18:1isomers contained isomers of 201 and 221,
inhibited the A6 as well as the A5 desaturation [36].
The influence of the dietary linoleic acid content on
the conversion has also been examined. Increasing
amounts of linoleic acid increased the desaturation
but could not completely inhibit the competitive
effect of the fatty acid isomers [27, 371. No adverse
effects from feeding the rats with partially hydrogenated fats were observed in these experiments.
Long-term trials with rats through many generations did not show any influence of trans fatty
acids on either growth, life span or reproduction
~381.
In conclusion, fatty acid isomers can compete
with PUFAs in a number of enzyme reactions,
whereby the formation of. long-chain . PUFAs is
decreased. The extent of inhibition depends on the
ratio between fatty acid isomers and PUFAs, but
cannot be completely removed, even at relatively
high PUFA concentrations.
IS Determination of
trans fatty acids
The fatty acid isomers may, as mentioned previously, have a different biological and thus nutrition-
380
S. Stender et al.
al significance. It is therefore necessary to be able to
determine the total content as well as the amount of
the different fatty acid isomers, especially the trans
isomers, in the fats included in our nutrition.
The majority of the trans bonds occur in the
monoene fatty acids, but in the partially hydrogenated fats, minor amounts of trans isomers of
dienoic and trienoic fatty acids may also occur. A
complete characterization will, therefore, be circumstantial and too time-consuming for general
purposes.
The techniques that can be used are based on
physical (IR spectroscopy, MS, UV spectroscopy,
NMR) as well as chromatographic (GC, TLC and
HPLC) and pure chemical principles (ozonolysis)
122, 39, 401.
Currently, the most common methods for determining the trans fatty acid content are IR spectroscopy and capillary GC. However, both have limitations regarding quantification.
1.5.1 IR spectroscopy. With this technique, the
absorbance of trans bonds in the infrared part of the
spectrum is being utilized for quantification, e.g.
elaidic acid will absorb at 967nm. A number of
internationally approved standard methods exist:
IUPAC, AOCS, AOAC C41-431. For samples with
a low trans content ((5%), the precision of the
determination is low. An optimized method described by Madison et al. [44] gives the possibility of
quantifying down to 2%. With the IR method, it is
not possible to distinguish between most of the
individual trans fatty acids. The intensity of the
absorbance is also influenced by the molecular
positions, i.e. a trans bond in linoleic acid will only
show 85% of the value for elaidic acid.
By using equipment with built-in Fourier transformation of the spectra as described by Sleeter and
Matlock [45], corrections that increase the accuracy
of the IR method can be made, but this equipment
is expensive and therefore not available for routine
analyses.
1.5.2 GC. GC of methyl esters is used as a
standard procedure for determination of the distribution of fatty acids in fats and oils. A number of
the polar stationary phases used, such as SIL 88,
OV 275, and SP 2340, also permit separation of
certain fatty acid isomers with cis as well as trans
configurations. For the partially hydrogenated
edible oils with a high content of positional fatty
acid isomers there will, however, be an overlap
between the cis and the trans fractions.
Sampugna et al. [46] used a 15m capillary
column with SP 2340 for analysing standard methyl
esters of fatty acids, and the method was then used
for determining the trans fatty acid content in 220
different foodstuffs [47]. The separation of the geometrical isomers of C1 was, however, not quite
satisfactory.
With the use of a lOOm capillary column, Slover
et al. [48] found that the geometrical isomers of
c l 8 : 2 could be quantified, whereas an overlap
between the monoene positional isomers from the
cis as well as the trans group still existed. Ratnayake
and Beare-Rogers [49, 501 have improved the separation, but they concluded that the amount of all
different trans isomers cannot be determined by GC
alone. Identification of the individual isomers is also
difficult because of the lack of standard substances.
In spite of this, the method is now officially
approved by the American Oil Chemists Society
c5 11.
Provided that GC is combined with a preliminary
separation of cis and trans fractions on silver
nitrate-impregnated carrier material, either by TLC
or HPLC, a better quantification of the individual
positional isomers of the fatty acids is achieved.
In conclusion, the present IR methodology does
not permit a routine determination of less than 2%
trans fatty acid in fats. Using GC, the large number
of fatty acid isomers, geometrical as well as positional, cannot be separated completely, and thus
cannot produce an unambiguous quantification of
the trans fatty acid content.
SECTION 2. TRANS FAlTY ACIDS A N D DISEASE
2.1 Trans fatty acids and the development of
arteriosclerosis
2.1.1 Epidemiological studies. A high average daily
consumption of hydrogenated fat and oil, and thereby of trans fatty acids, was linked with increased
standardized mortality from CHD in 14 regions in
Great Britain [52]. In this study, there was no
adjustment for other factors connected to the consumption of fat and oil.
In another British study of the fat tissue of
subjects who had died from CHD, the concentrations of trans 16:l and trans 18:l were found to
be higher than in subjects who had died from other
causes [53].
Willet et al. [l] have published the results from a
study regarding connections between daily consumption of trans fatty acids and CHD (fatal or
non-fatal coronary thrombosis) in 85 095 women at
the Nurses Health Study, during a follow-up period
of up to 8years. The consumption of trans fatty
acids was estimated on the basis of a ‘semiquantitative foods frequency’ questionnaire. The
study included all trans fatty acid isomers of c18.
The method for measuring the consumption of trans
fatty acids was partially validated by a fat tissue
biopsy study of 115 women [SS] and in a corresponding study of 118 men [54]. The statistical
analysis was carried out by means of a class of
models called ‘proportional hazard models’, in
which linearity between the effect purpose (CHD
event) and a number of observed factors (i.e. consumption of trans fatty acids, smoking, body mass
index, hypertension, genetic disposition etc.) was
assumed. In this way, it is possible to ‘adjust’ for
other risk factors and calculate the relative risk of
The influence of truns fatty acids on health: a report from The Danish Nutrition Council
trans fatty acid consumption. The energy-corrected
average consumption of trans fatty acids for these
women was 2.4, 3.2, 3.9. 4.5 and 5.7gJday in each
quintile. Corresponding to this, the cases of CHD
were 80, 89, 70, 86 and 106 respectively.
After adjusting for age and total energy consumption, a high consumption of trans fatty acids was
connected with an increased risk of CHD. The
relative risk of the top 20% in trans fatty acid
consumption versus the bottom 20% was 1.50 (95%
confidence limits 1.12-2.00). The connection between
the trans fatty acid consumption and the relative
risk was significant with P(O.001. By adjusting for
other known risk factors for developing CHD,
including smoking and fat consumption, but not
plasma cholesterol, the relative risk changed only a
little (1.35-1.57). The connection was strongest (95%
confidence limits 1.05-2.66) for the 69 181 women
who claimed they had not changed their consumption of margarine for the past 10years. Willet tried
with this procedure to distinguish women who,
because of vague heart symptoms, felt obliged to eat
‘more healthily’, i.e. more vegetable margarine.
The increased risk of CHD for women with a
high consumption of trans fatty acids was ascribed
to the consumption of hydrogenated vegetable fat,
which made up 60% of the total trans fatty acid
consumption. The remaining 40% came from the
animal kingdom (products from ruminants).
A reduction in the risk of developing CHD of the
magnitude described above has a determining
influence on the average life expectancy. Based on a
number of presumptions, the reduced risk of coronary thrombosis can be calculated to be two-thirds of
the present risk for Danish women, corresponding
to an average increase in life expectancy of half a
year, and for Danish men of almost a year [SS, 561.
The statistical analysis of Willet’s work does not
account for the effects of different model assumptions of the low risk, i.e. non-linearity regarding
trans fatty acid consumption and CHD occurrence.
Willet’s studies were criticized by Kuller among
others in a letter to the Lancet in 1993 [57]. Kuller
points out that the method used for estimating the
consumption of energy and fat, and thereby of trans
fatty acids, is imprecise and, further, underestimates
the trans fatty acid consumption. Kuller therefore
believes that it is impossible to state whether an
increased occurrence of CHD is related either to
trans fatty acids or other components in food.
London [58] examined the connection between
the daily consumption of different fatty acids and
the fatty acid composition of fat in the subcutaneous adipose tissue of 115 postmenopausal women
in the U.S.A. Biopsy studies showed that on average
the trans fatty acids made up 4.4% of the fatty acids
in fat tissue, whereas the trans fatty acids for women
on average made up 5.8% of the total consumption
of fatty acids. There was an increased content of
trans, n-3 and total PUFAs in the tissue for
women with a high consumption of the correspond-
38 I
ing fatty acids. This connection was most pronounced for the trans fatty acids. Corresponding
findings have been made for 118 men [54]. The
conclusion is that, based on fat biopsies, one can
roughly estimate the individual’s consumption of
trans fatty acids and n - 3 fatty acids.
Siguel and Lerman [59] found significantly higher
values for trans fatty acids and LDL cholesterol,
and lower values of HDL cholesterol, in the plasma
of 47 patients, than in the control material of 56
individuals. The patients had all developed arteriosclerosis in at least one coronary artery, established
by means of angiography. The authors point out
that a reason for the higher value of trans fatty
acids could be that the patients, due to the disease,
have changed their fatty acid consumption towards
more margarine and less butter.
Roberts et al. [60] have recently reported the
results of a study of the connection between sudden
cardiac death and the content of trans fatty acids in
fat tissue. Sixty-six subjects, who had suffered a fatal
myocardial infarction, were compared with 286
control individuals. The study found that the risk of
sudden death in those with the top 20% trans fatty
acid content in their fat tissue was significantly
higher (2.3-2.6) than in the bottom 20%.
Ascherio et al. [61] have published the results of
a case-control study (239 cases and 282 controls, of
which 78% were men in both groups) where the
connection between consumption of trans fatty acids
and the first coronary thrombosis was calculated.
After adjustment for age, sex and energy consumption, a high consumption of trans fatty acids was
directly connected to an increased risk of coronary
thrombosis (relative risk of highest/lowest 20% =
2.44, 95% confidence limits 1.42-4.19, P(O.0001).
The connection was mainly due to the risk ratio for
the top 20%, where the energy-adjusted consumption of trans fatty acids was 6.5gJday. The corresponding value for the consumption of trans fatty
acids in the bottom 20% was 3.1 gJday. The connection was still highly significant after adjusting for
the established risk factors of coronary thrombosis,
i.e. use of multi-vitamins and consumption of saturated fat, monounsaturated fat, linoleic acid, cholesterol, vitamins E and C, B-carotene and fibres.
Hydrogenated vegetable fat made up 74% of the
total consumption of trans fatty acids, the remaining
part coming from animal fat. The risk of coronary
thrombosis could almost exclusively be ascribed to
the trans fatty acids from hydrogenated vegetable
oil. There was found to be no significant connection
between the relatively low consumption of trans
fatty acids from animal fat and coronary
thrombosis.
The relatively few published epidemiological studies regarding the consumption of trans fatty acids
and the occurrence of CHD (Table 2) have all found
a positive connection. However, epidemiological studies of this type are difficult to interpret. This is due
to considerable uncertainties in the evaluated con-
S. Stender et al.
382
Table 2, Epidemiological studies regarding connection between tnns fatty acid parameters and chosen end points for
arteriosclerosis.
Study type
Trans fatty acid
parameters
End points
Result
Association on macro
level
Consumption of
hydrogenated fat
particularly from
partially hydrogenated
marine oils in different
regions
Mortality of CHD in
geographic regions
Positive connection
Case control
(I36 versus 95)
Trans fatty acid
content in fat
tissue
Death from CHD
Significantly higher trans
161 and trans 181 in
the CHD group
Cohort, 85 095 women
prospectively
Consumption of
trans fatty acid
measured by means of
a semiquantitative
questionnaire
CHD event
Positive connection
between consumption
and CHD event
Case control
(47 versus 56)
Plasma trans
fatty acid
Angiographic certified
coronary thrombosis
Plasma trans
fatty acid
significantly higher in
the disease group
Case control
(66 versus 286)
Trans fatty acid
content in fat tissue
Sudden (cardiac) death
Relative risk of
highest d lowest 20%
Ref.
2.3-2.6
Case control
(239 versus 282)
Consumption of
trans fatty acid
measured by means of
a semiquantitative
questionnaire
sumption of trans fatty acids and a number of
assumptions in connection with the use of the
statistical models. The biggest problem, however, is
that the cause-effect connection remains unknown.
The participants have chosen for themselves how
much vegetable fat they will eat. The consumption
of trans fatty acids, therefore, can either be a marker
or an active agent. The adjustment for known risk
factors is based on a number of assumptions, and
adjustments for unknown risk factors are, naturally,
not possible. It is clear though that the connection
pointed out cannot be overlooked, and that it
should be tested with more direct methods. The
problem is that a study where lots are drawn to
decide who should eat little and who should eat
more trans fatty acid, so that the only difference
between the two groups is the consumption of trans
fatty acids, would be extremely resource-consuming.
It must, therefore, be regarded as doubtful whether
such a randomized intervention study will ever be
carried out. An eventual cause-effect relation as a
basis for the statistical connection between the
consumption of trans fatty acids and the presence of
CHD can, however, be rendered by means of studies
into the effect of trans fatty acids on disease mechanisms. In this connection, it is of interest to examine
whether changes in the consumption of trans fatty
acids will cause changes in the levels of the lipoproteins of the blood which, based on other studies,
First coronary
thrombosis
Relative risk of
highest d lowest 20%
2.44
must be regarded as playing a causal role in
developing arteriosclerotic diseases.
2.1.2 Trans fatty acids and the lipoproteins of the
blood. An atherosclerotic lesion in the arterial wall
consists of large amounts of cholesterol, localized in
the so-called foam cells and outside as lipoproteins
or cholesterol crystals. This intra- and extra-cellular
cholesterol comes from the lipoproteins of the
blood. In the lesion of arteriosclerosis, as well as in
the healthy arterial wall, the lipoproteins of the
blood penetrate the endothelium and intima in a
process which depends on the penetrability of the
surface, the concentration of lipoproteins in the
blood and the size and polarity of the lipoproteins.
In the healthy arterial wall, the lipoproteins will
leave the vascular wall again. In connection with the
development of the lesion of arteriosclerosis, oxidized LDL and very-low-density lipoprotein particles are taken up by the macrophages of the arterial
wall, which are transformed into foam cells. HDL
seems to have a restraining effect on the oxidation
and is possibly capable of removing LDL cholesterol from the arterial wall. An increased concentration in the blood of LDL cholesterol and a
reduced concentration of HDL cholesterol will,
other things being equal, enhance the development
of arteriosclerosis. An increase in the penetrability of
the arterial wall in the surface layer could be
assumed to have the same effect. An increased
The influence of trans fatty acids on health: a report from The Danish Nutrition Council
resistance to oxidation of LDL, due to incorporation of antioxidants or less oxidizable fatty acids in
the LDL particle, must also be assumed to reduce
the risk of developing arteriosclerosis.
Lp(a) is a lipoprotein with the same structure as
LDL, but with an additional protein chain, apolipoprotein (apo) a. The latter protein has a number
of similarities with plasminogen. In a number of
studies, but not all, Lp(a) has been an independent
risk factor for developing arteriosclerosis. Due to
this fact, and due to the resemblance with plasminogen, Lp(a) has been considered a possible connecting link between the generation of coronary thrombosis and the fat deposit in the arterial wall. The
Lp(a) concentration shows large variations from one
individual to another, but the concentration of one
individual is very constant and, based on present
knowledge, more or less independent of food and
medicine. The so-called cholesterol ester transfer
proteins (CETPs) catalyse the transport of cholesterol esters between HDL and the other lipoproteins
in plasma. The protein is present in humans,
monkeys and rabbits, but not in pigs and rats. With
a lack of this protein in humans, an abnormally low
plasma concentration of LDL cholesterol is found.
It has recently been shown in incubation tests of
CETP and human lipoproteins that the activity of
CETP is increased by elaidic acid compared with
oleic acid. CETP in uitro causes a decrease of the
HDL cholesterol concentration and an increase of
the LDL cholesterol concentration [62].
Mattson et al. [63] compared the effect on
plasma concentration of cholesterol and triglycerides in healthy men on two different diets for
4 weeks. One diet contained approximately 21 E%
(energy percentage) trans fatty acids, the other diet
had 21 E% as oleic acid, with 17 and 13 subjects
respectively in each group. The diets caused no
changes in the plasma triglycerides or the plasma
cholesterol.
Mensink and Katan [9] examined the plasma
concentration of total cholesterol, LDL cholesterol,
HDL cholesterol and triglycerides in 25 men and 34
women following three different diets, all taken for
3 weeks without any intermediate ‘washing out’ period. The three diets were different regarding the
trans fatty acid content, oleic acid and saturated
fatty acids, but were otherwise identical regarding
energy, protein, total fat, carbohydrates, alcohol,
cholesterol and fibres. The diet with a high trans
fatty acid content (11% of the total energy content
or approximately four times as high as the mean
consumption in the Danish population) reduced the
plasma concentration of HDL cholesterol by
0.17 mmol/l (approximately 12%) compared with the
two other diets. The plasma concentration of LDL
cholesterol was increased by 0.37 mmol/l (approximately 14%) compared with the oleic acid diet and
reduced by 3% compared with the diet with a high
saturated fatty acid content.
The triglyceride concentrations were significantly
383
lower on the oleic acid diet than on the two others,
where the concentrations were similar.
Reeves et al. [64], in a letter to the New England
Journal of Medicine, pointed out that Mensink and
Katan [9] did not use hydrogenated fat but catalytically isomerized fats. The two different methods of
production result in very different distributions of
the trans double bonds in the molecules and, thereby, possibly different physiological effects. Reeves et
al. also pointed out that Mattson and colleagues
[63] used diets containing cis and trans fats, whose
fatty acid compositions were the same except for
presence or absence of trans fatty acids. Further, in
Mattson’s work, fat containing trans fatty acids
hydrogenated according to a technique used in the
U.S.A. was used. Kritchevsky [65], in a letter to the
New England Journal of Medicine, criticized the
work of Mensink and Katan with the same arguments as Reeves [64]. Mensink and Katan [66]
have rejected this criticism, in particular, that the
lack of effect on the plasma concentration of total
cholesterol in Mattson’s work was due to the low
statistical power in the design of the study.
Mensink [67] has compared the changes in the
plasma concentrations of Lp(a) in young, healthy
men and women, who were not overweight, after
3 weeks’ consumption (without ‘washing out’) of
diets with the same energy content (stearic or other
saturated fatty acids, linoleic or oleic and trans fatty
acids) but with a markedly different trans fatty acid
content. The diets, containing 8-1 1% trans fatty
acids (percentage of total energy consumption) or
oleic acid, gave rise to significant and pronounced
increases in Lp(a) concentrations compared with the
linoleic and stearic diet.
Nestel et al. [lo] examined the effect of four
different fats produced by mixtures of different solid
fats and oils [(i) lauric myristic palmitic acid, (ii)
oleic acid, (iii) elaidic acid and (iv) palmitic acid]
incorporated into normal food for 3 weeks. The fats
were calculated to make up 57% of the total fat
consumption (35 E%). The consumption of energy,
total fat, protein, carbohydrates and fibres from
3 days’ food record in each period were estimated at
the same level. The plasma concentrations of total
cholesterol, LDL cholesterol and Lp(a) of 27 men,
with initially slightly increased values of total
cholesterol, were measured. The elaidic-acidsupplemented diet [with a calculated trans consumption of approximately twice the average consumption of the population (Australia)] gave LDL
cholesterol concentrations much like the two diets
with saturated fatty acids, but higher than the one
supplemented with oleic acid. However, the HDL
cholesterol concentration was significantly lower
than in the period where palmitic acid was added.
The elaidic-acid-supplemented diet also caused a
significant increase in Lp(a) of 20-25% compared
with the palmitic acid period and saturated fatty
acid period, whereas the values were not significantly different from the oleic acid period.
384
S. Stender et al.
Lichtenstein et al. [68] measured LDL cholesterol
and apo B values in the plasma of 14 men and
women, aged 44-78 years, with slightly increased
LDL cholesterol values after three consecutive diet
periods (32 days with 1 week of ‘washing out’) with
isocaloric food consumption. Diet (i) was basic food
corresponding to the average American diet with
35E% fat, of which 12.9E% was saturated fatty
acids and 0.8E% trans 18:l A9. Diets (ii) and (iii)
both contained 30-35Ez fat, 20E% corn oil
(approximately 50 g/day) or a corn-oil-based margarine. Diet (ii) had a trans fatty acid content (18:l
A9) of 0.4% and diet (iii) of 4.2%. The average
concentrations of LDL cholesterol and apo B were
3.96mmol/l and 101mg/dl after diet (i), 18% and
22% lower respectively after diet (ii) and 12% lower
in both cases after diet (iii). The values of the corn
oil and margarine diets were not significantly different. No significant changes were found in Lp(a). It
was concluded that a trans fatty acid consumption
of the amounts seen in diet (iii) suppresses the
cholesterol-decreasing effect of diet (ii).
Wood et al. [12] have examined the effect of five
diets, with different amounts of different fats, on the
concentrations of plasma lipids, lipoproteins and
apolipoproteins in 38 healthy men. The various fats
were consumed as pure fat, cookies, ice cream and
milk, in amounts corresponding to 24% of the total
energy consumption, for 6 weeks, with a ‘washing
out’ period of 6 weeks between the diet periods. The
five fats were (i) ‘butter’, (ii) ‘butter-olive oil’, (iii)
‘sunflower oil’, (iv) ‘hard margarine’ with 3.5% 18:2
and (v) ‘soft margarine’ with 61% 18:2. The trans
fatty acid contents were 5.3%, 2.6%, 2.6%, 29% and
0% respectively. The food consumption during a 7day recording period showed no differences in
energy, total fat (38% of total energy), protein and
carbohydrate, but the consumption of cholesterol
was lower in (iv) and (v). Compared with ‘soft
margarine’, the ‘hard margarine’ showed a significantly higher value of total cholesterol expressed in
mmol/l (5.12 versus 4.8) and LDL cholesterol (3.47
versus 3.26), but no difference in HDL cholesterol,
apo AI, apo B or apo E. Lp(a) was not measured.
Zock and Katan [69] examined the effect of three
different 3-week diets (without ‘washing out’) on the
plasma concentrations of total cholesterol, HDL
cholesterol and LDL cholesterol in 26 men and 30
women, all of whom were healthy. The fatty acid
composition of the three diets was (i) ‘linoleic diet’
with 12% of the total energy consumption as linoleic acid, 2.8% as stearic acid and 0.1% as trans fatty
acids, (ii) ‘stearic diet’ with corresponding value of
3.9, 11.8 and 0.3% and (iii) ‘trans diet’ with the
values 3.8, 3.0 and 7.7%. The daily consumption of
trans fatty acids in diet (iii) was 24g. Other components in the diets were equal. Plasma LDL cholesterol was 2.83 mmol/l on the linoleic diet and significantly higher, 3.00 and 3.07 respectively, after
3weeks on diets (ii) and (iii). Plasma HDL cholesterol was significantly lower, 0.06 and 0.10 mmol/l
respectively, on diets (ii) and (iii) compared with diet
(i). The plasma lipids were not significantly different
after the stearic and trans diets. The results achieved
in this test with a trans fatty acid E% of 7.7, and the
results achieved in a previous test [9] with a trans
fatty acid E% of 11, are, according to the authors,
consistent with a linear correlation between the
consumption of trans fatty acids, measured in EX,
and both an increase in LDL cholesterol and a
reduction in HDL cholesterol. One of the three
points on the graph is, however, based on comparison with stearic acid, the other point is based on
comparison with oleic acid.
Judd et al. [8] examined the effect of four different diets in 6weeks on plasma concentrations of
blood lipids and lipoproteins in 58 subjects (29 men
and 29 women). Diet SAT contained 49g of saturated fat, 27.5g of oleic acid and 1.8g of trans fatty
acids (per lOOg of total fat). The corresponding
figures for diet OLEIC were 35.4, 42.4 and 1.9; for
diet MOD-TRANS 34.3, 35.7 and 9.7 and for diet
HIGH-TRANS 33.6, 29.2 and 17.0. The results are
given in Table 3. Plasma LDL cholesterol after
MOD-TRANS and HIGH-TRANS was significantly
higher than after OLEIC and lower than after SAT.
The difference in the plasma cholesterol between
MOD-TRANS and HIGH-TRANS was not
significant.
Troisi et al. [70] have, in a cross-sectional study,
measured the plasma lipid concentration and trans
fatty acid consumption in 748 men. After correction
for a number of factors known to influence the
plasma lipid concentration, including age, physical
activity, energy consumption and cholesterol and
linoleic acid consumption, the trans fatty acid consumption was directly related to plasma LDL cholesterol concentration and inversely related to the
HDL cholesterol concentration.
The study indicates that the effects of trans fatty
acid consumption on the lipoproteins of the blood
observed in metabolic studies under very controlled
circumstances are confirmed in a free-living population. Based on the results from other studies regarding the connection between the concentration of
lipoproteins and CHD, the authors conclude that
the difference in LDL and HDL cholesterol concentrations with a trans fatty acid consumption of
2.1 g/day and of 4.9g/day corresponds to an
increased risk of coronary thrombosis of 27%.
Based on the results of the above studies, which
are summarized in Table 3, it must be considered as
well documented that the consumption of trans fatty
acids compared with the consumption of oleic acids
and linoleic acids increases the plasma LDL cholesterol concentration. In three out of four studies, the
consumption of trans fatty acids reduces the LDL
concentration compared with a consumption of
saturated fatty acids (except stearic acid). The consumption of trans fatty acids further shows, regardless of which type of fatty acid it is compared with,
a lower or an unchanged HDL cholesterol concen-
Ref.
4
3
3
3
3
4s
30M
2SM
34F
1SM
34F
26M
30F
27M
6M
3
6
26M
30F
29M
29F
6
6
38M
8F
No. of weeks
Subjects, no.
and sex (M/Q
1
HDL-cholesterol
2
3
4
38 ; 10
38; 17
40;8
38 ; 6
30 ; 4
36 ; 7
40;8
+4
+6
+6
40; II
40; I 1
(0)
38 ; 21
-I
-2
-6
-6
(0)
+3
+6
-4
-2
-5
-7
(-4)
-12 - I 2
(-3)
-7
(0)
+8
+6
-3
-2
-8
+ I 4 -3
1
(+2) +8
+6
+3
(15)
(0)
(-4)
(+5)
+ 14
Triglyceride
2
3
4
+II +8
+6
+I6
(0)
1
LDL-cholesterol
2
3
4
Total cholesterol
2
3
4
(E%) in trans fatty
acid diet
1
Changes in plasma lipid fractions (%)
Trans fatty acid
intake in the diet:
(I)fat E% total;
(2) trons fatty acid
2
+23
LP(3
3
+41 +73
1
+23
4
~-
Table 3. Survey o f the percentage changes in various plasma lipid fractions when different acids in the diet are replaced by vegetable trans fatty acids. 1. Proportional t o the oleic acid diet,
2. Proportional t o the saturated fatty acid diet (palmitic acid, habitual diet or butter). 3. Proportional t o the stearic acid diet. 4. Proportional t o the linoleic acid diet. Abbreviations: E, energy. The values in
parentheses indicate noMignificant differences. A trans fatty acid E % of 10 corresponds to 26g trans fatty acids at an energ), intake of IOMJ.
a
6
E.
c
v)
z
0
Dl
2.
m
$
3
3
3
W
m
n
a
c
m
%
s
m
-.
386
S. Stender et al.
tration. The studies which reported that trans fatty
acids caused a decrease in HDL plasma cholesterol,
observed either no effect or an increase in the
triglyceride concentration. Only a small number of
studies are available where the effect of trans fatty
acids on Lp(a) was measured. The consumption of
trans fatty acids corresponding to 8-11% of total
energy consumption (approximately 20-30 g/day)
has shown an increase in Lp(a), while a lower
consumption had no significant effect. Although
stearic acid is a saturated fatty acid, it has a column
of its own in Table 3. This is due to the fact that
consumption of stearic acid seems to be more
cholesterol neutral than consumption of a number
of other saturated fatty acids, such as palmitic acid,
lauric acid and myristic acid.
Regarding the effect on the lipoproteins of the
blood, the studies available indicate that a margarine or shortening consisting of oleic acid and stearic
acid would decrease the LDL cholesterol concentration and increase the HDL cholesterol concentration compared with one containing trans fatty
acids. To summarize, the studies on lipoprotein
concentrations to some extent support the hypothesis of a cause-effect connection between high trans
fatty acid consumption and increased development
of arteriosclerosis.
Taken together, the few studies which appeared
during the last 5-6 years regarding CHD and
consumption of trans fatty acids, both epidemiological as well as studies of effect on lipoproteins in
plasma, support the assumption that an increased
consumption of trans fatty acids increases the occurrence of CHD. The studies suggest that the trans
fatty acids are just as, or perhaps even more,
atherogenic than saturated fatty acids.
2.2 Trans fatv acids and embryogeny
In a study of the influence of trans fatty acids on
the fertility of rats, Hanis et al. [13] found that the
group receiving trans fatty acids had a reduced litter
size, an increased percentage of abnormal sperm
cells and irregularities in the mating season compared with the other groups. These symptoms may
be ascribed fully to the lack of essential fatty acids.
The rats receiving trans fatty acids, approximately
2.5E%, received only 0 S E % linoleic acid. It is not
clear how large the consumption of essential fatty
acids must be to eliminate this effect of trans fatty
acids in rats.
On the basis of animal studies [71], it has
previously been assumed that trans fatty acids do
not penetrate the placenta and that the embryo is
therefore protected against trans fatty acids. Studies
of humans, however, show that trans fatty acids are
transferred passively to the embryo [72]. Koletzko
and Muller [73] measured the plasma fatty acid
composition of 30 mothers and their newborn
babies (from umbilical cord blood samples). The
same level of trans fatty acids was found in both
mother and baby. In another study by Koletzko
[14], premature babies who had a blood sample
taken 4 weeks after their birth had a trans fatty acid
content at the same level as in the previous study.
Houwelingen and Hornstra [l5] also found a strong
positive correlation between the trans fatty acid
content in the plasma of mothers and in the umbilical cord plasma of their babies. They could also
identify trans fatty acids in abortion tissue down to
a gestational age of 5 weeks.
In Koletzko’s study of premature babies [14], a
negative correlation was found between the birth
weight (which ranged from 1.3 to 2.1kg) and the
trans fatty acid content in plasma 4 days after birth.
There was no significant correlation between trans
fatty acids and gestational age. Houwelingen and
Hornstra [1 51 examined healthy babies born at
term and also found a negative correlation between
the trans fatty acid content in the artery wall of the
umbilical cord and both the birth weight and the
circumference of the head. In the study by Koletzko
[141 there was a significant negative correlation
between the concentrations of elaidic acid (trans
18:l) in the blood and the total amount of longchain unsaturated fatty acids. Houwelingen and
Hornstra [l5] also found a significant negative
correlation between trans fatty acids and PUFAs
with longer chains. In both studies it was concluded
that the trans fatty acids interfere with the elongation and the desaturation of the essential fatty acids,
and that this might be the reason for the negative
correlation between birth weight and the level of
trans fatty acids. Both studies are observational, and
although a significant correlation was found, it has
not been proved that the trans fatty acids caused
the low birth weight and the reduced circumference
of the head.
In other studies, the trans fatty acid content in
breast milk has been measured. In a study of
German mothers, Koletzko et al. [74] found that
the average trans fatty acid content was 4.4% of the
total fatty acid content. For mothers in Spain,
where the consumption of trans fatty acids is lower,
the content of the breast milk was on average 0.95%
of the total fatty acid content [75]. Two studies in
the USA, where the mothers were given diets with
different trans fatty acid levels, showed that the
trans fatty acid content in the food [72, 761 and the
trans fatty acid content in the fat tissue of the
mother [76] was of importance for the trans fatty
acid content in the milk. No studies have compared
the trans fatty acid content in breast milk with the
growth and development during the first months of
life.
Both the embryo and the breast-fed baby are in
this way exposed to trans fatty acids corresponding
to the consumption of the mother. It is still uncertain, however, whether the exposure to trans fatty
acids has negative consequences early in life. In
animal studies, a large consumption of trans fatty
acids influenced the synthesis of long-chain PUFAs
The influence of trans fatty acids on health a report from The Danish Nutrition Council
in a negative way. Theoretically, a corresponding
effect could manifest itself in humans. Long-chain
PUFAs have an impact on both the growth and
development of vision and the central nervous
system early in life. The amount of trans fatty acids
needed to affect the synthesis of long-chain PUFAs
is, however, unknown. The authors mentioned conclude that, since they cannot exclude a damaging
effect on the embryo and on the development of the
newborn baby, it must be reasonable to try to
reduce the consumption of trans fatty acids by
pregnant and breast-feeding women.
In their review article, Wahle and James [16]
emphasize that, due to a lack of conclusive research
results, only future research can confirm or deny
whether trans fatty acids have a negative impact on
the embryo and the newborn baby.
23 Trans fatty acids and cancer
2.3.1 Epidemiological studies. A high total consumption of fat is associated with a high prevalence
of certain types of cancer, including large intestinal
and breast cancer [77]. The co-incidence between
an increased occurrence of these types of cancer and
an increased fat intake, including an increased consumption of industrially produced vegetable fats,
has led to the suggestion that trans fatty acids could
play a pathogenetic role [78]. Epidemiological studies of the connection between the intake of trans
fatty acids and cancer, where the intake of trans
fatty acids has been validated (e.g. by fat biopsies),
are, however, lacking.
2.3.2 Studies of mechanisms. Carcinogenic effects
of hydrogenated vegetable fats or specific trans fatty
acids have been examined in rats and mice. Many
different test models have been used, e.g. measurements of activation of mutagenic substances [79],
uptake of fatty acids in tumor cells [SO], and
induction of tumors after administering carcinogenic
substances [Sly 821. The trans fatty acids have been
given in varying dietary levels from 5 to 43% by
weight. In some tests, the trans fatty acids have been
compared with corresponding cis fatty acids, and in
others, with saturated and polyunsaturated fatty
acids. At large intakes some differences in the
metabolism of the fatty acids tested in the cancer
cells are found, but the effect of trans fatty acids on
cancer development seems comparable to the effect
of saturated fat, or corresponding cis fatty acids. In
the absence of added chemical carcinogens, the trans
fatty acids show no mutagenicity [79]. No increased
tumor frequency or other changes of the cells have
been observed in mice, rats or rabbits after longterm consumption of partially hydrogenated vegetable oils [83].
During hydrogenation of vegetable fat, small
amounts ((1%) of conjugated dienes, in which the
two double bonds are separated by a single bond,
are formed. In animal tests, some of these fatty acid
307
< 10%
linoleic acid
H CI8Itrcms
IC18I
Minimum 10%
linoleic acid
0 C182
Minimum 20%
linoleic acid
Minimum 55%
linoleic acid
Butter
20
3 0 4 0
Weight % of total fatty acids
Fig. 4. Fatty acids in margarines and butter.
0
10
5
0
6
0
isomers have been found to inhibit the development
of chemically induced cancer [84].
Record [85] has examined the effect of isocaloric
diets containing 4 or 1 8 g of trans fatty acids per
day for 3 weeks on occurrence of micronuclei in the
lymphocytes of 17 healthy men with slightly
increased plasma cholesterol concentrations. No
differences in the micronucleus frequency were
observed, and the author concluded that the findings are in agreement with other data and give no
reason to believe that trans fatty acid isomers have
any effect on the genetic material.
In conclusion, dietary fatty acid isomers do not
seem to have any carcinogenic effect.
SECTION 3. THE CONSUMPTION OF TRANS
F A l l Y ACIDS IN DENMARK
The consumption of trans fatty acids is derived
mainly from two sources: (i) the fatty acids generated by biological hydrogenation in the gastrointestinal tract of ruminants (ox/lamb) and then deposited in the fat and secreted in the milk, and (ii) the
fatty acids generated from industrial hydrogenation
of liquid oil in the production of margarines, frying
oils etc. The consumption from other sources like
vegetables, game, pork, fish and egg is very limited.
?7.ans fatty acids in animal and vegetable products are mainly monoenes with trans 18:l A l l
(vaccenic acid) being the dominant fatty acid in
milk fat and meat products, and trans 18:l A9
(elaidic acid), trans 18:l AlO, trans 18:l A l l (vaccenic acid) in margarines (see also Section 1). In the
latter products, the composition of trans fatty acids
may vary considerably, depending on the oils used
as the basis for the production. The extent and
technique of hydrogenation also plays a role in this
variation.
3.1 Trans fatty acid content in foods
In a recent study [86], the trans fatty acid
content, apart from the long-chain isomers of
marine origin, has been determined in a number of
margarine products, covering the entire Danish
market (Fig. 4).It is seen that products containing a
minimum of 20% linoleic acid (vegetable frying
388
S. Stender et al.
Table 4. The average trans fatty acid content (trans 181) of some food
products. Data adapted from [W].
Product
Pastry
C r oiIIants
Cookies
Pizza base
Pitta bread
Potato chips
French fries, frozen, prefried
French fries (fast-food outlets)
Trans fatty acid
content (g/lOOg)
2.4
I.9
1.5
0
0
0. I
0.9
5
margarine) have the highest trans fatty acid content,
whereas products with 2 5 5 % linoleic acid only
have a small trans fatty acid content. The fact that
products with 20% linoleic acid have such a high
trans fatty acid content may be due to the fact that
strongly unsaturated oils are used for their production, demanding an intensive hydrogenation. Similar
levels of trans fatty acids have been found in solid
types of margarine (10-20% linoleic acid) in other
countries [87, 88, 481. It is characteristic that the
variation in trans fatty acid content between the
same type of products is large. Thus, the study from
the National Food Agency of Denmark [86] found
a variation in the trans fatty acid content of
between 0 and 23% (percentage of total fatty acids)
in margarine types with 20% linoleic acid. On the
contrary, there was only a small variation within the
individual brands (samples taken at an interval of
2months). In Fig. 4, the trans fatty acid content in
butter (milk fat) is also given. A Danish study [89]
has shown an average content of 4.7% (variation:
3.66.1%). Seasonal variation was pronounced, with
the highest content in summer butter.
There are no published data available of the trans
fatty acid content in cake and pastry margarines
(baking margarines for industrial use). Based on
analysis of the composition of fatty acids in cakes
[go], the content can be calculated to be about 10%
of the total fatty acid content (variation: 2-14%).
The trans fatty acid content of industrial margarines
given by the margarine industry is even larger, 17%
on average (variation: 628%).
As mentioned above, the solid margarines occur
in a number of manufactured foods and therefore
contribute to the consumption of trans fatty acids.
The products with the highest content of trans fatty
acids are usually products where margarine has
been used in the production. Liquid vegetable frying
fat contains a small amount of trans fatty acids
[9l]. Hardened (hydrogenated) frying fat (presently
used by the major burger chains) has a high content
of trans fatty acids (mean: 30% of total fatty acids).
The heating of frying fats will increase their trans
fatty acid content insignificantly. The trans fatty
acid content in some food products is given in
Table 4.
In mixed spreads of butter and vegetable oils,
typically with 25% vegetable oil in the butter, a
Table 5. Average consumption of fat containing trans fatty acids, based on
per capita statistics 1991. The values in parentheses are based on the margarine
industry's statement of a trans fatty acid content of 17% in their industrial products.
Foods
Fat consumption
(g/day per person)
Trans fatty acid
consumption
(E/daY Per Penon)
Table and frying margarine*
Baking margarine*
Milk fat
Fat from cow and lamb
Total
20
10
38
8
76
I.5
1.0 (1.7)
2.0
0.5
5.0 (5.7)
*Margarine supply is 37g/day per person corresponding to 30g fat/day per person
(solid margarines contain approximately 16% water).
smaller trans fatty acid content than in butter is to
be expected. Figures given by the industry show a
content of 4 5 % of the total fatty acid content.
Analyses of the trans fatty acid content in fat
from ruminants have shown figures of about 5% of
total fatty acids [21, 47, 921.
3.2 Consumption of trans fatty acids and foods containing
trans fatty acids
In Table 5, the average daily consumption of
trans fatty acids in Denmark, estimated from per
capita statistics of 1991, is given. Trans fatty acids
from margarines make up about half of the total
consumption, and within the margarine types it is
mainly the solid types that contribute to the consumption. As stated above, the consumption from
baking margarine is perhaps somewhat higher (see
Table 5). The consumption of deep-fat frying fat in
industry and households is unknown, but the average consumption of trans fatty acids from these
products is low. However, there may be groups
within the population with high intakes from these
sources (e.g. the fast-food generation).
It should be emphasized that calculations based
on per capita statistics do not reflect real consumption, but express the amounts available to the
Danish consumer. In the estimated consumption,
certain losses have been accounted for (i.e. by
estimating the contribution from meat products). In
general, the per capita statistics overestimate the
real consumption.
3.2.1 Distribution of intake by sex and age. It is
not possible to calculate the distribution of intake
between sexes and in different age groups. The food
intake study from 1985 [93, 941 showed that the
average daily consumption of margarine and milk
fat was lower in women than in men (30g/day
compared with 49 g/day for margarine, and 35 g/day
compared with 49g/day for milk fat). This means
that women consume less trans fatty acids than
men. However, based on the same data, the average
consumption of margarine per unit energy was
similar for men and women [93, 941.
3.2.2 Maximum consumption of trans fatty acids.
By extrapolating margarine intake in the 1985 study
The influence of trans fatty acids on health: a report from The Danish Nutrition Council
Table 6. Estimated average consumption of trans fatty acids during a period
of ISyears, based on per capita statistics
Trans fatty acids,
total consumption
(g/day Per person)
Trans fatty acids,
consumption from margarine
@/day per person)
*Data taken from
1976*
1985
1991
8.5
6.0
5.0
6.0
4.0**
2.5
[96].**Data from [97l were used t o calculate the consumption.
[93, 941, it can be estimated that 5% of the adult
population (150 000 Danes) will consume 5 g or
more of trans fatty acids from margarine. If an
individual’s consumption is derived solely from margarine products with a high trans fatty acid content
(20% of the total fatty acids), the consumption will
reach 15 g/day per person.
3.2.3 Consumption of trans fatty acids over time.
Even though the total fat consumption in Denmark
from milk fat, margarines and meat has not shown
marked changes during the last decade, relatively
large changes in the intake of foods essential to the
consumption of trans fatty acids have taken place
[95]. In the decade from 1980 to 1990, a pronounced decrease in the consumption of margarines
(from approximately 46 g/day per person to 40 g/day
per person), and a slightly smaller decrease in the
consumption of milk fat (from 44g/day per person
to 40gJday per person) has taken place, and has
been compensated for by an increased consumption
of animal fat. Figures given by the retailers indicate
that changes in the consumption of the different
margarine types have taken place. Table margarines,
with a higher trans fatty acid content, comprised a
larger part of the total consumption of margarine in
1985 (70%) compared with 1990 (58%). The consumption of baking margarines seems unchanged
during the same period. In Table 6, the estimated
consumption over a period of 15 years is given. A
pronounced decrease in the consumption of trans
fatty acids seems to have taken place. The decrease
is caused by a decrease in the consumption of table
and frying margarines and by a lower trans fatty
acid content in margarines, which may be explained
by improved processing conditions of hydrogenation
[98] or increased use of vegetable oils with a lower
degree of unsaturation.
3.2.4 Consumption of trans fatty acids in other
industrialized countries. Figures for the consumption
of margarine (Margarine Sector Organization) and
trans fatty acids are given in Table 7. The consumption of margarines is higher in the northern European countries compared with the southern European countries, where the liquid fats dominate.
From the figures it can be seen that there is no
correlation between the consumption of margarine
and the consumption of trans fatty acids. The
reason for this is probably first of all that the
calculation of the consumption is made on an
369
insecure basis. Other explanations may be the varying intake of the different types of margarine and
the fact that the years for calculating the consumption of margarine and trans fatty acids do not
correspond completely.
SECTION 4. SHOULD A MAXIMUM LIMIT FOR THE
CONSUMPTION OF TRANS F A l l Y ACIDS FROM
MARGARINE BE RECOMMENDED TO THE DANES?
Based on the information available on the correlation between the consumption of trans fatty acids
and the occurrence of CHD, and the uncertainty
regarding a harmful effect on the growth of the
fetus, it seems reasonable to reduce the consumption
of trans fatty acids as much as possible. No evidence
suggests that the consumption of trans fatty acids
has beneficial effects on health compared with the
consumption of saturated, monounsaturated or
polyunsaturated fatty acids.
The average consumption of trans fatty acids
from margarines in 1991 in Denmark was approximately 2.5 g/day per person. For about 150000
adult Danes, the consumption is estimated to be
more than 5g/day per person. Among these ‘high
consumers’ will be pregnant and breast-feeding
women. Such a high intake of trans fatty acids must
be regarded as unacceptable.
In order to reduce the risk of CHD, and due to
the uncertainty regarding the effect on the growth of
the fetus, it would be desirable to reduce the
consumption of trans fatty acids from margarines,
ensuring that no one consumes more than 2 g/day.
This amount corresponds to the consumption of
trans fatty acids found among the 20% who consumed the least trans fatty acids in the American
studies [l, 611. This can be achieved by reducing
the total fat content of the food to the recommended level and by decreasing the consumption of
margarine-containing products or by reducing the
trans fatty acid content in margarines.
In Table 8, it is shown how a reduction of the
content of trans fatty acids in margarine products
will affect the total consumption of trans fatty acids
from margarines. Even for the group in the population eating most margarine, a maximum daily consumption of 2 g of trans fatty acids per person is
achieved by reducing the total fat consumed and by
reducing the trans fatty acid content in margarine to
5% or less.
SECTION 5. NUTRITIONAL LABELLING
5.1 The rules in force
Rules for nutritional labelling are found in the
‘Legal notice of nutritional labelling etc. of prepacked foods’ [104]. The notice contains decisions
that carry through the EU council directive no.
901496 [l05]. As a basis, it is voluntary to make
nutritional declarations, but if a nutritional claim is
S. Stender et al.
390
Table 7. Consumption of margarine and t n n s fatty acids from all sources in some European countries
Country
Consumption of margarine
Consumption of
(g/day Per P e 4
trans fatty acids
Year*
Ref.
1991
I984
“1
klday Per person)
-
31
35
28
Denmark
Sweden
The Netherlands
Norway
England
Germany
Spain
5
7
[991
21
20
20
3
*States the year for calculating the consumption of trans fatty acids.
Table 8. Connection between the percentage trans fatty acids in table and
frying margarine, as well as in bakery margarine, and the consumption of
vegetable t a n s fatty acids in Denmark by unchanged total fat consumption.
The calculations are based on a consumption of fat from table and frying margarines
of 20g/day per person and from bakery margarine of IOg/day per person.
Trans fatty acid content (%)
Table and frying margarine
Bakery margarine
8
17
8
10
5
5
2
2
Trans fatty acid consumption (g/day per person)
Consumption of trans fatty acids
Average consumption
3.2
‘High consumption’ (5% or
6.4
I50 OOO adults consuming most)
2.5
5.0
1.4
2.8
0.6
1.2
stated, labelling is compulsory. The rules apply in
the case of both a voluntary or a compulsory
labelling.
According to the notice, the fat content can be
declared either as (i) the total lipid content per lOOg
in the product, or (ii) the total lipid content and the
content of (a) saturated fatty acids, meaning all fatty
acids without double bonds; (b) monounsaturated
fatty acids with cis double bonds, and (c) PUFAs,
meaning all fatty acids with cis& methyleneinterrupted double bonds (all per lOOg of the
article). Further components, including trans fatty
acids, can be stated (cf. EU council directive no.
901496).
A change of the above notice would call for a
change in the EU directive, which means that the
decision must be reached by the EU. Denmark
cannot, on its own, change the rules for nutritional
labelling, including the compulsory labelling of trans
fatty acids, but could pose the problem within the
terms of the EU.
The trans fatty acid content in the product is
included in the calculation of the total fatty acid
content. An indirect figure for the trans fatty acid
content is achieved by labelling as stated in (ii),
being the difference between the total fatty acid
content (minus the glycerol part) and the sum of (a),
(b) and (c). The use of this form of labelling is not
very widespread however.
A nutritional claim includes any statement on the
packaging or in advertisements designed to give the
consumer the impression that a food has certain
nutritional qualities. Nutritional claims of the type
‘low trans fatty acid content’ are possible with the
present legislation. In any case which mentions trans
fatty acids, they must be labelled separately, preferably under the fat content, which would involve a
nutritional labelling as follows:
Fat
g
- saturated fatty acids
g
- polyunsaturated fatty acids
g
- monounsaturated fatty acids
i2
- trans fatty acids
f3
This nutritional claim does not obviate the long
nutritional labelling, because the saturated fatty acid
content must be stated when the content of, for
example, PUFAs is labelled (cf. EU council directive
no. 90f496).
SECTION 6. SUGGESTION BY THE DANISH
NUTRITION COUNCIL
A daily consumption of 5 g of trans fatty acids or
more from margarine is at best neutral and at worst
suspected of increasing the risk for development of
CHD and of causing negative effects on fetal
growth. It is estimated that 150 000 Danes consume
more than 5g of trans fatty acid daily from margarine. Simply by reducing the total fat consumption to
the recommended level, a considerable reduction of
the trans fatty acid intake can be achieved. A
reduction of the trans fatty acid content of margarine products can immediately decrease the trans
fatty acid intake further (see Table 8).
On this basis, the Danish Nutrition Council
suggests that the trans fatty acid content in margarines should be reduced. It is suggested that the
content in all types of margarines should be reduced
to 10% or less before the end of this year (1994).
Within a period of 2-3 years, the content in all types
of margarines should be reduced to 5% or lower.
The Danish Nutrition Council encourages all
producers of margarines and margarine-containing
foods to produce products that can be labelled ‘free
of trans fatty acids’.
The influence of trans fatty acids o n h e a l t h a r e p o r t f r o m T h e Danish Nutrition Council
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