European Journal of Clinical Nutrition (2001) 55, 29±38
ß 2001 Macmillan Publishers Ltd All rights reserved 0954±3007/01 $15.00
www.nature.com/ejcn
Seasonal variations of antioxidant imbalance in Cuban healthy
men
J Arnaud1*, P Fleites2, M Chassagne3, T Verdura4, J Barnouin3, M-J Richard1, J-P Chacornac3, I Garcia
Garcia5, R Perez-Cristia2, AE Favier1 and the SECUBA group
1
Laboratoire de Biochimie C, CHUG, BP 217, Grenoble, France; 2Centro national de Toxicologia, La Habana, Cuba; 3INRA, UniteÂ
d'Ecopathologie, Saint GeneÁs Champanelle, France; 4Instituto Finlay, La Lisa, La Habana, Cuba; and 5Instituto Farmacia y Alimentos,
La Coronela, La Lisa, Ciudad de la Habana, Cuba
Objective: To determine the antioxidant imbalance in healthy Cuban men 2 y after the end of the epidemic
neuropathy (50 862 cases from 1991 to 1993) and to evaluate its change over 1 y.
Design: Prospective study.
Setting: La Lisa health centres (Havana, Cuba).
Subjects: One-hundred and ninety-nine healthy middle-aged men were selected and 106 completed the study.
Subjects were studied at 3 month intervals over 1 year.
Interventions: No invervention.
Main outcome measures: An assessment of dietary intake and the determination of blood lipid peroxides
(TBARS), glutathione, diglutathione, glutathione peroxidase, superoxide dismutase, vitamin E, carotenoids,
copper, zinc and selenium were performed at each period.
Results: While dietary zinc, vitamins C and E, carotenoids and fat dietary intakes and blood concentrations were
low for adult men compared to international reference ranges, serum TBARS concentrations were high at every
period. Some signi®cant seasonal variations were observed. The lowest carotenoids (P < 0.002) and vitamin
C(P ˆ 0.0001) intakes, serum b-carotene (P ˆ 0.0001) and lutein=zeaxanthin (P < 0.05) concentrations, and the
highest blood TBARS (P ˆ 0.0001) and diglutathione (P < 0.001) concentrations were observed at the end of the
rainy season (October). This period seemed to pose the greatest risk of antioxidant imbalance.
Conclusions: Cuban men still represent a vulnerable population in terms of antioxidant imbalance. A national
program of vegetable growing and increase in fruit and vegetable consumption is now evaluated in Cuba.
Sponsorship: Nestec-NestleÂ, Merck-Biotrol diagnostics, Trace Element Institute for Unesco, Grenoble University,
INRA.
Descriptors: oxidative stress; trace elements; antioxidant vitamins; carotenoids; season; glutathione
European Journal of Clinical Nutrition (2001) 55, 29±38
Introduction
*Correspondence: J Arnaud, CHUG, BP217, 38043 Grenoble, Cedex 9,
France.
E-mail: [email protected]
Guarantors: R Perez-Cristia, J Barnouin and J Arnaud.
Contributors: JA participated in the study design, carried out trace elements
determinations, participated in the discussion of results and wrote the paper.
PF participated in the study design, coordinated the study, examined the
subjects, participated in the discussion of results and reviewed the paper.
MC collected the data, did the statistical analysis, participated in the
discussion of results and reviewed the paper. TV participated in the study
design, collected the data, participated in the discussion of results and
reviewed the paper. JB participated in the study design, coordinated the
study, participated in the discussion of results and reviewed the paper. MJR carried out enzyme, glutathione compound and lipoperoxide
determinations and reviewed the paper. J-PC carried out liposoluble vitamin
determinations. IGG coordinated dietary records and participated in the
discussion of results. RP-C participated in the study design, coordinated the
study and participated in the discussion of results. AEF participated in the
discussion of results.
Received 20 January 2000; revised 15 September 2000; accepted 20
September 2000
From 1991 to 1993, 50 862 cases (461.4 per 100 000
inhabitants) of optic and peripheral neuropathy of unknown
etiology were reported in Cuba (Tucker & Hedges, 1993;
PeÂrez-CristiaÁ & Fleites-Mestre, 1994; Roman, 1994;
CNFIT, 1995; Bowman et al, 1996; Macias-Matos et al,
1996). The increase in physical exercise associated with
tropical weather, monotonous diet and dramatic limitations
in food availability as well as smoking appeared to be the
primary risk factors of the epidemic (Tucker & Hedges,
1993; Roman, 1994; CNFIT, 1995). Indeed, the number of
cases was reduced in children, pregnant women and elderly
people who received additional foods (Roman, 1994). There
was no evidence of infectious diseases (Roman, 1994), or
genetic defects (Tucker & Hedges, 1993). No particular
nerve-toxic chemical was identi®ed (Tucker & Hedges,
1993; Roman, 1994). Nutritional and biological studies
®rst suggested that vitamins were centrally important. A
Seasonal antioxidant imbalance
J Arnaud et al
30
contribution of the impairment of protective antioxidant
pathways in the Cuban epidemic neuropathy was also
suggested. Serum lycopene concentrations were found to
be dramatically decreased in patients vs controls (CNFIT,
1995; Bowman et al, 1995). Blood selenium, a- and bcarotenes were also decreased in patients vs controls, but to
a lesser extent) CNFIT, 1995). However, high concentrations of serum thiobarbituric acid reactive substances
(TBARS) were reported both in patients and controls
(PeÂrez-CristiaÁ & Fleites-Mestre, 1994).
The present results are part of a large multidisciplinary 1 y
prospective study performed in healthy middle-aged subjects
in order to improve long-term food safety in Cuba (Barnouin &
PeÂrez-CristiaÁ, 1998) and named `Seguridad Alimentaria y
Buena Alimentacion in Cuba (SECUBA)'. The ®rst aim of
this study was to determine the extent of the previously
observed health risks in Cuba 2 after the end of the epidemic
neuropathy. At that time, the Cuban population neglected to
take multivitamin supplement and the economic situation of
Cuba was particularly worrying, despite few new neuropathy
cases. The second aim of this survey was to evaluate the
variations in the studied parameters over 1 y because nutrient
intake largely depends on temperature and rainfall in tropical
countries (Bates et al, 1994; Cooney et al, 1995) and the
temporal distribution of cases showed an exponential increase
in March 1993. The ®nal aim of the study was to evaluate the
in¯uence of smoking on the risk factors as smoking was
strongly associated with the occurrence of the illness
(Tucker & Hedges, 1993; Roman, 1994; CNFIT, 1995).
However, the in¯uence of smoking was not within the scope
of the present paper and has been published elsewhere (Barnouin et al, 2000). Brie¯y, smokers had lower plasma bcryptoxanthin a- and b-carotene concentrations but higher
vitamin E intakes and serum copper concentrations than nonsmokers. These results suggest that smoking increases the
oxidative imbalance. The present work focuses on the changes
in antioxidant imbalance over 1 y. The imbalance between the
production of reactive oxygen species and the counteracting
antioxidant defence systems can result in oxidative stress,
unleashing a cascade of pathological processs. Cubans seem
particularly at risk of oxidative stress because of food shortage
(PeÂrez-CristiaÁ & Fleites-Mestre, 1994), a traditionally low
consumption of vegetables and fruits, exposure to tobacco
smoke (Rahman & MacNee, 1996) and to sunlight all year
long (Roe, 1987). Increased oxidative stress has been reported
in energy-protein malnutrition (Golden et al, 1991). However,
energy restriction without compromising essential nutrients to
avoid malnutrition induces an overexpression of superoxide
dismutase and catalase genes, suppresses iron accumulation in
tissue and therefore can protect against oxidative damage (Yu,
1996; Weindruch & Sohal, 1997).
Methods
Subjects
One-hundred and ninety-nine clinically healthy middle-aged
men (27 ± 59 y) living in Havana were randomly selected
European Journal of Clinical Nutrition
from the La Lisa health centres and agreed to participate in
the study. The age and sex of the volunteers were selected
because this population was the most sensitive to optic
neuropathy in 1993. Havana was selected for practical
reasons, but also because the neuropathy incidence in
1993 corresponded to the average of the island of Cuba.
Information regarding social and demographic characteristics, occupation and toxic exposure were obtained by interview. However, as exposure to asbestos, heavy metals,
radiation, pesticides and other chemicals did not change
over the year, these factors were not evaluated as potential
confounders in the present work.
One-hundred and forty-one subjects out of 199 completed the biological study. However, due to insuf®cient
blood available, the complete set of biological determinations was performed in 112 subjects, except for glutathione
and diglutathione which could be determined only in a subsample of 30 subjects. One-hundred and six subjects out
of the remaining 141 complete the dietary and life habit
questionnaire at the four periods.
The main characteristics of subjects at the inclusion,
those who completed the study and those who did not
complete the study, are indicated in Table 1. All subjects
were free of HIV infection and viral hepatitis.
Study design
Subjects were studied at four different times of three month
interval over 1 y (March ± April 1995 (period 1); June ± July
1995 (period 2), October 1995 (period 3) and January ±
February 1996 (period 4)). April corresponds to the end of
the dry season and October to the transition period between
rainy and dry season. A complete medical examination,
body mass index (BMI) and basal metabolic rate (BMR)
(Scho®eld, 1985) calculations, blood collection and assessment of dietary intakes were performed at each period. The
protocol was approved by the Cuban Ministry of Public
Health and all the selected subjects gave their informed
written consent. Procedures followed were in accordance
with the Helsinki Declaration of 1975, as revised in 1983.
Assessment of dietary intake
Daily dietary records were quanti®ed in household measures
and reported on a semiquantitative food-frequency questionnaire by the volunteers for 7 consecutive days at each
of the four periods. At the end of each day, 20 trained
dieticians from the `Instituto Farmacia y Alimentos'
checked information on the diet quality and quantity
record for accuracy, completeness and clarity. The essential
antioxidant nutrients (vitamin C and E, total cartotenoids,
zinc (Zn) and copper (Cu)) and macronutrient intakes were
calculated using the Cuban NUTRISIS food composition
database provided by the Instituto de Nutricion e Higiene
de los Alimentos, Havana, Cuba (Rodriquez et al, 1992).
NUTRISIS contains the nutrient content of the most commonly consumed foods (n ˆ 628) in Cuba. This method had
been used before during the epidemic neuropathy (PeÂrezCristiaÁ & Fleites-Mestre, 1994; CNFIT, 1995).
Seasonal antioxidant imbalance
J Arnaud et al
31
Table 1 Characteristics of the 199 Havana volunteers at their enrolment in the study (March ± April 1995) and comparison between those who completed
(n ˆ 106) and those who did not complete the study (n ˆ 93)
Ageb (y)
Body mass indexb (kg=m2)
Energy intake=basal metabolic rate ratiob
Race:d Caucasian=black=mixed
Smoking status:d nonsmokers=former smokers=current smokers
Current smokers:
number of cigarettes smoked=daye
number of cigars smoked=daye
smoking yearse
Former smokers:
stopping duration (y)b
smoking yearse
Years of educationb
Employment:d
employed=retired or unemployed
day-time=night-time=both
Hours of work=dayb
Occasional use of vitamin supplementd
Albuminb (g=l)
Transthyretinb (mg=l)
Cholesterolb (mmol=l)
Alanine aminotransferaseb (U=l)
Aspartate aminotransferaseb (UI=l)
gamma-glutamyl transferasee (UI=l)
Creatininb (mmol=l)
Ureab (mmol=l)
Glucoseb (mmol=l)
Hemoglobinb (g=l)
n ˆ 199
n ˆ 106
n ˆ 93
Pa
39.1 (6.6)
23.9 (3.6)
1.05 (0.30)
58.8=18.6=22.6
32.7=16.1=51.3
39.8 (6.5)
23.9 (3.1)
1.06 (0.32)
62.3=17.0=20.7
31.1=17.0=51.9
38.3 (6.9)
23.9 (4.1)
1.03 (0.26)
54.8=20.4=24.7
34.4=14.0=51.6
20 (2 ± 70)
1 (1 ± 6)
23 (1 ± 36)
19 (2 ± 70)
1 (0 ± 5)
23 (3 ± 49)
17 (2 ± 35)
1 (0 ± 6)
21 (1 ± 36)
9.5 (6.6)
15 (2 ± 35)
12.0 (2.6)
11 (6.9)
17 (2 ± 35)
12.1 (2.5)
8 (6.1)
12 (2 ± 29)
11.8 (2.7)
93=7
70=4.5=18.6
6.8 (3.0)
16.6
48 (2)
327 (67)
4.39 (1.01)
18 (11)
28 (11)
20 (1 ± 293)
88 (10)
4.38 (1.09)
5.56 (0.56)
142 (20)
92=7
72.6=2.8=15.1
6.5 (2.9)
15.1
48 (2)
333 (65)
4.46 (1.00)
17 (10)
27 (10)
21 (1 ± 139)
88 (11)
4.33 (1.08)
5.59 (0.52)
143 (20)
94=6
63.4=6.4=22.6
7.2 (3.1)
16.1
48 (2)
320 (69)
4.36 (1.02)
19 (11)
29 (12)
19 (1 ± 293)
88 (9)
4.44 (1.10)
5.53 (0.59)
141 (21)
NSc
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
a
P ˆ signi®cant probability between volunteers who completed and those who did not complete the study.
Expressed as mean (s.d.).
c
NS ˆ nonsigni®cant, P > 0:05.
d
Expressed as percentage.
e
Expressed as median (range).
b
Blood collection, storage and transport
Venous blood samples (14 ml) were obtained after an overnight fasting in free trace element tube and heparin Vacutainer1 tubes (Becton Dickinson, Pont de Claix, France).
Tubes were immediately protected from light and stored on
ice. An aliquot of whole blood was deproteinized with
metaphosphoric acid within half an hour from venipuncture
(glutathione (GSH) and diglutathione (GSSG) determinations). Supernatants were isolated by centrifugation at
1700 g for 10 min at ‡ 4 C and were immediately stored
at 7 80 C (GSH and GSSG determinations). The remaining blood was centrifuged at 1700 g for 10 min at ‡ 4 C to
separate serum or plasma. Aliquots of serum, plasma and
homogenized erythrocytes were frozen and stored at
7 80 C (thiobarbituric acid reactive substances (TBARS),
enzymes, vitamins and carotenoids) or 7 20 C (trace elements) within 2 h of sampling. Precautions were taken to
prevent trace element contamination of the sample. Once
a week, the tubes were delivered on dry ice to French
laboratories for analyses. Samples were kept frozen until
analysis.
Blood analyses
The essential antioxidant nutrient (vitamin E, carotenoids,
Zn, Cu and selenium (Se)), the principal antioxidant
enzymes (Cu ± Zn superoxide dismutase (SOD; EC
1.15.1.1) and Se ± glutathione peroxidase (GPX; EC
1.11.1.9)) and the main non-enzymatic scavengers (reduced
GSH; Jones et al, 1995) as well as two parameters of
oxidative stress (TBARS, the most commonly used screening parameters in epidemiological studies (McCall & Frei,
1999) and GSSG, an early index of oxidative stress (Sen,
1997)) were measured. Erythrocyte SOD and GPX were
measured as previously described (Preziosi et al, 1998) on
an RA1000 autoanalyzer (Bayer diagnostics, Puteaux,
France) using a pool of human erythrocytes as internal
precision quality control. Total glutathione (tGSH) and
diglutathione (GSSG) were measured in whole blood as
previously described (Preziosi et al, 1998) using a Kontron
spectrometer (Rotkreuz, Switzerland) and a pool of deproteinized human whole blood as internal precision quality
control. Reduced glutathione (GSH) was calculated by
difference (tGSH 7 2GSSG). Plasma a-tocopherol and carotenoids were quanti®ed by reverse-phase HPLC with
spectrometric detection (Kontron, Rotkreuz, Switzerland)
using commercial solutions as internal quality control. Total
carotenoids were calculated as the sum of lutein=zeaxanthin,
b-cryptoxanthin, lycopene and a- and b-carotenes. Serum
trace elements (Zn, Se, Cu) were measured by atomic
absorption spectrometery (Perkin Elmer, Norwalk, CT)
European Journal of Clinical Nutrition
Seasonal antioxidant imbalance
J Arnaud et al
32
Table 2
Daily dietary intake according to season in male Havana volunteers
na
Period b 1
Period 2
Period 3
Period 4
Pc
106
7.0 (2.0)
1676 (473)
61 (16)
41 (18)
65 (33)
7.1 (4.8)
1028
(150 ± 13 515)
2.9 (1.1)
8.5 (2.5)
6.8 (2.1)
1628 (493)
67 (37)
42 (23)
32 (17)
7.0 (5.0)
779
(88 ± 5030)
2.8 (1.0)
8.6 (2.7)
6.7 (1.8)
1615 (437)
62 (16)
44 (18)
31 (17)
8.2 (4.0)
586
(44 ± 2480)
2.4 (0.8)
8.3 (2.4)
6.6 (1.8)
1580 (442)
62 (16)
41 (18)
51 (31)
7.9 (3.9)
1395
(144 ± 5065)
2.6 (0.9)
8.9 (2.3)
NSd
Energy (MJ)
(kcal)
Protein (g)
Fat (g)
Vitamin C (mg)
Vitamin E (mg)
Total carotenoids (mg)
106
106
106
106
106
Copper (mg)
Zinc (mg)
106
106
&e
&
&
&
%
%
NS
NS
0.001
NS
0.001
0.001
NS
Results are expressed as mean (s.d.) except for other than normal distribution parameters, expressed as median (range).
n ˆ number of volunteers.
b
Period 1 ˆ March and April 1995; Period 2 ˆ June and July 1995; Period 3 ˆ October 1995; Period 4 ˆ January and February 1996. Periods 2 and
3 ˆ rainy season.
c
P ˆ signi®cant period effect probability adjusted for smoking as a ®xed effect and individual as a random effect.
d
NS ˆ nonsigni®cant, P 0:05.
e
% and &ˆ signi®cant between period variation (for P-values, see text, Results section).
a
using Seronorm1 Trace Element (Nycomed, Oslo, Norway)
as precision and accuracy internal quality control. Serum
TBARS analysis was performed by ¯uorometry (Richard et
al, 1992) on a Perkin Elmer ¯uorometer (Norwalk, CT)
using commercially available lyophilized serum as internal
precisioin quality control.
Statistics
Statistical analyses were performed using SAS software
(SAS Institute, Cary, NC). For each parameter, means,
median and standard deviations were calculated at the four
time periods. Response trends over time for the studied
parameters were assessed by variance ± covariance analyses
on repeated measures using the mixed model methodology
of SAS. This procedure computes ef®cient estimates of
®xed (ie time and smoking) and random (ie individual)
effects when a covariance structure, referring to variances at
individual times (variation between volunteers) and a correlation between measures at different times on the same
subject (covariation within volunteers), characterizes the
data. Missing data do not cause serious problem with this
procedure (Littell et al, 1998, 1999). Consequently, the time
analyses were run for all subjects whatever the period,
except for the glutathione compounds. For these parameters,
we considered only the subjects with values at the four
periods because missing values were too numerous. Individual data were normalized by logarithmic or square root
transformation when necessary before statistical analysis.
Chi-square tests were performed to evaluate seasonal differences in sub-de®cient value frequencies and the
Mann ± Whitney test was used to compare volunteers who
completed and those who did not complete the study. The
strength of relationships between intakes and biological
status were further evaluated by Pearson correlations
before and after adjustment for age, BMI, smoking habits,
energy and alcohol intakes and either fat intakes and plasma
cholesterol concentrations (for vitamin E and carotenoids)
European Journal of Clinical Nutrition
or protein intakes (for copper and zinc). All tests were
considered signi®cant at P < 0.05.
Results
As indicated in Table 1, the volunteers who completed and
those who did not complete the biological and nutritional
study were similar. In addition, the 141 volunteers who
completed the biological study were similar to the 58
volunteers who did not (results not shown).
Seasonal variations of nutritional dietary intakes
No differences were found between periods concerning the
nutritional intake of energy, fat, protein, vitamin E and Zn
(Table 2). Carotenoids decreased from March ± April to
October 1995 (P < 0.002) and then increased between
October 1995 and January ± February 1996 (P ˆ 0.0001).
Vitamin C decreased between March ± April and June ± July
1995 (P ˆ 0.0001), remained stable between June ± July and
October 1995 and increased between October 1995 and
January ± February 1996 (P ˆ 0.0001). Copper intake
decreased between June ± July and October 1995
(P ˆ 0.0023).
Percentages of volunteers with values under two-thirds of
the United States recommended daily allowances or estimated safe and adequate daily dietary intakes of 1989
(National Research Council, 1989) are indicated in Table
3. A greater percentage of subjects with low vitamin C
intake was observed in the rainy than in the dry season
(P ˆ 0.001). The frequency of Cu intake under the estimated
safe and adequate daily dietary intakes (National Research
Council, 1989) was higher in periods 3 and 4 than in periods
1 and 2 (P ˆ 0.002).
Seasonal variations of blood antioxidant defences
Periodic means (or medians) of the studied blood parameter
concentrations are presented in Tables 4 and 5. All were
Seasonal antioxidant imbalance
J Arnaud et al
33
Table 3 Percentage of male Havana volunteers with daily intakes under two-thirds of the
recommended dietary allowances or under estimated safe and adequate daily dietary intakes
(National Research Council, 1989) at different periods
Period a 1
(nc ˆ 189)
Period 2
(n ˆ 153)
Period 3
(n ˆ 164)
Period 4
(n ˆ 145)
Pb
24.9
58.7
4.8
70.4
75.8
60.1
9.1
75.2
76.2
48.8
15.8
80.5
41.4
53.8
15.2
71.7
0.001
NSd
0.002
NS
Vitamin C < 40 mg
Vitamin E < 7 mg
Copper < 1:5 mg
Zinc < 10 mg
a
Period 1 ˆ March and April 1995; Period 2 ˆ June and July 1995; Period 3 ˆ October 1995; Period
4 ˆ January and February 1996. Periods 2 and 3 ˆ rainy season.
b
P ˆ signi®cant period effect probability.
c
n ˆ number of volunteers.
d
NS ˆ nonsigni®cant, P 0:05.
affected by season. Plasma a-tocopherol values decreased
between October 1995 and January ± February 1996
(P < 0.0001). The variations of the ®ve plasma carotenoids
concentrations were not exactly similar but the lowest
plasma concentrations were observed in rainy season for
all of them except b-cryptoxanthin. Plasma a-carotene
concentrations decreased between March ± April and
June ± July 1995 (P < 0.0001), remained low in October
1995 and increased between October 1995 and January ±
February 1996 (P < 0.0001). Plasma b-carotene concentrations decreased between June ± July and October 1995
(P < 0.0001) and then increased between October 1995
and January ± February 1996 (P < 0.0001). Plasma b-cryptoxanthin concentrations increased slowly but regularly
from March ± April to October 1995 and then decreased
between October 1995 and January ± February 1996
(P < 0.05). Plasma lutein=zeaxanthin concentrations
decreased slightly between June ± July and October 1995
(P < 0.05). Plasma lycopene concentrations decreased
between March ± April and June ± July 1995 and then
increased regularly from June ± July 1995 to January ±
February 1996 (P < 0.0001). Serum Cu concentrations
increased from March ± April to October 1995
(P ˆ 0.0001) and then remained similar between October
1995 and February 1996. Serum Se values decreased
between March ± April and June ± July 1995 (P < 0.002),
then increased between June ± July and October 1995
(P < 0.001) and remained similar between October 1995
and January ± February 1996. Serum Zn concentrations
increased between March ± April and June ± July 1995
(P ˆ 0.0003) and then remained similar from June ± July
1995 to January ± February 1996. Erythrocyte GPX activity
Table 4 Blood antioxidants according to season in male Havana volunteers
a-Tocopherol (mmol=l)
a-Tocopherol=cholesterol
(mmol=mmol)
a-Carotene (nmol=l)
b-Carotene (nmol=l)
b-Cryptoxanthin (nmol=l)
Lutein-zeaxanthin (nmol=l)
Lycopene (nmol=l)
Total carotenoids (nmol=l)
Copper (mmol=l)
Selenium (mmol=l)
Zinc (mmol=l)
GPXe (UI=gHbf)
SODg (UI=mgHb)
Glutathione (mmol=l)
Total glutathione (mmol=l)
na
Period b 1
Period 2
129
127
21.3 (8.4)
4.75 (1.45)
21.5 (8.0)
4.99 (1.64)
128
126
129
128
130
123
138
131
138
136
112
30
30
51 (2 ± 466)
95 (13 ± 1088)
93 (67)
563 (163 ± 3503)
348 (41 ± 2130)
1427 (702)
13.3 (2.4)
1.19 (0.22)
12.6 (2.9)
46 (9)
1.21 (0.11)
1197 (296)
1265 (281)
&
%
&
&
%
&
%
24 (2 ± 192)
106(6 ± 1011)
117 (91)
494 (128 ± 7766)
74 (4 ± 492)
1206 (978)
15.1 (2.6)
1.12 (0.18)
13.5 (2.3)
45 (9)
1.20 (0.09)
1231 (392)
1299 (390)
Period 3
&
&
%
&
%
%
%
&
%
Period 4
Pc
0.001
0.001
0.001
0.001
0.001
0.001
0.017
0.001
0.001
0.001
0.001
0.001
0.001
0.006
0.001
0.001
20.9 (7.1)
4.70 (1.12)
&d
&
18.6 (5.2)
4.06 (0.90)
32 (6 ± 155)
60 (6 ± 415)
132 (85)
486 (156 ± 2228)
120 (2 ± 1713)
1037 (554)
16.5 (2.8)
1.19 (0.15)
14.0 (2.4)
42 (9)
1.21 (0.10)
1190 (312)
1295 (315)
%
%
&
43 (6 ± 685)
90 (13 ± 909)
112 (85)
492 (163 ± 3459)
310 (19 ± 2533)
1243 (868)
16.7 (3.4)
1.23 (0.18)
13.7 (2.7)
40 (9)
1.20 (0.10)
925 (248)
955 (246)
%
%
&
&
&
&
Results are expressed as mean (s.d.) except for other than normal distribution parameters, expressed as median (range).
n ˆ number of volunteers.
Period 1 ˆ March and April 1995; Period 2 ˆ June and July 1995; Period 3 ˆ October 1995; Period 4 ˆ January and February 1996. Periods 2 and
3 ˆ rainy season.
c
P ˆ signi®cant period effect probability adjusted for smoking as a ®xed effect and individual as a random effect.
d
% and & ˆ signi®cant between period variations (for P-values, see text, Results section).
e
GPX ˆ glutathione peroxidase.
f
Hb ˆ hemoglobin.
g
SOD ˆ copper zinc superoxide dismutase.
a
b
European Journal of Clinical Nutrition
Seasonal antioxidant imbalance
J Arnaud et al
34
Table 5
Seasonal variations of blood oxidative stress indices in male Havana volunteers
na
Period b 1
Period 2
Diglutathione (mmol=l)
Diglutathione=total glutathione (%)
30
30
TBARSe (mmol=l)
TBARS=cholesterol (mmol=mmol)
132
130
31 (13)
1.94
(1.40 ± 22.9)
3.15 (0.55)
0.72 (0.15)
34 (15)
2.62
(0.60 ± 5.89)
2.96 (0.48)
0.69 (0.13)
&
&
Period 4
Pc
20 (7)
2.03
(0.91 ± 4.04)
3.12 (0.47)
0.70 (0.15)
0.001
0.001
0.001
0.001
0.001
Period 3
%d
%
%
%
51 (29)
4.29&
(0.74 ± 8.47)
3.33 (0.47)
0.78 (0.17)
&
&
&
&
Results are expressed as mean (s.d.) except for other than normal distribution parameter, expressed as median (range).
n ˆ number of volunteers.
Period 1 ˆ March and April 1995; Period 2 ˆ June and July 1995; Period 3 ˆ October 1995; Period 4 ˆ January and February 1996. Periods 2 and
3 ˆ rainy season.
c
P ˆ signi®cant period effect probability adjusted for smoking as a ®xed effect and individual as a random effect.
d
% and & ˆ signi®cant between period variations (for P-values, see text, Results section).
e
TBARS ˆ thiobarbituric acid reactive substances.
a
b
decreased from June ± July 1995 to January ± February 1996
(P < 0.0001). Erythrocyte SOD activity increased between
June ± July and October 1995 (P < 0.001) and decreased
from October 1995 to January ± February 1996 (P < 0.01).
A signi®cant decrease in whole blood GSH concentrations
was observed between October 1995 and January ± February
1996 (P < 0.004). Whole blood GSSG concentrations and the
ratio GSSG=tGSH increased between June ± July and October 1995 (P < 0.01) and decreased between October 1995
and January ± February 1996 (P < 0.01). Serum TBARS
concentrations were the lowest in June ± July 1995 and the
highest in October 1995. The decreases between March ±
April and June ± July 1995 and between October 1995 and
January ± February 1996, as well as the increase between
June ± July and October 1995 were signi®cant (P ˆ 0.0001).
The percentages of volunteers with blood concentrations
under de®cient cut-off for vitamin and trace elements are
presented in Table 6. Plasma b-carotene values were under
0.37 mmol=l in more than 89% of the volunteers. Frequencies of b-carotene and Zn sub-de®ciencies were lower,
respectively, in June ± July 1995 and in October 1995 than
in the other periods.
Relations between intakes and status
Simple Pearson correlations between nutrient intakes and
blood concentrations are indicated in Table 7 and adjusted
Pearson correlations in Table 8. Copper intakes and serum
Table 6
concentrations were correlated in January ± February 1996
(P < 0.05). The simple correlation between zinc intakes and
serum concentrations (P < 0.05) observed in March ± April
1995 was lost after adjustment by BMI, smoking, energy,
protein and alcohol intakes. Vitamin E intakes and plasma
concentrations were not related whatever the correlation
performed. Total carotenoids intakes and plasma concentrations were correlated in October 1995 whatever the correlation performed and in March ± April 1995 when plasma total
carotenoid concentrations were expressed as mmol=l and
simple Pearson correlation was used.
Discussion
The energy (Ascherio et al, 1992; Cooney et al, 1995),
Zn (Parr et al, 1992), carotenoids (Guilland et al, 1986;
Ascherio et al, 1992) and vitamin C and E (Guilland et al,
1986; Herbeth et al, 1988) intakes of the studied Cuban
population were low whereas Cu intake was rather high
(Parr et al, 1992) compared to reported values for adult men
in other countries. Energy and vitamin C intakes reported in
the present work were lower whereas protein and fat intakes
were higher than the values reported in 1992, at the beginning of the epidemic neuropathy (PeÂrez CristiaÁ & FleitesMestre, 1994) using the same method for dietary assessment. This method is regularly used for Cuban dietary
Percentage of male Havana volunteers with blood values under the de®cient cut-off according to season
a-tocopherol <11.6 mmol=l
a-tocopherol=cholesterol <2.22 mmol=mmol
b-carotene <0.37 mmol=l
Selenium <0.75 mmol=l
Zinc <10.7 mmol=l
Period a 1
(nc ˆ 199)
Period 2
(n ˆ 176)
Period 3
(n ˆ 163)
Period 4
(n ˆ 162)
Pb
6.3
0
96.3
1.5
23.7
3.0
0.6
89.1
1.7
13.6
3.7
0
98.7
0
6.1
7.5
0
99.4
0.7
10.7
NSd
NS
0.001
NS
0.001
a
Period 1 ˆ March and April 1995; Period 2 ˆ June and July 1995; Period 3 ˆ October 1995; Period 4 ˆ January and February
1996. Periods 2 and 3 ˆ rainy season.
b
P ˆ signi®cant period effect probability.
c
n ˆ number of volunteers.
d
NS ˆ nonsigni®cant.
European Journal of Clinical Nutrition
Seasonal antioxidant imbalance
J Arnaud et al
Table 7 Seasonal simple Pearson correlations between micronutrient
intakes and corresponding blood concentrations
Period a 1
b
Vitamin E
Total carotenoidsd
Total carotenoidsf
Copper
Zinc
c
NS
re ˆ 0:148
P ˆ 0:048
n ˆ 179
NS
NS
r ˆ 0:161
P ˆ 0:028
n ˆ 188
Period 2
Period 3
Period 4
NS
NS
NS
NS
NS
NS
r ˆ 0:200
P ˆ 0:017
n ˆ 142
r ˆ 0:216
P ˆ 0:010
n ˆ 142
NS
NS
NS
NS
NS
r ˆ 0:225
P ˆ 0:010
n ˆ 145
NS
a
Period 1 ˆ March and April 1995; Period 2 ˆ June and July 1995; Period
3 ˆ October 1995; Period 4 ˆ January and February 1996. Periods 2 and
3 ˆ rainy season.
b
Same results were obtained with plasma a-tocopherol concentrations
expressed in mmol=l and mmol=mmol cholesterol.
c
NS ˆ nonsigni®cant.
d
Plasma total carotenoid concentrations expressed in mmol=l.
e
r ˆ correlation coef®cient, P ˆ signi®cant probability of the correlation,
n ˆ number of subjects involved in the correlation.
f
Plasma total carotenoid concentrations expressed in mmol=mmol
cholesterol.
Table 8 Seasonal adjusted Pearson correlations between micronutrient
intakes and corresponding blood concentrations
Period a 1
Period 2
Period 3
Period 4
Vitamin Eb
Total carotenoidsd
NSc
NS
NS
NS
NS
NS
Total carotenoidsf
NS
NS
Copperg
NS
NS
NS
re ˆ 0:194
P ˆ 0:026
n ˆ 137
r ˆ 0:203
P ˆ 0:019
n ˆ 137
NS
Zincg
NS
NS
NS
a
NS
r ˆ 0:212
P ˆ 0:017
n ˆ 131
NS
Period 1 ˆ March and April 1995; Period 2 ˆ June and July 1995; Period
3 ˆ October 1995; Period 4 ˆ January and February 1996. Periods 2 and
3 ˆ rainy season.
b
Pearson correlation was adjusted by BMI, number of cigarettes smoked=day,
energy, fat and alcohol intakes, plasma cholesterol concentrations when
plasma a-tocopherol concentrations were expressed in mmol=l and by BMI,
number of cigarettes smoked=day, energy, fat and alcohol intakes, when
plasma a-tocopherol concentrations were expressed in mmol=mmol
cholesterol.
c
NS ˆ nonsigni®cant.
d
Pearson correlation was adjusted by BMI, number of cigarettes smoked=day,
energy, fat and alcohol intakes, plasma cholesterol concentrations and plasma
total carotenoid concentrations were expressed in mmol=l.
e
r ˆ correlation coef®cient, P ˆ signi®cant probability of the correlation,
n ˆ number of subjects involved in the correlation.
f
Pearson correlation was adjusted by BMI, number of cigarettes
smoked=day, energy, fat and alcohol intakes and plasma total carotenoid
concentrations were expressed in mmol=mmol cholesterol.
g
Pearson correlation was adjusted by BMI, number of cigarettes
smoked=day, energy, protein and alcohol intakes.
intake estimates and the average daily energy intakes
reported in La Lisa were, respectively 11.9, 7.8 and
9.6 MJ in 1989, 1993 and 1998 (unpublished observations).
Nevertheless, it has been demonstrated that reliable diet
records are dif®cult to obtain, especially from randomly
selected subject (Black et al, 1991; Golberg et al, 1991;
Scott et al, 1996). In particular, the energy intakes=BMR
ratio indicated in Table 1 suggest an underestimation of the
energy intakes (Black et al, 1991), probably explained by an
underestimation of the consumption of food and beverage
rich in carbohydrates. However, the low energy intakes
could also be the consequence of the very severe economic
crisis in Cuba at that time. Decrease in cooking time,
because of fuel shortages, has been proposed as a possible
contributing factor to the epidemic neuropathy (Tucker &
Hedges, 1993) and could partly explain the low energy
intakes reported in Table 2. Indeed, the energies of raw and
thoroughly cooked polished rice, which is the most consumed food in Cuba (from 210 to 260 g=day according to
period) are, respectively, 1.53 and 0.46 MJ=100 g (Rodriguez et al, 1992). Therefore, a dramatic decrease in cooking
time could signi®cantly underestimate the energy intakes.
Low energy intake makes the attainment of requirements of
vitamin and Zn intakes dif®cult, which contributes to the
high frequencies of Cuban subjects with intake of vitamin C,
vitamin E and Zn lower than two-thirds of the RDA
(National Research Council, 1989). The low antioxidant
intakes in the studied Cuban population support the hypothesis that this population is still at risk of oxidative stress.
The rainy season in Cuba (periods 2 and 3) appears the
most likely to contribute to antioxidant vitamin C and
carotenoid de®ciencies as their intakes were the lowest.
These low intakes are probably the consequences of the
dramatically lower fruit and vegetable consumption during
the rainy than the dry season (Barnouin & PeÂrez-CristiaÁ,
1998). Decline of vitamin C intakes has been previously
reported during the rainy season in Gambia (Bates et al,
1994). To our knowledge, seasonal variations of dietary
carotenoids have not been reported in tropical countries. In
more temperate countries, results are discrepant (Saintot et
al, 1995; Scott et al, 1996). As previously described (Scott
et al, 1996, Tucker et al, 1999), correlations between total
carotenoid intakes and plasma concentrations depended on
period and adjustments. The in¯uence of period could be
related to differences in the carotenoid bioavailability from
foods (Ascherio et al, 1992). However, whatever the adjustment used, total carotenoid intakes and plasma concentrations were correlated in October 1995 when they were
at their lowest values. This constant relation could be
explained by reduced individual variations in dietary
intake and plasma carotenoid concentrations in this de®cient
period and contrasts with the lack of association between
b-carotene, lutein or lycopene dietary intakes and plasma
concentrations observed in developing countries (Thurnham
et al, 1998). Comparison with previous works is dif®cult
(Asherio et al, 1992; Rautalahti et al, 1993; Pamuck et al,
1994; Yong et al, 1994; Cooney et al, 1995, Saintot et al,
1995, Tucker et al, 1999) because the degree of correlation
35
European Journal of Clinical Nutrition
Seasonal antioxidant imbalance
J Arnaud et al
36
between intakes and blood carotenoid concentrations largely
depends on the type of carotenoid (Asherio et al, 1992;
Yong et al, 1994, Tucker et al, 1999), carotenoid biovailability from food sources (Asherio et al, 1992; Yong et al,
1994), season (Saintot et al, 1995; Scott et al, 1996),
smoking (Yong et al, 1994), the method of dietary assessment (Scott et al, 1996) and other unknown factors (Yong et
al, 1994). Moreover, the discrepancies in the relation
between carotenoid intakes and plasma concentrations,
reported in different studies, may re¯ect a problem in the
accurate measurement of the intakes of micronutrients and
the variability in the dietary and biochemical measurements
(Scott et al, 1996). Concerning the vitamin E intakes, which
remained similar all year long, our results agree with
previous work (Guilland et al, 1986). No direct correlation
was found between intakes and plasma concentrations,
although vitamin E dietary intake is known to be one of
the determinants of serum a-tocopherol concentrations and
the positive relation reported between vitamin E intakes and
status in almost all studies (Ascherio et al, 1992; Rautalahti
et al, 1993; Pamuck et al, 1994; Cooney et al, 1995).
However, some studies conducted in France do not ®nd
link between intake and plasma concentrations for vitamin E
(Herbeth et al, 1988; Costa de Carvahlo et al, 1996).
Moreover, the relation between vitamin E intakes and
status has been reported to be weak in non-supplement
users (Ascherio et al, 1992) and smokers (Coates et al,
1991). However, the lack of relation between vitamin E
intakes and plasma concentrations observed in this study
could also be related to an inaccurate measurement of
vitamin E intakes. The seasonal changes in Cu intakes
contrast with the results obtained in the United States,
where intakes remained similar all year long (Patterson
et al, 1984). Furthermore, no correlation between dietary
intakes and serum concentrations of Cu or Zn were found in
the United States (Patterson et al, 1984) in contrast with the
correlation between copper intakes and serum concentrations found in January and February 1996. The relation
between zinc intakes and serum concentrations depended on
season and adjustment, suggesting the determining role of
factors, ie smoking, energy, protein and alcohol intakes on
serum zinc concentrations (Kant et al, 1989; Hashim et al,
1996).
The plasma lutein=zeaxanthin concentrations were relatively high compared to those reported in developed countries (Ascherio et al, 1992; Olmedilla et al, 1994; Pamuch et
al, 1994; Scott et al, 1996; Van de Vijver et al, 1997),
whereas the plasma concentrations of a-tocopherol, a- and
b-carotenes, b-cryptoxanthin and lycopene were in the
lower range of previous studies whatever the period (Herbeth et al, 1992; Ascherio et al, 1992; Olmedilla et al, 1994;
Scott et al, 1996). These results agree with studies conducted in developing countries (Thurnham et al, 1998).
Moreover, the plasma concentrations of b-cryptoxanthin
and a- and b-carotenes were lower than during the Cuban
epidemic neuropathy in 1993, whereas plasma lycopene
concentrations were similar (PeÂrez-CristiaÁ & FleitesMestre, 1994; CNFIT, 1995). These low plasma carotenoid
European Journal of Clinical Nutrition
and a-tocopherol concentrations emphasize the risk of antioxidant imbalance in the studied population. Although not
correlated to the corresponding dietary intakes, they could
be related to the low carotenoids and vitamin E supplies
from diet. Whatever the season, the serum trace elements
(Zn, Se and Cu) concentrations were in the range of
international reference ranges (Iyengar, 1989) and similar
to those observed during the Cuban epidemic neuropathy in
1993 (PeÂrez-CristiaÁ & Fleites-Mestre, 1994; CNFIT, 1995).
Erythrocyte GPX and SOD activities depend largely on the
method used for their determination. The activities obtained
in Cuban subjects were in the range of previous values
observed in French adults using the same methods (Hininger
et al, 1997; Preziosi et al, 1998; Roussel et al, 1998). These
results agree with the reported lack of effect of energyrestricted diet on GPX and SOD activities in experimental
conditions (Velthuis-te Wierick et al, 1995). Oxidation of
GSH to GSSG is an early index of oxidative stress (Sen,
1997) and TBARS is a commonly used screening parameters to assess oxidative stress in epidemiological studies,
although it lacks speci®city. In Cuban subjects, serum
TBARS concentrations were higher than those found in
French adults (Preziosi et al, 1998; Roussel et al, 1998) and
similar to those during the epidemic neuropathy in 1993
(PeÂrez-CristiaÁ & Fleites-Mestre, 1994). Moreover, the
TBARS=cholesterol ratio was very high compared to
French adults (Roussel et al, 1998), suggesting an intensive
lipid peroxidation. Whole blood GSSG concentrations were
higher or similar, depending on season, than values
observed in French adults (Preziosi et al, 1998) and whole
blood GSH concentrations were relatively high compared to
previous works (Michelet et al, 1995: Preziosi et al, 1998).
Ninety percent of plasma b-carotene individual values
were under the de®cient cut-off values. These results con®rm the carotene de®ciency in the Cuban studied population. Indeed, these percentages are dramatically higher than
those reported elsewhere (Pamuck et al, 1994; Preziosi et al,
1998). The percentage of subjects at risk of Zn de®ciency is
also higher than those reported elsewhere (Roussel et al,
1998).
The seasonal patterns of plasma carotenoids observed in
Cuba were dif®cult to compare to those of previous studies
performed in countries where seasonal changes in diet,
temperature and sunlight exposure are completely different
(Rautalahti et al, 1993; Olmedilla et al, 1994; Winklhofer-Roob et al, 1997). Moreover, fruit and vegetable
consumption, the major sources of carotenoids, largely
depends on the population lifestyle, food distribution
system and culture and is traditionally low in Cuba. The
dramatic increase in some plasma carotenoids (a-carotene,
lycopene) concentrations and decrease in plasma b-cryptoxanthin concentrations during the dry season agree in part
with previous works (Thurnham & Flora, 1988; Olmedilla
et al, 1994; Scott et al, 1996) and could be the consequence
of fruit and vegetable consumption changes (Barnouin
& PeÂrez-CristiaÁ, 1998). Plasma a- and b-carotene
concentrations generally increase in summer and=or fall
(Olmedilla et al, 1994; Saintot et al, 1995; Scott et al,
Seasonal antioxidant imbalance
J Arnaud et al
1996), whereas plasma b-cryptoxanthin concentrations
increase in winter (Thurnham & Flora, 1988; Olmedilla et
al, 1994). No clear seasonal patterns of change for plasma
lutein and lycopene concentrations have been reported
(Olmedilla et al, 1994; Cooney et al, 1995; Scott et al,
1996; Van de Vijver et al, 1997). In Hawaii, an island at the
same latitude as Cuba, large increase in plasma a- and bcarotene concentrations were reported in the dry season,
whereas lutein=zeaxanthin concentrations decreased
(Cooney et al, 1995). The within-subject variability may
be the main determinant of these discrepancies. Previous
works on plasma a-tocopherol concentration seasonal ¯uctuation are also discrepant (Rautalahti et al, 1993; Olmedilla
et al, 1994; Maes et al, 1996; Van de Vijver et al, 1997;
Winklhofer-Roob et al, 1997). The decrease in plasma atocopherol concentrations observed in January ± February
1996 could not be explained by difference in vitamin E
intakes, which remained similar during the entire study. To
the best of our knowledge, seasonal variations of serum Cu
and Se concentrations have not been described before. No
difference in serum Zn concentrations was reported between
winter and summer in Japan (Ohno et al, 1988). The
observed variations in serum Zn and Cu concentrations in
Cuban subjects were not related to the variations observed
in the corresponding dietary intakes. To our knowledge,
seasonal changes in whole blood glutathione concentrations,
erythrocyte GPX and SOD activities have not been reported
previously.
Conclusion
The overall results suggest that this Cuban population
represents a vulnerable population group, especially in
terms of antioxidant vitamin, carotenoids and Zn de®ciencies. Although no strong correlation exists between plasma
carotenoids, vitamin E and Zn concentrations and the
corresponding dietary intake, the studied Cuban population
demonstrated inadequate carotenoids, vitamin E and Zn
intakes and blood concentrations. Moreover, the rainy
season appears to present a greater risk of oxidative stress
than the dry season. Indeed, vitamin C intakes and total
carotenoid intakes and plasma concentrations were lower,
whereas GSSG concentrations were higher in the rainy than
in the dry season. The Cuban Ministries of Public Health
and Agriculture started a national research program on the
effects of vegetable cultivation development and increase in
fruit consumption on antioxidant imbalance of the Cuban
population.
Acknowledgements ÐNestec-NestleÂ, Merck-Biotrol Diagnostics and the
Trace Element Institute for Unesco are thanked for their ®nancial support.
Pharmaciens sans FrontieÁres are acknowledged for the logistical help. The
authors thank the Cuban volunteers who participated in this study. The
technicians, nurses, physicians and researchers involved in this study are
also gratefully acknowledged.
37
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