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Articles |
1
Northern Ireland Centre for Diet and Health, School of Biomedical Sciences, University of Ulster, Coleraine, County Londonderry, Northern Ireland BT52 1SA, United Kingdom.
2
Institute of Food Research, Colney, Norwich NR4 7UA,
United Kingdom.
a Author for correspondence. Fax 44-2870-324965; e-mail M.Chopra{at}ulst.ac.uk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Methods: After a depletion period of 8 days, 34 healthy females (18 nonsmokers, 16 smokers) were supplemented with ß-carotene- and lutein-rich (green) and lycopene-rich (red) vegetable foods, each for 7 days.
Results: Baseline concentrations (mean ± SD) of plasma ß-carotene (0.203 ± 0.28 µmol/L vs 0.412 ± 0.34 µmol/L; P <0.005) and lutein (0.180 ± 0.10 vs 0.242 ± 0.11 µmol/L; P <0.05) but not lycopene (0.296 ± 0.10 vs 0.319 ± 0.33 µmol/L) were significantly lower in smokers compared with nonsmokers. After supplementation, the change (supplementation minus depletion) in plasma ß-carotene (0.152 ± 0.43 vs 0.363 ± 0.29 µmol/L in smokers vs nonsmokers; P = 0.002) and LDL lutein (0.015 ± 0.03 vs 0.029 ± 0.03 µmol/mmol cholesterol; P = 0.01) was significantly lower in smokers than nonsmokers. Green-vegetable supplementation had no effect on the resistance of LDL to oxidation (lag-phase) in either group. After red-vegetable supplementation, plasma and LDL lycopene concentrations were increased in both groups, but only nonsmokers showed a significant increase in the lag-phase (44.9 ± 9.5 min at baseline, 41.4 ± 6.5 min after depletion, and 49.0 ± 8.9 min after supplementation; P <0.01) compared with depletion.
Conclusions: In this short-term intervention study, a dietary intake of >40 mg/day of lycopene by a group of nonsmoking individuals significantly reduced the susceptibility of LDL to oxidation, whereas an equivalent increase in lycopene by a group of smokers showed no such effect.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The formation of oxidized LDL is believed to be important in the etiology of atherosclerosis and hence vascular diseases, including cardiovascular disease (9). Supplementation studies with respect to the effects of ß-carotene on LDL oxidation have produced conflicting results (10)(11)(12)(13), whereas others have raised concerns regarding possible toxic effects of ß-carotene supplements in smokers (14), especially because two large-scale intervention trials have reported an increased incidence of cancer in smokers and asbestos workers (15)(16). However, both epidemiological and intervention studies continue to point to the benefits of carotenoid-rich fruits and vegetables. In addition, whole foods contain many nutritive and nonnutritive components that may add to, or modify, the antioxidant effects of carotenoids.
In previous studies (17)(18), volunteers
received a mixed diet of vegetables providing an intake of 
30 mg of
total carotenoids for 2 weeks. In these studies, susceptibility of
lipoproteins to oxidation was reduced in both smokers and nonsmokers.
However, food consumed by the volunteers contained a mixture of
carotenoids; therefore, it was not possible to assess whether the
beneficial effects on lipoprotein oxidation were attributable to a
specific carotenoid component.
The objective of the present research program was to study in greater detail the response of smokers and nonsmokers to a carotenoid-rich diet. The protocol differed from previous studies in that the volunteers received either a diet predominantly rich in lycopene (red vegetables) or one rich in ß-carotene and lutein (green vegetables) for a period of 7 days. A supplementation period of 7 days was selected because in our previous study (17) we did not find a significant difference in plasma carotenoids between days 7 and 14 of supplementation. In addition, to increase the sensitivity of the response, a short vegetable-depletion period was introduced before the commencement of vegetable intervention. Plasma antioxidant concentrations and oxidative resistance of LDL to ex vivo copper-initiated oxidation were measured before and after dietary intervention.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The average cigarette consumption by the smokers was 16 ± 9 (SD) cigarettes/day. Smokers were asked to continue their customary cigarette smoking habits, which included smoking at least one cigarette after the meal. With the exception of one volunteer who was on vitamin C supplements, subjects did not consume vitamin supplements during the study. Data from this subject were included because her plasma vitamin C concentrations remained within the population range throughout the study. Seven volunteers took oral contraceptives, one volunteer was undergoing hormone replacement therapy, and four women were postmenopausal. Menstrual cycle information was provided by 24 women (13 nonsmokers and 11 smokers). At the baseline blood sampling, 14 volunteers (8 nonsmokers and 6 smokers) were in the follicular phase, and 10 (5 nonsmokers and 5 smokers) were in the luteal phase of the menstrual cycle.
Prestudy blood samples (baseline) were collected and analyzed for plasma lipid concentration, full blood count, plasma glucose concentration, and liver function (liver enzymes). With the exception of one smoker with a plasma triglyceride concentration of 3.63 mmol/L, all measurements were within the reference interval. The body mass index and all variables were monitored when volunteers returned to provide their blood samples.
Two volunteers failed to complete the study: one smoker withdrew from the study during the "green week" (see below), and results from another smoker whose combined intake of ß-carotene and lutein was only 1.5 mg/day during the green week were also excluded. Thus, 32 subjects completed the whole study, and during the green week, the dietary intake data were available for 31 subjects only.
Ethical approval for the study was obtained from the University of Ulster, Ethical Committee, and the subjects gave written informed consent.
intervention study
The study lasted 4 weeks: week 1, dietary monitoring; week 2,
dietary vegetable depletion (8 days); and weeks 3 and 4, red- or
green-vegetable intervention for 7 days each. Throughout the study,
starting from 1 week before the first blood sampling, volunteers were
asked to keep a record of their daily intake of food. The information
was used to monitor their dietary intake. At the end of the first week,
the first blood sample was taken. During week 2, volunteers were asked
to exclude most carotenoid-containing foods from their diet. Such foods
included red, green, yellow, and orange vegetables; egg yolk; colored
cheese; and fish such as salmon, rainbow trout, crawfish, lobster, and
shrimp. During the depletion week, the only vegetables that the
subjects were allowed to consume were potatoes, mushrooms, and
cauliflower, and the only fruits were bananas and pears. After the
depletion period (8 days), another blood sample was taken, and
volunteers were divided in two groups: one group (6 smokers and 10
nonsmokers) was given green vegetables and subjects were asked to
refrain from red vegetables (green week), whereas the second group (10
smokers and 8 nonsmokers) received red vegetables and was asked to
refrain from green, orange, and yellow vegetables (red week). At the
end of the third week, another blood sample was taken and volunteers
were asked to change to the alternative vegetable group. At the end of
the fourth week, the final blood sample was taken. The blood samples
collected are referred to as baseline, depletion, green, and red
according to the week at the end of which blood was collected.
The possibility of a second depletion period between red and green vegetables was considered but was not well received by subjects and would have required additional blood sampling. Therefore, a washout period between the red and green weeks was not introduced.
dietary aspects
Volunteers were asked to keep as precise a record as possible of
their daily intake of foods, especially carotenoid-rich food. They were
asked also to record a brief description of cooking methods, drink
accompanying the meal, and leftovers. They also kept a record of their
sports activities and alcohol and cigarette consumption. Volunteers
completed a lifestyle questionnaire, and a food diary was given each
week to record dietary intake. The dietary intake of carotenoids was
assessed from the food diaries on four separate occasions (days 7, 14,
21, and 28), with day 7 being the habitual, or background diet.
Volunteers were provided with vegetable food packs (see below)
of known weight and were asked to specify on their dietary sheets
whether they consumed all, one-half, or one-fourth of the pack. All
subjects were instructed to consume at least 200 g of creamed
spinach and 100 g of mango puree/day in the green week and
200 g of tomato puree and 100 g of watermelon/day in the
"red week". They were also provided with additional weighed foods,
and were given the following list of food items to choose from and an
indication of the amounts required to provide 25 mg carotenoids/day.
This amount of carotenoids was obtained by consuming 300–400 g of
vegetables and fruits/day. The foods included processed frozen foods
(creamed spinach, peas, corn on the cob, sweet corn, broccoli, parsley,
mixed vegetables, and mango puree), canned foods (tomato soup, tomato
juice, tomato puree, tomato sauce, and baked beans), and fresh food
(iceberg lettuce, spring greens, tomatoes, and watermelon). The daily
intake of ß-carotene, lycopene, and lutein consumed was calculated
using a computerized nutrient analysis program, CompEat (Lifetime
Nutritional Services Ltd, London), to which an extended carotenoid
database was added (19). The carotenoid content of some of
the major foods consumed by the volunteers is shown in Table 1
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blood sampling
Blood samples (30 mL) were collected in plain syringes at the
following time points: baseline, after 8 days of depletion, and at the
end of each supplementation week. Blood was transferred into
appropriate tubes for different analyses. Aliquots of blood for LDL
isolation and micronutrient analysis were transferred to tubes
containing 1 g/L EDTA. Plasma was obtained after centrifugation at
1000g for 10 min at 10 °C and centrifuged at
1000g for an additional 45 min to remove any remaining cell
debris (Mistral 3000 centrifuge). Plasma for ascorbate analysis was
diluted with 2 parts 100 g/L meta-phosphoric acid and frozen at
-70 °C within 1 h of separation.
biochemical measurements
The fresh blood samples collected were routinely screened for a
full blood count, lipid profile, liver function tests, and serum
creatine kinase activity at the local hospital laboratory to monitor
the health of subjects during the study period.
lipoprotein isolation
Lipoproteins were prepared from freshly isolated plasma. Briefly,
the density of plasma was adjusted by adding 0.32 g of KBr per mL
of plasma (Sigma). The density-adjusted plasma ( 4 mL) was layered
below 7 mL of a KBr solution (density = 1.005 kg/L), pH 7.4,
containing 1 g/L disodium EDTA in 11-mL Optiseal polyallomer tubes
(Beckman). Lipoproteins were isolated by centrifugation in a Beckman
XL-70 ultracentrifuge equipped with a NVT60 rotor at 300 000gfor 2.5 h at 7 °C. LDL and HDL layers were aspirated by
syringe and stored at 4 °C under nitrogen until required for
analysis. Electrophoresis of LDL and HDL on Paragon lipoprotein gels
(Beckman) showed that the lipoprotein preparations were free of
contamination from plasma proteins (checked using albumin calibrator
and Coomassie blue protein stain). LDL oxidation and micronutrient
analyses were completed on the freshly isolated LDL and plasma within 2
days of collection of blood.
ldl oxidation
Before oxidation, 0.5 mL of LDL was dialyzed under nitrogen for
24 h at 4 °C in 700 volumes of degassed, phosphate-buffered
saline, pH 7.4, containing 10 µmol/L EDTA. The cholesterol content of
the dialyzed LDL (DLDL) was measured by the CHOD-PAP method
(Boehringer), which entailed adding 10 µL of sample to 1 mL of
cholesterol reagent. The absorbance was measured after 15 min at 500
nm. The average absorbance of duplicate measurements was multiplied by
a factor of 5.75 to calculate cholesterol concentration of DLDL in
mg/mL. For oxidation experiments, total (mass) DLDL/mL was calculated
by multiplying the DLDL cholesterol concentration by the previously
published factor of 3.16 (20). The final volume in the
cuvette, 1 mL, contained 0.25 mg of DLDL mass and was equivalent to 0.1
µmol/L DLDL.
Oxidation was initiated by the addition of copper chloride (final concentration in the cuvette, 11.7 µmol/L), and the formation of lipid diene conjugates was monitored at 234 nm. The calculated precision for the measurement of lag-phase after copper-initiated oxidation of LDL was 5.6% (n = 5). The intraassay precision of the cholesterol assay was 3% (n = 6).
micronutrient analysis
Reversed-phase HPLC was used to measure the fat-soluble
micronutrient content of the plasma and lipoprotein fractions
(21). The assay was standardized with pure calibrators of
ß-carotene, lutein, lycopene, and 
-tocopherol, and response
factors were calculated to estimate the micronutrient concentrations of
the plasma and lipoproteins. The following procedure was used to
measure micronutrients in the samples.
The sample (0.1 mL) was mixed with 0.1 mL of 100 g/L sodium dodecyl
sulfate (Sigma) and 0.2 mL of ethanolic tocopherol acetate (85
µmol/L; Sigma), and vortex-mixed for 1 min. To this mixture, 1 mL of
heptane containing 500 mg/L butylated hydroxytoluene (Sigma) was added,
and samples were vortex-mixed for 3 min. Samples were centrifuged at
1000g for 10 min at 10 °C. The upper heptane layer (0.7
mL) was removed, evaporated to dryness, and reconstituted in 0.1 mL of
reconstitution mobile phase [acetonitrile:methanol:dichloromethane,
500:500:128 (by volume) and 100 mg/L butylated hydroxytoluene]. An
aliquot (50 µL) of the sample was injected onto a 3 µm Spherisorb
ODS2 column (10 cm x 4 mm) on a Millipore-Waters HPLC system. The
samples were eluted with mobile phase (as above except that it
contained 10 mg/L butylated hydroxytoluene) at 1 mL/min. Data were
collected and integrated using Maxima software (Millipore-Waters).
Lipoprotein micronutrient and plasma -tocopherol concentrations were
expressed in µmol/mmol cholesterol. The intraassay CVs for the
micronutrient analyses in plasma and LDL, based on six measurements of
the same sample, are shown in Table 2
.
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statistical analysis
Data were skewed and thus were transformed using logs (ln).
Parametric tests were used on log-transformed data. The lag-phase,
dietary intake, and plasma, LDL, or HDL data for each carotenoid (-
and ß-carotene, lutein, and lycopene) or tocopherol (- and
-tocopherol) concentration were examined in smokers and nonsmokers
separately. Two-way repeated-measures ANOVA was used to determine the
main effects of time (i.e., the effect of depletion and
supplementation) and smoking. For those variables that showed a
significant effect of time, within-subject comparisons were done by
paired t-test to detect which periods differed significantly
from each other at P <0.05. Values shown in
Tables 2–7
and Fig. 1
are geometric means and standard deviations.
Standard deviations were calculated from the formula: antilog (log mean
+ log SD) - geometric mean. The effect of smoking on the change
in carotenoids (supplementation minus baseline/depletion) was
analyzed using one-way ANOVA. The statistical analysis was
carried out with the statistical package for Social Sciences (SPSS,
Ver. 6; SPSS Inc) and STATISTICA (Statsoft).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. The groups were of similar age, and the only differences were
the higher serum triglycerides (P = 0.044) and alkaline
phosphatase activity (P = 0.029) in the smokers. The
white blood cell counts in smokers also tended to be higher but were
not significant (P = 0.073). There was no change in any
of the biochemical variables tested over the 4-week study period
(results not shown). Subjects maintained consistent habits, i.e.,
physical exercise and alcohol intake, throughout the study period.
There was no significant difference in plasma or LDL carotenoid
responses between the women taking contraceptives and those who were
not (results not shown). The LDL carotenoids of 24 women in different
phases of the menstrual cycle were compared at all time points. Only
lycopene was significantly different at the depletion time point
(results not shown).
dietary intakes of carotenoids at the four time points
Analyses of the dietary ß-carotene, lycopene, and lutein intake
of the two groups of subjects at the four time points are shown in
Table 4
. Repeated-measures ANOVA analysis for time and smoking showed a
significant effect of time but no effect of smoking. At the end of the
depletion week, intake of all carotenoids (ß-carotene, lutein, and
lycopene) was significantly lower than the baseline (P
<0.01) in both smokers and nonsmokers. In weeks 3 and 4, after dietary
intervention, both groups showed a significant increase in the dietary
intake of lycopene in response to red fruits and vegetables
(P <0.001, paired t-test) and for ß-carotene
and lutein in response to green vegetables (all P <0.001)
compared with both the baseline and depletion values. In addition,
during the red week, the intake of ß-carotene and lutein was restored
to baseline values.
The smokers and nonsmokers were randomly allocated to receive the red
and green vegetables during the third and fourth weeks. An independent
t-test was used to investigate whether the changes in plasma
or LDL carotenoid concentrations were influenced by the order in which
the vegetables were given to the volunteers. No difference was found
between the group receiving red vs those receiving green vegetables
first. Therefore, the results obtained from subjects consuming each
specific vegetable supplement were combined for the subsequent analyses
shown in Tables 5
and 6
and Fig. 1
.
comparison of plasma and lipoprotein micronutrients in smokers and
nonsmokers at baseline and depletion
One-way ANOVA of the baseline and depletion data showed that
plasma ß-carotene (P <0.005; data shown in Table 5
) and lutein (P <0.05) concentrations were
significantly different between smokers and nonsmokers. In LDL, the
effect of smoking was seen only for ß-carotene at baseline
(P <0.01) and for both ß-carotene (P <0.005)
and lutein (P <0.01) at depletion. In HDL, the effect of
smoking was seen for lutein at both baseline (P <0.02) and
depletion (P <0.002), and only at depletion for
ß-carotene (P <0.01). No effect of smoking was observed
for lycopene in plasma or lipoprotein fractions. Plasma ascorbate and
the -tocopherol:cholesterol ratio were not different between smokers
and nonsmokers.
changes in plasma and lipoprotein micronutrient concentrations
during the study period
Two-factor repeated-measures ANOVA showed a significant effect of
time on the plasma and lipoprotein concentrations of carotenoids and a
significant effect of smoking on plasma lutein only. A pair-wise
comparison on the log-transformed data was performed to detect which
periods differed significantly from each other.
effect of depletion diet
The depletion diet produced a decrease in plasma carotenoids and
ascorbic acid. Combined data for smokers and nonsmokers showed that the
depletion diet produced a significant decrease in plasma ß-carotene
(P = 0.04, t-test; Table 5
), lycopene
(P = 0.001), and ascorbic acid (P =
0.02; Table 6
). When smokers and nonsmokers were analyzed separately, the
results were significant only for plasma lycopene (P =
0.001 for nonsmokers; P = 0.03 for smokers) and
ascorbic acid (P = 0.01 for smokers; Table 6
). In LDL,
the depletion diet produced a significant decrease only in ß-carotene
(P = 0.05 for nonsmokers; P = 0.01 for
smokers), and there was no significant change in carotenoids in HDL.
The depletion diet produced a increase in the
-tocopherol:cholesterol ratio in plasma (P = 0.03
for nonsmokers; P = 0.01 for smokers) and HDL
(P = 0.001 for nonsmokers; P = 0.06 for
smokers) but not in LDL (Table 6
).
effect of vegetable supplementation
After vegetable supplementation, both smokers and nonsmokers
showed significant increases in plasma (P = 0.001,
paired t-test; Table 5
) and lipoprotein (P =
0.001) carotenoids at the end their respective weeks, i.e.,
ß-carotene and lutein at the end of the green week and lycopene at
the end of the red week. The increase in carotenoids was significant
compared with both the baseline and depletion values. The distribution
of plasma carotenoids in lipoproteins post vegetable supplementation
were 70% ß-carotene, 19% lutein, and 57% lycopene in LDL and 25%
ß-carotene, 52% lutein, and 22% lycopene in HDL.
After both red- and green-vegetable supplementation, the plasma and
lipoprotein -tocopherol:cholesterol ratios were not different from
depletion values. Plasma ascorbate (Table 6
) was significantly higher
than after depletion in both group of subjects after red
(P = 0.002 for nonsmokers; P = 0.02 for
smokers) and green (P = 0.01 for nonsmokers;
P = 0.03 for smokers) vegetable supplementation. The
supplementation and baseline ascorbate concentrations were not
significantly different.
One-way ANOVA was performed on the change (supplementation minus
baseline/depletion) in plasma and lipoprotein micronutrients to examine
whether smokers and nonsmokers responded differently to increased
vegetable intake (Table 5
). When compared with the depletion point, a
significant effect of smoking was observed for ß-carotene in plasma
(P = 0.01; Table 5
) and for lutein in both the LDL
(P = 0.02; Table 5
) and HDL (P = 0.03)
fractions. When the change in micronutrients was compared using
baseline values, i.e., supplementation minus baseline, ß-carotene did
not reach significance (0.14 ± 0.43 µmol/L in smokers,
0.26 ± 0.5 µmol/L in nonsmokers), but both LDL (0.013 ±
0.03 µmol/mmol cholesterol in smokers vs 0.03 ± 0.01
µmol/mmol cholesterol in nonsmokers; P = 0.011) and
HDL (0.035 ± 0.18 µmol/mmol cholesterol in smokers vs 0.14
± 0.09 µmol/mmol cholesterol in nonsmokers; P =
0.007) lutein remained significantly different between two groups.
These results show that there were some differences in the response of
smokers and nonsmokers to increased fruit and vegetable
supplementation.
Both ß-carotene and lutein were significantly different between
smokers and nonsmokers at the depletion point; further statistical
analysis was therefore carried out using analysis of covariance to
compare the change in carotenoids (supplementation minus depletion)
between smokers and nonsmokers with depletion value used as the
covariate. The results showed that smoking had a significant effect on
the change in plasma ß-carotene (P = 0.01) and lutein
in LDL (P = 0.02) but not in HDL (Table 5
).
impact of dietary intervention on susceptibility of ldl to
oxidation ex vivo
The lag-phase results for both nonsmokers and smokers at the four
time points are shown in Table 7
. Repeated-measures ANOVA showed a significant effect of time
and smoking on the LDL lag-phase. Pairwise comparisons showed that
there was no evidence of any change in the smokers at any time point.
In nonsmokers, there was a significant increase in the lag-phase after
red-vegetable supplementation only (P = 0.01 compared
with depletion). The marginal mean (smokers plus nonsmokers) was
significantly increased after red-vegetable supplementation compared
with the depletion point only (P = 0.042, paired
t-test).
comparative impact of dietary vegetable supplementation in smokers
and nonsmokers
The responses in plasma carotenoids and lag-phase related to the
actual intake of carotenoids in the smokers and nonsmokers are shown in
Fig. 1
. The ratio of change in plasma and dietary intake of
carotenoids was not significantly different between smokers and
nonsmokers. Only nonsmokers showed a positive mean change in the
lag-phase:dietary intake ratio after red-vegetable supplementation;
however, the difference in response between smokers and nonsmokers did
not reach statistical significance (one-way ANOVA).
When the plasma:dietary intake ratios of subjects (both smokers and nonsmokers) were compared between the green and red weeks, plasma concentrations of both ß-carotene and lutein (as µmol/mg of dietary intake) were significantly higher than for lycopene (P = 0.001).
correlations between dietary intake, ldl carotenoids, and lag-phase
There were positive correlations (Pearson) between the dietary
intake and LDL concentrations of respective carotenoids. The dietary
intake of ß-carotene from green vegetables positively correlated with
the LDL ß-carotene for all subjects (r = 0.35;
P = 0.05; n = 30); likewise, there was a
significant correlation between the dietary intake and LDL lutein
concentrations (r = 0.76; P = 0.0001;
n = 31). In LDL, ß-carotene correlated positively with the
lutein (r = 0.61; P = 0.001; n =
31). When analyzed separately, the significance of correlations between
dietary intake and LDL ß-carotene was lost in both smokers
(r = 0.29; not significant; n = 12) and nonsmokers
(r = 0.29; not significant; n = 18), but
correlations between dietary intake and LDL lutein remained significant
(r = 0.75; P = 0.001; n = 18 for
nonsmokers; and r = 0.88, P = 0.001;
n = 13 for smokers).
For lycopene, there was a significant positive correlation between the dietary and LDL lycopene concentrations for 34 subjects (r = 0.36; P = 0.03), but the significance was lost when smokers and nonsmokers were analyzed separately.
No correlation was observed between the LDL carotenoid concentrations and the lag-phase. However, the increase in dietary intake of lycopene (supplementation minus depletion) correlated positively with the change in the lag-phase (lag-phase after red-vegetable supplementation minus depletion week): r = 0.55; P = 0.001; n = 34.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The objective of the vegetable-supplementation regimen was to increase
the intake of vegetables to 300–400 g/day to provide at least 25 mg of
carotenoids/day. The intake of vegetables was very similar to those
proposed by the various national advisory groups for cardiovascular
disease prevention (22)(23). Green vegetables
contained predominantly ß-carotene and lutein (Table 1
)
(24). The red vegetables selected for the study were mainly
tomato-based products and contained predominantly lycopene. Therefore,
the vegetable supplements provided different carotenoid profiles. Soups
and pureed forms of spinach and tomatoes are reported to increase the
bioavailability of carotenoids (25)(26)(27), and such foods were
the major source of carotenoids in the present study. The carotenoid
concentrations of lycopene in the 300 g of tomato products used in
the present study were higher than the combined concentrations of
ß-carotene and lutein in the green vegetables (see Table 1
). In
addition, subjects in the present study found red vegetables more
palatable than green; therefore, the dietary intake of lycopene was
greater (40 mg/day) than ß-carotene and lutein combined (21 mg/day).
Direct comparison between the two diets, therefore, was not possible;
however, it was possible to compare the plasma response to the two
diets by taking a ratio of plasma concentrations to dietary intake in
the respective supplementation weeks, i.e., red and green weeks. The
results showed that the concentrations of ß-carotene and lutein
achieved in the plasma/mg dietary intake were significantly higher than
for the lycopene. In contrast to a previously published report
(28), the relative bioavailability of lutein was not
significantly different from ß-carotene.
The lag-phase was significantly increased after red-vegetable
supplementation only. On the basis of the LDL carotenoid data (Table 5
), the mean lycopene concentration was increased by 0.115 µmol/mmol
cholesterol in nonsmokers and 0.089 µmol/mmol cholesterol in smokers,
compared with ß-carotene concentrations of 0.067 µmol/mmol
cholesterol (nonsmokers) and 0.024 µmol/mmol cholesterol (smokers)
and lutein concentrations of 0.029 µmol/mmol cholesterol in
nonsmokers and 0.015 µmol/mmol cholesterol in smokers in the green
week. Thus, although the data suggested that the increase in plasma
lycopene per mg increase in dietary intake was lower for lycopene than
for ß-carotene and lutein, in absolute terms, the LDL carotenoid
concentrations achieved were higher for lycopene than the other
carotenoids. Our results are in agreement with two previously published
reports that showed increased resistance of LDL to oxidation after
supplementation of healthy subjects with tomato products
(29) and diabetic patients with tomato juice
(30).
The third objective of the study was to compare the various responses
to the vegetable treatments of smokers with nonsmokers. At baseline, in
agreement with previously published reports
(5)(18)(31)(32),
ß-carotene and lutein concentrations in the plasma were significantly
lower in smokers compared with nonsmokers. The plasma
-tocopherol:cholesterol ratio and ascorbic acid were not
significantly different between smokers and nonsmokers. In the
supplementation weeks, smokers did not consume as many vegetables as
nonsmokers. Thus, the dietary intake of carotenoids were slightly lower
in smokers (14% lower in the red week and 23% in the green week; not
statistically significant) than nonsmokers. The plasma and LDL
concentrations of carotenoids achieved were also lower in smokers than
nonsmokers, but the result was significant only for ß-carotene and
lutein (Table 5
). However, when the ratios of plasma (Fig. 1
) and LDL
(results not shown) concentrations to dietary intake were compared
between smokers and nonsmokers, no differences emerged between the two
groups. In the case of LDL lag-phase, only nonsmokers showed an
increase in the resistance of LDL to oxidation after red-vegetable
supplementation (Table 7
). However, when the increase in the
lag-phase/mg of dietary intake of carotenoids was compared between
smokers and nonsmokers, no differences were observed.
In the present study, there was no significant difference in the mean
dietary intakes of the respective carotenoids between smokers and
nonsmokers, but the increases in plasma and LDL carotenoids were
different between the two groups. In plasma, the increase in
ß-carotene was greater in nonsmokers than smokers (P
= 0.01; Table 5
). A similar trend was observed for ß-carotene,
lutein, and lycopene in LDL, but it reached statistical significance
only for lutein. Two-factor repeated-measures ANOVA indicated a
significant effect of smoking on the LDL-lutein. We previously have
shown that there was no difference in the absorption of ß-carotene,
lutein, or lycopene between smokers and nonsmokers (33).
However, in that study subject numbers were small, and the results from
the present study indicate that there are some differences in the
response of smokers and nonsmokers to increased vegetable intake,
suggesting that the absorption and/or turnover of carotenoids might be
different in the two groups.
The results of the present study differed from our previous study. One
difference was that, in the previous study (18), an increase
in the lag-phase was associated with increased plasma - and
ß-carotene, and plasma lycopene did not change. The increase in
plasma ß-carotene was greater in the present study than observed in
the previous studies (17)(18), but there was no
apparent effect on LDL oxidation in the green week. The subjects in the
previous study received a mixed diet of red and green vegetables and
mostly consumed fresh tomatoes as a source of lycopene. Lycopene is
reported to be less bioavailable from fresh tomatoes than the processed
ones (27). In the present study, plasma concentrations for
lycopene achieved after red vegetables were higher in the red week
compared with ß-carotene and lutein in the green week, and the lag
phase was increased only in the red week. A second difference between
the results of the two studies was that in the previous study, both
smokers and nonsmokers showed an increase in the lag-phase
(16), but only nonsmokers showed a positive lag-phase
response in the present study. The average cigarette consumption by
smokers in the present study was 16 cigarettes/day compared with 10
cigarettes/day in the previous study. In addition, the information
obtained from the food diaries of subjects from the present study
showed that subjects smoked at least one cigarette after the vegetable
meal, and smoking has been reported to reduce the plasma concentration
of carotenoids (2). It is not known whether the smokers in
the previous study had smoking habits similar to those of the subjects
of the present study. The third difference between the studies was that
the previous study was conducted in France and the present study in
Northern Ireland. Therefore, differences in subject populations,
smoking habits (different brands as well as number of cigarettes
smoked), and food preferences in the two countries may have accounted
for the differences in the results obtained from the two studies.
In the previous studies (17)(18), the volunteers were from both sexes. To reduce variations between subjects, it was decided to use only one sex, and women were selected for their greater reliability in compliance with the dietary regimes (M. Chopra, personal observation). Some authors have reported that menstrual cycle changes affect the carotenoids in lipoproteins (34)(35), whereas others have reported no effect (36). However, even when shown to have an effect, there is only a 5% variation in plasma (34), a 7–10% variation in LDL (35), and a 3–7% variation in HDL carotenoids during the menstrual cycle. The changes in LDL carotenoids after vegetable supplementation in the present study were in the order of 70–140%. The small fluctuations attributable to the menstrual cycle were, therefore, not deemed a potential confounder in this study.
The vegetable supplementation had no effect on the
-tocopherol:cholesterol ratio in the plasma, LDL, and HDL. Indeed,
significant increases in the plasma, LDL, and HDL
-tocopherol:cholesterol ratios were observed after the depletion
diet. Because the dietary intake of vitamin E was not different at the
baseline and depletion (5.6 ± 2 and 5.3 ± 3 mg/day,
respectively), the changes in plasma and lipoproteins are unlikely to
be attributable to the diet. It is possible that there is a release of
-tocopherol from storage tissues because its concentration was
significantly increased in the HDL fraction, but this increase occurred
in nonsmokers only. Plasma ascorbate was significantly increased after
the supplementation diets (both red and green) in both smokers and
nonsmokers, suggesting that the plasma response of smokers to ascorbate
from vegetables is not different from nonsmokers.
Vegetables and fruits are also a good source of flavonoids (37)(38), which are reported to show antioxidant activity both in vitro (39) and in vivo (40)(41). Changes in plasma flavonoids were not measured in the present study; therefore, the possibility of any synergism between dietary flavonoids and carotenoids on LDL oxidation could not be tested.
In conclusion, an increase in the vegetable intake of 300–400
g/day increases the plasma and LDL concentrations of carotenoids in
both smokers and nonsmokers. The increases in plasma ß-carotene and
LDL lutein were lower in smokers. After an increase in LDL lycopene,
there was an increase in the resistance of LDL to ex vivo oxidation,
but only in nonsmokers. These results, however, differ from those
obtained in an earlier study when the effects on LDL oxidation were
associated with increases in plasma - and ß-carotene. Together,
both studies suggest that vegetable supplements can increase the
resistance of LDL to oxidation, but at present it is still not possible
to characterize the specific component responsible for the observed
antioxidant effects.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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