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*
Division of Human Nutrition and Epidemiology and
Food Science Group, Department of Food Technology and Nutritional Sciences, Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands;
Institute of Food Safety and Toxicology, Danish Veterinary and Food Administration, DK-2860 Søborg, Denmark; and
**
Unilever Research Vlaardingen, 3130 AC Vlaardingen, The Netherlands
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol, FRAP (ferric reducing ability of plasma)
and uric acid and erythrocyte enzyme activities were assessed, and
differences among experimental groups were tested. Consumption of
spinach resulted in greater (P < 0.01) erythrocyte
glutathione reductase activity and lower (P < 0.05) erythrocyte catalase activity and serum -tocopherol
concentration compared with the control group. Consumption of the
carotenoid supplement led to lower -tocopherol responses
(P = 0.02) compared with the basic diet only. Our
data suggest that the short-term changes in erythrocyte glutathione
reductase activity and serum -tocopherol concentration can be
attributed to an increased carotenoid (lutein and zeaxanthin) intake,
but ß-carotene is unlikely to be a causative factor. Lower
erythrocyte catalase activity after intervention with spinach products
may be related to other constituents in spinach such as
flavonoids.
KEY WORDS: • carotenoids • antioxidants • -tocopherol • humans • spinach
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-carotene,
zeaxanthin, cryptoxanthin and lycopene. A number of carotenoids are
precursors of retinol and retinoids, but carotenoids also have several
other functions in humans, including protecting against oxidation by
quenching singlet oxygen (Stahl et al. 1997
). In
addition, ß-carotene reacts chemically with peroxyl radicals to
produce epoxide and apocarotenal products (Canfield 1992
). A low level of carotenoids is associated with poor
cognitive performance (Berr et al. 1998
), and higher
plasma ß-carotene levels are associated with better memory
performance in elderly people (Perrig et al. 1997
). On
the basis of intake or biomarkers of intake, carotenoids have been
postulated to play a protective role in angina pectoris
(Riemersma et al. 1991
), cardiovascular disease
(Gaziano et al. 1995
, Kardinaal et al. 1993
, Knekt et al. 1994
, Street et al. 1994
) and cancer (Stahelin et al. 1991
,
van Poppel 1996
), particularly cancer of lung
(Dartigues et al. 1990
, Knekt et al. 1990
) and stomach (Chen et al. 1992
). However,
intervention studies have not borne this out (The ATBC Cancer Prevention Study Group 1994
, Blot et al. 1993
, Blot 1997
, Greenberg et al. 1996
, Omenn et al. 1996
, Rapola et al. 1997
). These intervention
studies used ß-carotene supplements rather than a mixture of
carotenoids as present in fruits and vegetables. The positive effects
on health postulated for the carotenoids have been attributed largely
to their antioxidant actions. However, studies linking higher
carotenoid intakes to better antioxidant defense and to a decrease in
oxidative damage in the body are very few and have generally used
indirect methods (Esterbauer 1996
, Miller and Rice-Evans 1997
, Puhl et al. 1994
).
Therefore, we conducted a dietary controlled intervention study to
investigate the effects of intake of a carotenoid supplement dissolved
in oil and three differently processed spinach products on a range of
enzymatic and nonenzymatic antioxidant parameters in human blood. The
effect of consumption of the differently processed spinach products on
the bioavailability of carotenoids has been published separately
(Castenmiller et al. 1999
). The primary aim of this
study was to evaluate whether a spinach intervention had an effect on
markers of antioxidative status and whether this effect could be
attributed to carotenoids present in spinach. |
SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The subjects were 72 healthy, nonsmoking, normolipidemic
volunteers; there were 42 women and 30 men, aged 18–58 y. The
subjects, students of Wageningen Agricultural University and other
inhabitants in the Wageningen area, were recruited through local
advertisements. None of the subjects were taking oral medications,
except for oral contraceptives, or supplements of any kind during the
last 3 mo before the study started or during the study. The screening
procedures included a test for elevated glucose and protein levels in
urine and a check for abnormal hematology or low hemoglobin
concentrations. All subjects completed a medical and a general
questionnaire in addition to food-frequency questionnaires to
estimate their intakes of energy, and carotenoids and vitamin A. The
subjects had normal body mass indices (18–28 kg/m2); their
fasting serum cholesterol concentrations were <6.5 mmol/L and
triacylglycerol concentrations were <2.8 mmol/L. During the study, two
male subjects withdrew from the study for personal reasons. For
characteristics of the subjects, see Table 1
. The protocol for this study was approved by the Medical-Ethical
Committee of Wageningen Agricultural University and all subjects gave
their written informed consent.
|
The study started in January 1997 with a 3-wk run-in period, during
which subjects chose their own diets but were instructed to avoid foods
rich in carotenoids and retinol. Then, subjects were stratified
according to age, sex, cholesterol concentration and energy intake and
assigned to six experimental groups. The six treatment groups were fed
the same basic diet throughout the study and the menu was changed daily
on a weekly cycle. The basic diet did not include fruits and vegetables
with moderate or high amounts of carotenoids and met the requirements
of the Dutch Recommended Daily Allowances (Netherlands Food and Nutrition Council 1992
). In addition to the basic diet, four groups
received daily a spinach product; one group received a supplement of a
suspension in vegetable oil of microcrystalline ß-carotene (40 g/kg;
Hoffmann-La Roche, Basel, Switzerland) and crystalline lutein and
zeaxanthin derived from marigold flowers (60 g/kg and 3 g/kg,
respectively; FloraGLO, kindly supplied by Kemin Industries, Des
Moines, IA). The carotenoid supplement was suspended in sunflower oil;
for the carotenoid supplement group, some of the sunflower oil used in
salad dressing fed to the control and spinach groups was replaced by
the carotenoid supplement suspension. The spinach groups received 20
g/MJ of whole-leaf spinach, minced spinach, liquefied spinach
or liquefied spinach to which dietary fiber was added. All spinach
products originated from one batch and were provided, prepared and
subsequently frozen by Langnese-Iglo (Wunstorf, Germany) for
Unilever Research (Vlaardingen, The Netherlands). Whole-leaf
spinach was washed and blanched for 90 s and cooled down quickly;
minced spinach was minced to 5 mm after blanching. An enzymatic
preparation with pectinase, hemicellulase and cellulase activities
(Rapidase LIQ+, kindly supplied by Gist-Brocades, Seclin, France)
was used for the liquefaction of minced spinach. After the enzyme
treatment, the spinach was boiled for 5–10 min to inactivate the
enzymes. One group received the liquefied spinach plus fiber prepared
from beetroot pulp (10 g/kg, Fibrex 600, kindly supplied by TEFCO Food
Ingredients b.v., Bodegraven, The Netherlands). The energy content of
the diets of the control group and carotenoid supplement group was
adjusted to that of the spinach groups by extra amounts of appropriate
foods. All frozen spinach was thawed and heated by microwave before
consumption. The spinach products contained no measurable nitrite and
<1000 mg nitrate/kg, thus ensuring that the nitrate intake of the
subjects was below the Acceptable Daily Intake for nitrate (Joint FAO/WHO Expert Committee on Food Additives 1995
). Microbiological
counts showed normal values and confirmed that the spinach products
were safe for human consumption. Subjects were supplied with total
diets, except for a limited choice of free products (~10 energy%).
Twenty vegetarians took part in the experiment. They received the same
foods as the nonvegetarian subjects (n = 50),
except for the meat, which was replaced by a vegetarian substitute with
similar nutrient composition. On week days, subjects received a hot
meal at the Division of Human Nutrition and Epidemiology; foods for
their other meals and snacks were packed to be taken home. For weekend
days, all foods were packed together with instructions and suggestions
for preparation and consumption. The daily selection of free-choice
foods was recorded in a diary and the nutrient content was calculated
(NEVO Foundation 1995
). The free items did not contain
carotenoids or retinol. A maximum of two alcoholic consumptions per day
was allowed but was not to be consumed together with the hot meal.
Individual body weights throughout the study were maintained at ± 2 kg. Subjects were asked not to change their usual pattern of
activities.
Blood measurements.
Blood was taken from fasting subjects at 0715–1000 h by venipuncture
on d 0, 1, 8, 15, 21 and 22 of the intervention period for serum
analyses of carotenoids and -tocopherol concentrations, on d 0, 8,
15, and 22 for plasma analysis of ferric reducing ability
(FRAP),3
uric acid and vitamin C concentrations and on d 0 and 22 for analysis
of erythrocyte enzyme activities and oxidatively modified amino acids
in plasma proteins.
Carotenoids and -tocopherol.
Samples, to which no anticoagulant was added, were left to clot; within
1 h after being drawn, they were centrifuged and stored at
-80°C. Serum carotenoids and -tocopherol were measured by
HPLC. To avoid day-to-day analytical variations, all samples from an
individual were analyzed as a set. After precipitation with ethanol,
extraction followed with hexane twice, samples were evaporated under
nitrogen and injected into the HPLC system (Craft and Wise 1992
). All sample preparations and extractions were conducted
in duplicate and under subdued yellow light with minimal exposure to
oxygen. The intra-assay CV for serum analysis of -carotene,
ß-carotene, lutein and zeaxanthin in control pools averaged 7.4, 3.9,
3.6 and 8.7%, respectively.
Erythrocyte antioxidative enzymes.
Antioxidant enzyme activities were determined in erythrocyte lysates.
Heparinized blood samples were centrifuged at 1500 x g for 10 min and plasma was removed. The erythrocytes
were washed twice in 4 vol sterile physiological buffered saline,
resuspended in 1 vol of sterile, deionized water for lysis and
immediately frozen at -80°C. Automated assays were performed on a
Cobas Mira analyzer (Roche, Basel, Switzerland) to determine the
activity of the antioxidant enzymes glutathione peroxidase (GPx),
glutathione reductase (GR), superoxide dismutase (SOD) and catalase
(CAT). The activities of the enzymes were related to the amount of
hemoglobin in the blood hemolysates. SOD (Randox cat. no. SD 125,
Ardmore, UK), GPx (Randox cat. no. RS 05) and hemoglobin (Randox cat.
no. HG 980) were determined using commercially available kits. GR
activity and CAT activity were determined according to methods
described previously (Wheeler et al. 1990
). All analyses
were conducted within 20 d. The intraday CV of SOD, CAT, GR and
GPx averaged 1.9, 1.4, 3.1 and 1.9%, respectively. NADPH, glutathione,
FAD, purpald and potassium periodate were purchased from Sigma Chemical
(St. Louis, MO).
Determination of adipic semialdehyde (AAS).
Oxidatively modified amino acids in plasma proteins were analyzed
by HPLC (Daneshvar et al. 1997
). The protein fractions
were reacted with fluoresceinamine before the hydrolysis, and the
decarboxylated fluoresceinamine derivatives of adipic and glutamic
semialdehydes were measured by HPLC using a diode array detector. The
intraday CV for this determination was 5.2%.
FRAP, uric acid and vitamin C.
The antioxidant activity of samples, to which EDTA was added as an
anticoagulant, was assessed as their FRAP (Benzie and Strain 1996
). Uric acid concentration was measured in plasma samples
using enzymatic colorimetric methods (Boehringer Mannheim, Germany).
Vitamin C concentration in plasma treated with trichloroacetic acid was
determined fluorimetrically as ascorbate plus dehydroascorbate
(Vuilleumier and Keck 1996
). The intra-assay,
interday CV for plasma analysis of FRAP, uric acid and vitamin C was
3.5, 2.0 and 5.6%, respectively.
Food measurements.
The duplicate portions of the daily food intake of one subject
collected throughout the study were mixed thoroughly as weekly
portions; subsequently, pooled samples were stored at -20°C until
analysis. The moisture level and the ash content in each weekly portion
were determined using a vacuum oven at 85°C and a muffle furnace at
550°C. The protein concentration was determined by the Kjeldahl
method using a conversion factor of 6.25. The method of Folch et al. (1957)
was used to extract fat. Dietary fiber was analyzed
according to the AOAC (1996)
Official Method 992.16 for
total dietary fiber. Digestible carbohydrate was calculated by
difference. Carotenoids were extracted from wet material after
homogenization using tetrahydrofuran (THF), redissolved in THF/methanol
(1:1 v/v) and injected into the HPLC system (Hulshof et al. 1997
). The CV within runs of -carotene, ß-carotene and
lutein in control pools averaged 5.7, 6.8 and 8.8%, respectively.
Samples for vitamin C determination, to which metaphosphoric acid was
added, were collected from a number of daily homogenized duplicate
portions and immediately frozen. Vitamin C was determined
fluorimetrically, after extraction with metaphosphoric acid/acetic acid
(60:80, wt/v), as ascorbate plus dehydroascorbate (CV was 5.6%,
Vuilleumier and Keck 1996
).
Statistical analysis.
Responses to the dietary intervention were averaged for carotenoids and
-tocopherol for d 0 and 1 and for d 21 and 22 for each subject. All
other variables were measured once at the beginning and end of the
dietary intervention period. The response to treatment was calculated
for each person as the change in serum or plasma concentration or
enzyme activity from the start to the end of the dietary intervention
period. ANOVA was used to test equality of mean elevation for the
different treatment groups, thereby controlling for several factors and
covariables (SPSS Advanced Statistics, Chicago, IL). When all
significant predictors of antioxidant levels are included in a model,
it can be shown whether a particular variable is an independent
predictor of the response after treatment. For example, for the ferric
reducing ability of plasma (FRAP), uric acid should be included as a
covariable because the uric acid concentration determines ~60% of
the antioxidant activity of plasma (Benzie and Strain 1996
). To evaluate the effect of one of the erythrocyte enzyme
activities, all other activity responses were included in the model as
covariables. Because initial values may have affected the magnitude of
changes, effects of treatment were also assessed after adjustment for
baseline values by analysis of covariance, where applicable.
Significant F-tests were followed by Tukey studentized
range tests of pairwise differences and Least Significant Difference
(LSD) tests to evaluate differences among the four means of each
spinach treatment. Pearson’s correlations were calculated for the
responses of several variables. P-values < 0.05
were regarded as significant. Values are means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and results of analyses of various erythrocyte enzymatic activities and
nonenzymatic, antioxidant concentrations in serum or plasma are
described in Table 3
for the control, carotenoid supplement and pooled spinach groups. At
baseline (wk 0), there were no significant differences among the
erythrocyte antioxidant enzyme activities or serum or plasma
antioxidant concentrations of the various experimental groups, except
for erythrocyte SOD and GR activities. Results of the consumption of
differently processed spinach products on the bioavailability of
carotenoids are described elsewhere (Castenmiller et al. 1999
). The serum concentration of ß-carotene increased from
0.253 ± 0.130 to 2.608 ± 1.06 µmol/L in the
carotenoid supplement group and from 0.283 ± 0.141 to 0.445
± 0.172 µmol/L in the pooled spinach groups. For
lutein, the increase was from 0.218 ± 0.064 to 0.983 ± 0.287 µmol/L in the carotenoid supplement group and from
0.216 ± 0.072 to 0.915 ± 0.272 µmol/L in the
pooled spinach groups. The serum zeaxanthin response was higher in the
carotenoid supplement group than in the control group (P
= 0.006) or the pooled spinach groups (P < 0.001;
independent t test) but was not different between the pooled
spinach and control groups (P = 0.07). The serum
triacylglycerol and cholesterol concentrations did not change over the
experimental period or among the experimental groups (data not shown).
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Duplicate portions of the food provided to the participants were
analyzed as pooled weekly portions; results of chemical analysis are
shown in Table 2
. For macronutrients and ß-carotene, the
concentrations measured were consistent among the treatment groups and
for the 3 wk; the intake of lutein in the carotenoid supplement group
(0.6 mg/MJ) was lower than in the spinach groups (1.1 mg/MJ). The
vitamin C content of the diets with a carotenoid supplement,
whole-leaf spinach, minced spinach, liquefied spinach and liquefied
spinach plus added dietary fiber were 83, 75, 61, 68 and 48%,
respectively, that of the control group. The -tocopherol content of
the diets was not measured. However, we expected that the supply of
-tocopherol would have been similar for all treatment groups, but
may have been different for the vegetarian diet, in which meat was
replaced by a vegetarian product. Therefore, when analyzing the
-tocopherol responses, an adjustment was made for the factor
vegetarian diet.
Spinach products and carotenoid supplement vs. control diet.
The erythrocyte GR activity response, adjusted for responses of SOD and
CAT, was higher (P < 0.01), whereas the CAT, adjusted
for responses of GR, GPx and SOD, and -tocopherol responses,
adjusted for vegetarian diet (P = 0.02 and P
= 0.04, respectively), were lower in the pooled spinach groups
than in the control group. The response of erythrocyte GR activity,
adjusted for responses of CAT and SOD, was higher (P = 0.02), whereas the serum -tocopherol response was lower
(P = 0.02) in the carotenoid supplement group than in
the control group. Introducing the baseline value into the model, the
difference between GR activity of the control and carotenoid supplement
group was no longer significant, whereas the difference between the
control and spinach groups remained significant. The change in serum
-tocopherol concentration in the control group, which had the lowest
mean baseline (wk 0) serum -tocopherol concentration, was greater
than that for the carotenoid and pooled spinach groups. These data are
difficult to interpret. Adjustment of serum -tocopherol
concentrations for concentrations of cholesterol and triacylglycerol
may provide a better reflection of dietary intake (Willett et al. 1983
). The relative serum -tocopherol concentration is
the concentration of -tocopherol in serum related to that of the sum
of the concentrations of cholesterol and triacylglycerol in serum. The
relative serum -tocopherol responses also showed significant
differences between the control and pooled spinach groups (P
< 0.001) and tended to be different between the control and
carotenoid supplement group (P = 0.06; P
= 0.006 when adjusted for vegetarian diet). Analysis of covariance
with baseline values (wk 0) and vegetarian diet as covariable and the
serum concentration at wk 3 as dependent variable showed that the
difference between the pooled spinach group and the control group was
no longer significant, but the difference between the control and
carotenoid supplement group remained significant. This study could not
demonstrate any effect of intake of spinach or the carotenoid
supplement on plasma concentrations of AAS, vitamin C, FRAP or uric
acid.
Carotenoid supplement vs. spinach products.
Although the bioavailability of ß-carotene from the carotenoid
supplement was much higher than that from the spinach products, no
significant differences could be detected for any of the responses in
enzymatic erythrocyte activities, plasma AAS, FRAP, uric acid and
vitamin C concentrations or serum -tocopherol concentration between
carotenoid supplement and pooled spinach groups.
Results among various spinach products.
The differences among the four spinach groups tended to be different
(P = 0.07) only for uric acid responses (see
Table 4
). Whole-leaf spinach resulted in a higher uric acid response than
consumption of liquefied spinach (Tukey method, P = 0.06; LSD method, P = 0.01). These results indicate
that consumption of whole-leaf spinach, which resulted in lower
serum carotenoid responses than consumption of other spinach products,
may enhance oxidative defense more than consumption of liquefied
spinach.
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Pearson correlation coefficients for significant correlations of
responses among variables of antioxidant activities and concentrations
were calculated. Significant and meaningful correlations (r
> 0.30, P < 0.05) were observed among the
responses of erythrocyte enzymatic antioxidant activities of GPx, GR,
SOD and CAT (all positive), with an exception of the correlation
between SOD and CAT. Positive correlations existed between plasma
responses of FRAP and uric acid, plasma FRAP responses and serum
-tocopherol concentrations, plasma AAS and vitamin C responses. The
serum zeaxanthin responses were positively correlated with responses in
serum concentrations of -tocopherol (r = 0.39), and
a positive correlation was observed between erythrocyte GR responses
and serum lutein responses (r = 0.29). There were no
significant correlations between the serum ß-carotene response and
that of any of the other markers of antioxidative status measured.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol responses compared with the control group.
Consumption of the carotenoid supplement increased erythrocyte GR
activity response and decreased the serum -tocopherol response
compared with the control group. However, evidence for the lower
-tocopherol response in the spinach groups and greater GR response
in the carotenoid supplement group is not conclusive. Because the diets
supplied to the carotenoid supplement group and the control group were
similar except for the carotenoid supplement, differences between these
groups, as were found for responses of erythrocyte GR activity and
serum -tocopherol, can be attributed to the intake of ß-carotene,
lutein and a small amount of zeaxanthin as a carotenoid supplement
dissolved in oil. It should be noted that the lutein is obtained from a
natural source (marigold) and may thus contain some flavonoids and
other phytochemicals. A small but significant correlation was observed
between the responses of GR activity and serum lutein concentrations
(Pearson correlation coefficient: 0.29; P < 0.05),
suggesting that the greater responses of GR in the carotenoid
supplement and spinach groups compared with the control group were
related to the greater responses of serum lutein concentrations. The
antioxidant responses in the pooled spinach group were not
significantly different from those in the carotenoid supplement group.
This suggests that the observed changes in antioxidant activities or
concentrations were not caused by the increased absorption of
ß-carotene because the ß-carotene response, but not that of lutein,
was much higher in the carotenoid supplement group than in the spinach
groups. In cases in which significant differences between the pooled
spinach and control group were present but not between the carotenoid
supplement and the control group as we found for the response in
erythrocyte CAT activity, the difference was probably due to
constituents other than carotenoids present in spinach (Dragsted et al. 1997
). Antioxidant enzyme activities.
GR activity increased after 3 wk of dietary intervention with
carotenoids. Diminished degradation of the antioxidant enzymes because
of a protective role of carotenoids, known to deactivate singlet
oxygen, could explain the relative increase in enzyme activity in the
carotenoid supplement and spinach groups compared with the control
group. Alternatively, carotenoids might act by inducing the enzymes.
Although induction of an antioxidant enzyme theoretically cannot occur
in erythrocytes, induction may occur during erythropoiesis. Moreover,
activity of enzymes located in erythrocytes has been reported to change
within hours after eating (Saghir et al. 1997
).
GPx catalyzes the degradation of peroxides with concomitant oxidation
of glutathione. In the presence of GR and NADPH, the oxidized
glutathione is immediately converted to the reduced form. Thus, it is
not surprising that we found that the activities of GPx and GR were
correlated. SOD catalyzes the dismutation of superoxide anion radicals
and CAT catalyzes the reduction of hydrogen peroxide to water. From a
kinetic point of view, CAT and GPx are both able to destroy hydrogen
peroxide, but GPx has a much higher affinity for hydrogen peroxide than
does CAT, suggesting that hydrogen peroxide is degraded mainly by GPx
under normal conditions (Delmas-Beauvieux et al. 1996
).
In a study conducted in France, elderly hospitalized subjects were
supplied daily with a placebo; 20 mg zinc plus 100 µg
selenium (mineral group); 120 mg vitamin C and 6 mg ß-carotene and 15
mg vitamin E (vitamin group); or 20 mg zinc, 100 µg
selenium, 120 mg vitamin C, 6 mg ß-carotene and 15 mg vitamin E
(mineral and vitamin group). After 6 mo of supplementation
(Monget et al. 1996
), significant effects of vitamin
supplementation on GPx and SOD activities were reported, whereas after
1 y of supplementation (Galan et al. 1997
),
significant increases were observed in GPx activity in the groups
receiving minerals alone or in combination with vitamins, but there was
no effect on SOD activity or thiobarbituric acid-reactive
substances production. Omaye and co-workers (1996)
fed nine women a low carotenoid diet, followed by the same diet
supplemented with 15 mg ß-carotene daily for 28 d and found a
positive correlation between CAT and GPx activities; they concluded
that ß-carotene deficiency does have an effect on erythrocyte
antioxidant status. In our study, a correlation between CAT and GPx
activities was also found. Dixon et al. (1994)
reported
that erythrocyte SOD activity was depressed in carotene-depleted
women, but it recovered with repletion. In a later study
(Delmas-Beauvieux et al. 1996
), supplementation of 60 mg
ß-carotene daily for 1 y did not result in a significant
difference in SOD activity compared with baseline, whereas GPx activity
slightly increased, and a significant increase in glutathione status
values was observed after 12 mo compared with baseline. Thus, there is
some previous evidence that ß-carotene might influence the activity
of antioxidant enzymes. Our study now suggests that not only
ß-carotene but also lutein has an effect on antioxidant enzyme
activities.
Antioxidant vitamins.
After 3 wk of dietary intervention, the serum -tocopherol response
was lower in the groups given the carotenoid supplement or spinach
products than in the control group. The evidence for this was not
completely conclusive for the difference between the pooled spinach and
control groups. The lower response of -tocopherol may reflect its
increased utilization as an antioxidant, in combination with the lower
vitamin C content of the diets in the spinach groups. Furthermore, a
significant correlation was found between serum -tocopherol and
zeaxanthin responses.
Xu and co-workers (1992)
supplied subjects with a
placebo or 15–60 mg ß-carotene daily for 9 mo and found that all
ß-carotene doses resulted in similar decreases in plasma levels of
-tocopherol after 6 mo. On the other hand, the CARET study, after up
to 6 y of supplementation, found a small but significant increase
in the serum concentration of -tocopherol in the participants taking
30 mg ß-carotene and 7.5 mg retinyl palmitate (as retinol) per day
(Goodman et al. 1994
). Other studies have suggested that
oral supplements of ß-carotene do not change serum levels of
-tocopherol. The Polyp Prevention Study Group (Nierenberg et al. 1994
) supplied 505 patients with a placebo or 25 mg
ß-carotene daily and found that serum concentrations of vitamin E
were not altered after 9 mo. The Alpha-Tocopherol, Beta-Carotene
Cancer Prevention Study Group (Albanes et al. 1997
)
studied 491 men in Helsinki. After an average of 6.7 y, the group
supplemented with 20 mg ß-carotene/d did not have different
-tocopherol concentrations than the control group. Nierenberg et al. (1997)
conducted a supplementation study in which
subjects received a placebo (n = 54) or 25 mg
ß-carotene daily (n = 54) for 4 y and found that
supplementation with ß-carotene given orally did not alter serum
concentrations of -tocopherol. In an experiment in New Zealand
(Zino et al. 1997
), increased dietary intake of fruit
and vegetables in the intervention group for 8 wk did not change
concentrations of -tocopherol. We conclude that most studies in
which synthetic ß-carotene is given as a supplement observe no effect
on serum concentrations of -tocopherol. Our study now suggests a
relationship between serum zeaxanthin and -tocopherol responses.
Other antioxidants.
The higher response of plasma uric acid in the whole-leaf spinach
group compared with the liquefied spinach group is noteworthy. Urate
not only behaves as a radical scavenger but also stabilizes ascorbate
in biological fluids, e.g., human serum. This effect is due largely to
iron chelation by urate. Unlike radical-scavenging reactions, this
protective effect of urate is not associated with its depletion because
a stable, noncatalytic urate-iron complex is formed
(Sevanian et al. 1991
). This phenomenon may explain why
we did not find changes in responses of plasma vitamin C. The
antioxidative effect of spinach and carotenoid supplement consumption
could not be observed by measurements of FRAP. A study on the effect of
juice intervention on markers of antioxidative status found that after
1 wk of intervention with increasing amounts of black currant and apple
juice, only GPx activity had significantly increased with dose and FRAP
remained unchanged (Young et al. 1999
).
Conclusions.
Consumption of a supplement of ß-carotene, lutein and zeaxanthin, and
spinach products resulted in changes in erythrocyte enzyme activities
of GR and CAT and serum -tocopherol concentrations. Antioxidants and
antioxidant enzyme systems play key roles in the protection of
biological membranes, lipoproteins, and DNA and in the prevention of
protein damage. However, these defense systems are very complex, and
changes in one antioxidant may result in changes in concentrations of
other antioxidants or erythrocyte enzymes. We have documented the
changes that occur after intake of a pure carotenoid supplement
containing ß-carotene and lutein, and a small amount of zeaxanthin,
during a 3-wk controlled human dietary intervention study. The results
were compared with those obtained after consumption of spinach products
during the 3-wk study. The consumption of spinach resulted in greater
responses of erythrocyte GR activity and lower erythrocyte CAT and
serum -tocopherol responses. Consumption of the carotenoid
supplement increased erythrocyte GR activity and lowered the serum
-tocopherol response. Our data suggest that carotenoid intake of
lutein and zeaxanthin, but not ß-carotene, is positively associated
with erythrocyte GR activity and negatively with serum -tocopherol
concentration, respectively. The effect of spinach consumption on CAT
activity is most likely not related to its carotenoid content. If
analytical assays are improved in the near future and more information
on antioxidant enzyme activity becomes available, we may be able to
achieve a better understanding of the kinetics and mechanisms of the
complex antioxidant defense systems.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AAS, adipic semialdehyde; CAT, catalase; FRAP, ferric reducing ability of plasma; GPx,
glutathione peroxidase; GR, glutathione reductase; SOD, superoxide dismutase; THF, tetrahydrofuran.
Manuscript received April 28, 1999. Initial review completed May 28, 1999. Revision accepted August 5, 1999.
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