© 1995 American Heart Association, Inc.
Articles |
Supplementation With Low Doses of Vitamin E Protects LDL From Lipid Peroxidation in Men and Women
From the Gaubius Laboratory, TNO-PG, and the Department of Cardiology, University Hospital (A. van der L.), Leiden; and TNO Nutrition and Food Research, Zeist (G. van P.), the Netherlands.
Correspondence to Dr Hans M.G. Princen, Gaubius Laboratory, TNO-PG, PO Box 430, 2300 AK Leiden, the Netherlands.
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Abstract |
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Abstract There is accumulating evidence that oxidative modification of LDL is an important step in the process of atherogenesis and that antioxidants may protect LDL from oxidation. We and others have previously shown that ingestion of pharmacological doses of the antioxidant D,L-
-tocopherol (vitamin E),
far above the recommended daily intake (ie, 12 to 15 IU/d for adults),
increases the oxidation resistance of LDL. In this study, we
ascertained the minimal supplementary dose of vitamin E necessary to
protect LDL against oxidation in vitro. Twenty healthy volunteers (10
men and 10 women, aged 21 to 31 years) ingested consecutively 25, 50,
100, 200, 400, and 800 IU/d D,L--tocopherol acetate
during six 2-week periods. No changes were observed in LDL triglyceride
content, fatty acid composition of LDL, or LDL size during the
intervention. Concentrations of -tocopherol in plasma and LDL were
both 1.2 times the baseline values after the first period (25 IU/d) and
2.6 and 2.2 times, respectively, after the last period (800 IU/d).
There was a linear increase in LDL -tocopherol levels up to an
intake of 800 IU/d (r=.79, P<.0001) and a good
correlation between -tocopherol in plasma and LDL (r=.66,
P<.0001). Simultaneously, the resistance of LDL to
oxidation was elevated dose-dependently (+28% after the last period)
and differed significantly from the baseline resistance time even after
ingestion of only 25 IU/d. Correlation between -tocopherol
content of LDL and resistance time for all data was r=.57
(P<.0001), whereas the correlation between plasma
-tocopherol and resistance time was r=.69
(P<.0001). The rate of oxidation was decreased
significantly at 400 and 800 IU/d (-13% and -17%, respectively).
Baseline resistance time was not significantly different between men
and women, but propagation rate was higher with LDL from men at
baseline and after intake of 25 and 50 IU/d. Minor differences in LDL
vitamin E levels and resistance time were found between men and women
in response to vitamin E intake. Statistical evaluation of the
relations between -tocopherol content of LDL and resistance time in
each of the 20 individual subjects showed strong and significant
correlations for 14 individuals, indicating that vitamin E was the most
important parameter that determined the oxidation resistance of LDL in
these subjects. ANOVA indicated that LDL -tocopherol content (47%)
and interindividual variation (39%) were the most prominent parameters
that contributed to the total variance in resistance time.
Key Words: LDL oxidation • atherosclerosis • lag time • vitamin E • antioxidants
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Introduction |
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There is increasing experimental and epidemiological evidence that oxidative modification of LDL plays an important role in the development of atherosclerosis.1 2 3 Oxidized LDL is taken up more avidly by macrophages than is native LDL,4 5 6 which may contribute to the change of macrophages into foam cells in the atherosclerotic intima. Oxidatively modified LDL has been found in atherosclerotic lesions of humans and experimental animals,7 8 9 and elevated plasma levels of autoantibodies to oxidized LDL have been reported in patients with atherosclerosis.10 Oxidized LDL is cytotoxic to endothelial cells11 12 and can inhibit arterial relaxation.13 Oxidized LDL is chemotactic for monocytes,14 may prevent efflux of macrophages from the artery,15 and induces production of cytokines and growth factors by cells of the vascular wall.16 17 These cells, ie, endothelial cells, smooth muscle cells, and macrophages, have been implicated in the free radical–induced lipid peroxidation of the unsaturated fatty acids in LDL.1 2 3 18 This process can be mimicked in an in vitro assay by incubation of LDL with transition metal ions.5 18 19 With this assay the susceptibility of LDL to oxidation was found to be correlated with the severity of atherosclerosis.20 21 22 Lipid peroxidation occurs only after depletion of endogenous antioxidants in LDL and plasma.18 23 24 This may explain the inverse relation between plasma levels of dietary antioxidants and the risk of cardiovascular disease observed in several epidemiological studies.25 26 27 28 29
Vitamin E (-tocopherol) is a highly efficient, lipid-soluble
antioxidant present in LDL.18 30 31 In animal models
of atherosclerosis, addition of vitamin E to the diet has been reported
to reduce the development of atherosclerosis,32 33 34
although the data are not consistent.35 36 37 Recent data
from two large epidemiological studies show an association between the
use of vitamin E supplements and a reduced risk of coronary heart
disease (CHD) in both men and women.28 29 We and others
have shown previously that ingestion of vitamin E strongly protects LDL
against oxidative modification.38 39 40 41 42 43 In these studies
pharmacological doses of -tocopherol far exceeding the recommended
daily intake of 12 to 15 IU/d for adults were applied.
The aims of this study were to assess the minimal dose of vitamin E
supplementation needed to protect LDL against lipid peroxidation and
second, to evaluate whether there were differences in response to
increasing doses of vitamin E between men and women and between
individual subjects. We found that supplementation with as little as 25
IU -tocopherol per day increased the resistance of LDL to oxidation
and that the response to a vitamin E challenge displayed marked
interindividual variation.
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Methods |
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Experimental Protocol
Twenty healthy, nonsmoking volunteers (10 men and 10 women), aged 21 to 31 years, were recruited from the community to participate in this study. The subjects were normolipidemic and did not use any medication (other than oral contraceptives), vitamin supplements, or other dietary antioxidants. During 2-week periods, they ingested consecutively 25, 50, 100, 200, 400, and 800 IU vitamin E per day in the form of D,L-
-tocopherol acetate (Roche). The study
design is depicted in Fig 1
. One IU vitamin E equals 1
mg D,L--tocopherol acetate. During each 2-week period
the indicated dose of vitamin E was taken in capsule form in three
equal parts just before breakfast, lunch, and supper. Vitamin E intake
during the trial was monitored by counting unused capsules and
measurement of plasma -tocopherol levels.
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All participants completed the trial. During the trial they were
instructed by a dietitian to adhere to their normal eating habits. All
were on a normal diet as monitored during the first, second, and last
intervention periods by making use of 3-day food records. Dietary
intake of vitamin E and other relevant nutrients was calculated in the
low-dose supplementation periods, during which 25 and 50 IU/d,
respectively, were ingested and at the end of the study, when 800 IU/d
was ingested. For this purpose the frequency with which each food item
was consumed was multiplied by its vitamin E content or its content of
other nutrients, as derived from the Netherlands Food Composition
Table.44
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Blood was collected in EDTA-containing Vacutainer tubes (1 mg/mL) in the morning before the start of the intervention and at the end of each 2-week period. The blood was immediately placed on ice and cooled to 4°C. Plasma was prepared, frozen in liquid nitrogen in small portions (leaving as little empty space as possible in the tubes), and stored at -80°C. This procedure was completed within 1 hour.
All participants in this study gave their informed consent. The study was approved by the Medical Ethics Committee of the Netherlands Organisation for Applied Scientific Research TNO, Leiden, the Netherlands.
Preparation and Oxidation of LDL
The procedure for preparation and lipid peroxidation of LDL was
adapted from the method described by Esterbauer et al45
with some major modifications as described previously.38
In detail, from each subject 2 mL frozen EDTA-plasma (1 mg/mL) stored
at -80°C was rapidly thawed and used for isolation of LDL by
ultracentrifugation (18 hours at 40 000 rpm [285 000g]
in a Beckman SW40 rotor in a Beckman L7-55 ultracentrifuge at 4°C)
according to Terpstra et al46 without prestaining with
amido black. To protect LDL against oxidative modification during
isolation, 10 µmol/L EDTA was added to each density solution. The
entire procedure until the beginning of the oxidation experiment was
performed at 4°C. LDL was isolated from the appropriate density
fraction (d=1.019 to 1.063 g/mL) of the gradient, and a
sample was taken for cholesterol and protein determinations, during
which time the rest of the fraction was stored under nitrogen in the
dark at 4°C. To make use of the protective effect of EDTA against LDL
oxidation23 47 and to minimize the time between isolation
and the oxidation experiment, LDL was not extensively
dialyzed45 but instead was used immediately in the
oxidation assay after measurement of protein content. Because dialysis
was omitted, we did not add the lipophilic antioxidant butylated
hydroxytoluene to plasma after it was collected. The LDL-containing
fraction was diluted with a solution of the same density (1.18 mol/L
NaCl, 10 µmol/L EDTA) to a protein concentration of 0.17 mg/mL, and
sodium phosphate, pH 7.4, was added to a final concentration of 10
mmol/L. The assay mixture was brought to 37°C, and oxidation was
initiated by addition of 40 µmol/L CuSO4. The kinetics of
LDL oxidation was followed by continuously monitoring the change in
absorbance at 234 nm45 in a thermostat-controlled
(37°C), computerized Kontron Uvikon 930 spectrophotometer equipped
with a 10-position automatic sample changer (Tegimenta AG). After
setting the initial absorbance to zero, the increase in 234-nm
absorption was recorded every 3 minutes during a 5-hour period.
Absorbance curves of the seven consecutive LDL preparations obtained
from one subject before and at the end of each 2-week period of vitamin
E ingestion were determined in parallel. The time profile of the 234-nm
absorption curve shows three distinct phases: a lag phase, during which
absorption does not increase or increases only slightly, indicating
that the LDL is resistant to oxidation; and a propagation phase, during
which absorbance at 234 nm rapidly increases to a maximum. This period
is indicative of the autocatalytic chain reaction of lipid
peroxidation. After reaching a maximum, conjugated diene content slowly
decreases due to decomposition, with the resultant formation of
aldehydes (decomposition phase).18 45 A tangent is drawn
to the steepest part of the propagation phase curve and extrapolated to
the horizontal (time) axis. The interval between the addition of
Cu2+ ions and the x intercept of the tangent
line is defined as the lag time and is expressed in minutes. In some
cases, if the slope of the lag phase deviates from baseline, lag time
is obtained by dropping a perpendicular line from the intercept of the
tangents of the lag and propagation phases to the x axis.
The propagation rate is calculated from the slope of the tangent to the
curve during the propagation phase and a molar extinction coefficient
for conjugated dienes of 
234=29 500
L · mol-1 · cm-1,45
expressed as nanomoles of dienes formed per minute per milligram of LDL
protein. A representative graph showing the kinetics of LDL
oxidation is presented in Fig 4.
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) and
after (
) supplementation with vitamin E (800 mg/d) are shown. A
234 indicates absorbance at 234 nm.Each LDL preparation was oxidized in three consecutive oxidation runs on the same day. The values shown for lag time and propagation rate are means of the values thus obtained. The intra-assay coefficients of variation for lag time and propagation rate were 2.6% and 3.1%, respectively, on the basis of oxidation of the same LDL in three consecutive oxidation runs on one day.38
The interassay coefficients of variation for lag time and propagation rate were 4.9% and 7.4%, respectively, and were obtained by determining LDL oxidation for the same subject prepared on different days. In every oxidation run, one reference LDL, prepared from a reference plasma stored at -80°C, was used as a control. Oxidation runs with deviation higher than 10% from the mean lag time and propagation rate of former measurements were omitted.38 By omitting extensive dialysis, a more stable LDL preparation is obtained, which can be stored in the dark at 4°C under nitrogen for several days without affecting resistance time and propagation rate. This improves the precision of the method because each LDL preparation can be oxidized consecutively in triplicate. Oxidation of LDL under hypersaline conditions (1.18 mol/L NaCl) results in a higher lag time compared with oxidation in physiological saline (0.15 mol/L NaCl; data not shown). Because of this and to overcome the 10 µmol/L EDTA background (see Reference 4747 , for example), 40 µmol/L CuSO4 was added to initiate lipid peroxidation.
By using this highly standardized method, we found no differences in lag time and propagation rate between LDL prepared from plasma frozen in liquid nitrogen and those from freshly collected plasma from the same subject. In addition, no differences in these parameters were found after storage of plasma at -80°C for as long as 6 months.
Analytical Measurements
Cholesterol and triglyceride concentrations were determined
enzymatically with commercially available reagents (CHOD-PAP kit No.
236.691 and triglyceride kit No. 701.904, Boehringer-Mannheim). HDL
cholesterol was measured after removal of VLDL, IDL, and LDL by
precipitation with sodium
phosphotungstate–Mg2+.48 LDL
cholesterol concentrations were calculated by the formula of Friedewald
et al.49
LDL size was determined by electrophoresis of 7.5 to 10 µL plasma on 2% to 16% nondenaturing polyacrylamide gradient gels (Pharmacia LKB).50 High-molecular-weight standards (Pharmacia) were used together with a reference serum. After being stained with Sudan black B, gels were scanned with an LKB 2202 Ultrascan laser densitometer (LKB).
Fatty acid composition of LDL was determined by gas-liquid chromatography on a Chrompack gas chromatograph (model 438S) equipped with a CP-Sil88 column (50 mx0.25 mm [inner diameter]) and a flame ionization detector. LDL samples (0.1 mL of 0.25 mg protein per milliliter) were saponified by incubation with 0.4 mL of 0.3 mol/L NaOH in 90% (vol/vol) ethanol for 1 hour at 37°C. After dilution with 0.5 mL water and addition of 50 µg pentadecanoic acid (15:0) and 50 µg cis-13-docosenoic acid (22:1) as internal standards, the samples were acidified with 50 µL of 12 mol/L HCl, and the fatty acids were extracted twice with 2 mL hexane. The pooled hexane layers were evaporated under a stream of nitrogen at room temperature, and the fatty acids were esterified in 1.5 mL 100% methanol, to which 2.5% (wt/vol) acetyl chloride (Lipopure instant methanolic HCl kit, Applied Science) was added for 30 minutes at 60°C. After cooling, the fatty acids were extracted by consecutive addition of 3 mL chloroform and 0.9 mL water; the chloroform layer was washed once with water and evaporated under a stream of nitrogen at 37°C. The residue was dissolved in 100 µL hexane, and aliquots of 1 µL were injected with an automated liquid sampler (Chrompack model 911). Hydrogen was used as the carrier gas and samples were split in a 1:10 ratio. The injector temperature was 270°C and the detector temperature 300°C. Initial oven temperature was 200°C, and the temperature was programmed for 20 minutes at 200°C, from 200°C to 225°C at a rate of 5°C/min, and for 5 minutes at 225°C. Fatty acids were quantified by peak area comparison with the internal standard.
The protein content of the LDL preparations was measured according to
Lowry et al.51 -Tocopherol contents in plasma and LDL
(stored at -80°C) were assayed by high-performance liquid
chromatography with colorimetric detection.52 Vitamin C
(the sum of L-ascorbic + dehydro-L-ascorbic
acids) was assessed by high-performance liquid chromatography with
fluorometric detection in plasma (stored at -80°C), which was
supplemented with 10 mg/mL glutathione for stabilization directly after
blood collection.53
Statistical Analysis
Values in a treatment period were compared with those of the
previous period by Student's paired two-tailed t test.
Values are given as mean±SD. Differences between men and women were
evaluated statistically by Student's nonpaired two-tailed t
test. Linear correlations between the parameters considered were
calculated by the Pearson correlation test. Results were considered
significant if P<.05. All data analyses were performed with
the NCSS software package (version 5.01) developed by Dr
J.L. Hintze, Kaysville, Utah.
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Results |
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The study design is illustrated in Fig 1
. All participants
completed the study and compliance was very good, as monitored by pill
counts of unused capsules (5% of the capsules were returned) and
measurement of plasma vitamin E levels. The composition of the diet at
baseline (as relevant to this study) was derived from the 3-day food
records and is shown in Table 1. Men had a higher
caloric intake than women, with 35.6±6.2% versus 33.4±3.4% of
calories, respectively, in the form of fat. The ratio between
polyunsaturated and saturated fats was not significantly different
between men and women. Dietary vitamin E intake per day was higher for
men than for women and remained constant throughout the trial. For the
entire group, it was calculated to be 17.7±5.9 mg/d during the first
period and 14.8±6.9 and 16.1±6.8 mg/d, respectively, during the
second and last periods. The food records also showed no marked changes
in dietary intake of other nutrients during the trial.
No changes were observed in plasma concentrations of cholesterol, HDL
cholesterol, LDL cholesterol, and triglycerides during the intervention
(Table 2). Plasma HDL cholesterol levels in women were
significantly higher than those in men at all time points (data not
shown). Because triglyceride content and fatty acid composition of
LDL20 54 55 56 and LDL particle size57 58 have
been implicated as important parameters in determining the
susceptibility of LDL to oxidation, these parameters were measured. All
three parameters remained unchanged during vitamin E supplementation
(Tables 2 and 3). No differences were observed between
men and women (data not shown), although the 18:2/18:1 ratio in LDL
tended to be higher (P=.10) in men (2.18±0.33) than
in women (1.85±0.51) at the start but not at the end of the
intervention. Furthermore, plasma levels of the water-soluble
antioxidant ascorbic acid (vitamin C) did not change consistently
during vitamin E supplementation, indicating that vitamin E had no
"sparing" effect on vitamin C levels. The same holds true for the
reverse, since we found no relation between plasma vitamin E and
vitamin C levels.
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Ingestion of increasing amounts of vitamin E resulted in a gradual
increase in the concentration of -tocopherol in plasma and in LDL
(Fig 2 and Table 4). The total amount of
vitamin E contained in LDL before the trial (49±8% of total plasma
vitamin E) did not differ significantly from the value after
supplementation with 800 mg/d (46±10%). No differences in LDL
-tocopherol content were found between men and women. The rest
of vitamin E is contained in other lipoproteins (VLDL and
HDL59 ); no vitamin E is found in the lipoprotein-free
fraction (data not shown). Concentrations of -tocopherol in plasma
and in LDL for the whole group were both 1.2 times the baseline values
after the first period (25 mg/d) and 2.6 and 2.2 times, respectively,
after the last period (800 mg/d). Correlation for all data between
plasma -tocopherol and dose was .82 (P<.0001) (for men,
.87 and P<.0001; for women, .79 and P<.0001)
and between LDL -tocopherol and dose, .79 (P<.0001)
(for men, .85 and P<.0001; for women, .73 and
P<.0001). The correlation between -tocopherol
concentration in plasma and LDL was .66 (P<.0001) for the
whole group (Fig 3), .75 (P<.0001) for men,
and .59 (P<.0001) for women. The ratio of polyunsaturated
fatty acids (mol/mol) in LDL and -tocopherol in LDL decreased
from 90±13 before the trial to 43±7 after the last period, during
which 800 mg/d was ingested, and did not differ between men and
women.
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) and vitamin E
levels in LDL (nmol/mg LDL protein) (
) were measured as described
in "Methods." Values are mean±SD. *Indicates a significant
difference between the indicated dose and the previous dose.
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Effect of Vitamin E Supplementation on LDL Oxidation
Resistance of LDL against oxidative modification was assessed by
determination of the lag time and propagation rate of formation of
conjugated dienes, which are formed by conversion of polyunsaturated
fatty acids to fatty acid hydroperoxides with conjugated double
bonds.38 45 Representative oxidation curves are shown
in Fig 4. The lag phase increased gradually with
increasing dose of vitamin E (Fig 5A and Table 5). The increase was already significant
at 25 mg/d compared with the baseline value for the whole group
(P<.005). Resistance time for the whole group was elevated
by 28% at the end of the last period. The maximum rate of oxidation
was significantly reduced only at the end of the periods during which
400 (-13%) and 800 (-17%) mg vitamin E per day were ingested (Fig 5B). Propagation rate was significantly higher for LDL from men
compared with that for women at baseline and after supplementation with
25 and 50 mg/d. Propagation rate was correlated significantly with the
18:2/18:1 ratio (r=.56, P<.0003) and LDL
-tocopherol content (r=.44, P<.0001) for the
whole group.
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The vitamin E content of LDL and the lag time for all data were
correlated significantly (r=.57, P<.0001; Fig 6). The correlations for men and women were
r=.65 (P<.0001) and r=.53
(P<.0001), respectively. The correlations between
resistance time and plasma vitamin E level or ingested dose of vitamin
E were r=.69 (P<.0001) (for men, .64 and
P<.0001; for women, .73 and P<.0001) and
r=.68 (P<0.0001) (for men, .72 and
P<.0001; for women, .66 and P<.0001),
respectively. No significant relations between LDL or plasma
-tocopherol levels and lag times were found prior to
supplementation.
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Minor differences in LDL vitamin E levels and resistance time were
observed between men and women during the intervention (Tables 4 and 5). However, statistical evaluation of the correlation
between the -tocopherol content of LDL and resistance time at the
seven time points for each of the 20 individuals separately showed
marked interindividual differences. Strong and significant correlations
were found in 14 subjects (r=.82 to .94, .002<
P<.02), but nonsignificant relations between lag time and
vitamin E levels in LDL were observed in the remaining 6, 3 men and 3
women (r=.49 to .68, NS). The relation was not significant
in 8/20 individuals when the change in LDL vitamin E content was
compared with the change in lag time. When plasma vitamin E levels were
compared with resistance times at the seven time points, the number of
individuals showing a significant correlation increased to 16
(r=.78 to .95, .001<P<.04). Comparison of
ingested doses with lag times at the seven time points also showed a
significant association for 16 subjects (r=.77 to .97,
.001< P<.05). In both cases significant correlations were
found in 8 men and 8 women.
ANOVA for all data showed that variations in LDL vitamin E (47%) and between individual subjects (39%) were the most prominent parameters that contributed significantly to the variance in lag time. For the variance in propagation rate, the parameters were LDL vitamin E (37%), individual subjects (37%), and sex (12%) (P<.0001 for all parameters mentioned in this paragraph).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this article we have shown that ingestion of low supplementary doses of vitamin E protects LDL from lipid peroxidation in vitro. This effect can be attributed entirely to vitamin E, because during the intervention trial, no changes were found in other factors that have been shown to be important in determining the susceptibility of LDL to oxidation, such as LDL particle size,57 58 fatty acid composition,54 55 56 and triglyceride content.20 Minor differences were observed between men and women in plasma and LDL
-tocopherol concentrations and in the efficacy of vitamin E to
prolong the resistance time of LDL. In contrast, marked interindividual
differences were found; ie, the efficacy of vitamin E in LDL to
increase its resistance against oxidation varied considerably from
person to person. Furthermore, the maximal rate of oxidation was higher
in men than women at the start of the intervention trial, possibly
because the 18:2/18:1 ratio in LDL tended to be higher in
men.55 56 60
Vitamin E is a potent lipid-soluble antioxidant that has been shown to
be the major oxidation chain reaction–breaking compound in
membranes30 and an important factor in the protection of
polyunsaturated fatty acids in LDL against lipid
peroxidation.18 23 It has been shown previously that
administration of high, pharmacological doses of vitamin E elevate the
-tocopherol content in LDL and prolong the lag phase of
oxidation.38 39 40 41 42 43 Because these dosages highly exceed the
recommended daily intake for adults (12 to 15 mg/d),61 we
decided to assess whether ingestion of much lower doses of vitamin E
could also increase the resistance of LDL to oxidation. This may be
relevant, in view of the growing evidence from a number of
epidemiological studies that there is an association between intake or
plasma levels of vitamin E and a lower risk of
CHD.25 26 28 29 Such an association was found both in
populations showing differences in plasma -tocopherol levels due to
habitual differences in dietary intake of vitamin E25 26
and in large groups of men and women, some of whom used vitamin E
supplements.28 29
Our finding that supplementation with even 25 mg vitamin E per day leads to significant protection of LDL against oxidation in vitro and that intake of increasing doses of vitamin E gradually improves protection may provide a biochemical explanation for the observations in epidemiological studies. This view is supported by recent reports showing that the susceptibility of LDL to lipid peroxidation, as measured with the same type of oxidation assay used in our study, is correlated with the severity of coronary atherosclerosis in humans.20 21 22 On the other hand, it is conceivable that intracellular levels of antioxidants rather than plasma levels are more important in inhibiting cell-mediated LDL oxidation.62 63 Furthermore, it should be noted that the above-mentioned data from the literature are associative and that an effect of confounding factors cannot be ruled out.
In contrast to the significant effect of low-dose vitamin E on
resistance time, the progression of lipid peroxidation in LDL was
reduced only after intake of high doses of vitamin E (400 and 800
mg/d). Abbey et al64 also reported no significant effect
after a daily supplement of 200 mg -tocopherol. We suggest that only
at high dosages does -tocopherol become incorporated into the
interior of the LDL particle in sufficiently high amounts to retard the
autocatalytic chain reaction of the propagation
phase.38
The above-mentioned results of this study may be important with
regard to the atherogenicity of LDL. LDL particles are continuously
entering and leaving the arterial intima.65 66 During this
dynamic process, some of them may become entrapped for a shorter or
longer time in the extracellular matrix, where they are exposed to free
radicals generated by cells of the vascular wall.1 2 3 The
capability to resist oxidative modification may depend on, among
others, the amount of antioxidants within the particle. We have shown
that ingestion of low supplementary doses of vitamin E increases the
-tocopherol content of LDL and improves resistance to oxidation
and that increased protection is achieved at higher doses. This would
imply that with consumption of increasing dosages, the LDL particle has
a higher chance of escaping the vessel wall undamaged and even more
importantly, without generating harmful products that produce diverse
biological effects in the vascular wall.1 2 3 18 The finding
that the rate of lipid peroxidation is also reduced would imply that
-tocopherol in the core of the LDL particle, after surface
antioxidants have been consumed, may still be able to limit the burst
of noxious biological mediators. This may give the cells of the vessel
wall a better opportunity to respond adequately to oxidative
stress.
On the other hand, care should be taken in extrapolating these results to the in vivo situation in humans. Although supplementation with vitamin E in several32 33 34 but not all35 36 37 experimental animal studies has been shown to decrease the development of atherosclerosis, a causal relation between vitamin E intake and reduction of atherosclerosis has not been proven in humans. Recently, no beneficial effect on mortality due to CHD or stroke was observed in a large primary prevention trial with low-dose vitamin E (50 mg/d).67
Surprisingly, we found a higher correlation between plasma
-tocopherol level or ingested dose of vitamin E and lag time than
between the -tocopherol content of LDL and resistance time. This was
the case for data of the whole group and for the correlations within
each study subject separately. This observation suggests that
-tocopherol confers protection to LDL not only after incorporation
into the LDL particle but also by its presence in plasma and tissues.
How this may happen remains unclear. The mechanisms by which LDL is
oxidized in vivo are only poorly understood.1 2 3 It is
possible that a higher amount of -tocopherol in plasma and tissues,
eg, in cells of the vessel wall, protects LDL against subtle changes in
vivo, thereby rendering the particle less susceptible to oxidative
modification in vitro. In fact, the reverse has been reported to occur
with LDL shortly after subjects smoked cigarettes.68 These
subtle changes in LDL may include formation of low amounts of lipid
hydroperoxides, ie, possibly lower amounts than are present in
minimally modified LDL,16 17 69 which are a prerequisite
for copper ion–induced lipid peroxidation.70
In LDL prior to supplementation, we found no significant relation
between -tocopherol levels and lag times. A similar finding has been
reported previously by us and others38 39 40 41 42 43 57 58 71 and
indicates that under nonsupplemented circumstances, other factors
besides vitamin E determine the oxidation resistance of LDL. LDL of
vitamin E–deficient patients was shown to be even less susceptible to
oxidation in vitro than control LDL, despite lower vitamin E
contents.60 This observation led to the suggestion that
there may be a threshold level of vitamin E particles per LDL necessary
to substantially retard LDL oxidation.72 After enhancing
the LDL -tocopherol content to values normally not observed among
the population, resistance times correlated moderately with LDL
-tocopherol levels for the whole group (r=.57),
indicating that still other factors in LDL in addition to vitamin E
content influence LDL's susceptibility to oxidation. A similar though
somewhat higher correlation (r=.71) was found in a study by
Dieber-Rotheneder et al,40 in which higher doses of
vitamin E were given. Statistical evaluation of the relations for all
subjects separately revealed strong correlations in 70% of the
individuals (r=.82 to .94). This finding indicates that in
these subjects, -tocopherol becomes the most important parameter in
determining the oxidation resistance. Nonsignificant but still positive
associations between lag time and LDL vitamin E levels were found in
the other individuals. The latter group did not differ from the former
group in plasma or LDL -tocopherol concentrations before and during
the intervention trial, the latter indicating that vitamin E was taken
up and metabolized equally well in both groups. Similarly, no
differences were found in resistance times or propagation rates at the
seven time points in both groups. In addition, no differences were
found in LDL particle size, triglyceride content, fatty acid
composition, and the polyunsaturated fatty acid/-tocopherol ratio of
LDL. It is possible that these groups are too small to identify factors
that differ significantly or that other unknown factors are involved.
Nonetheless, it is clear from ANOVA that the factor "individual"
contributes markedly to the variance in lag time and propagation
rate.
It should be noted that although no significant linear association was
found between lag time and LDL -tocopherol level, all subjects in
the latter group (as defined above) showed a significant increase in
lag time. In addition, it is interesting to note that the number of
individuals showing an association between resistance time and either
plasma -tocopherol level or ingested dose of vitamin E was higher
than that with LDL -tocopherol levels. These findings indicate that
persons showing no significant correlation between LDL
-tocopherol levels and resistance times may also benefit by
intake of vitamin E in providing enhanced antioxidant protection of
their LDL.
In conclusion, this study demonstrates that intake of low-dose vitamin E, which may be achieved by consumption of vitamin E–enriched foods or vitamin E supplements, protects LDL against oxidation. Whether this finding may have implications for lowering the risk of CHD must await the results of primary and secondary intervention trials. The data presented here may be helpful in selecting the appropriate dose for those studies.
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Acknowledgments |
|---|
This work was supported by the Praeventiefonds, grant No. 28-1642-2, The Hague, the Netherlands. We thank Anneke Kramp-van Doorduin and Frank van Schaik for skillful technical assistance; Sanne Hulshoff for dietary supervision and calculation of dietary vitamin E intake; and Marisa Horsting for typing the manuscript. D,L-
-Tocopherol acetate was generously provided by F.
Hoffmann–La Roche Ltd. Received December 19, 1994; accepted December 29, 1994.
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 J. Virtamo, J. M. Rapola, S. Ripatti, O. P. Heinonen, P. R. Taylor, D. Albanes, and J. K. Huttunen Effect of Vitamin E and Beta Carotene on the Incidence of Primary Nonfatal Myocardial Infarction and Fatal Coronary Heart Disease Arch Intern Med, March 23, 1998; 158(6): 668 - 675. [Abstract] [Full Text] [PDF]
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L. P. L. van de Vijver, A. F. M. Kardinaal, W. van Duyvenvoorde, D. A. C. M. Kruijssen, D. E. Grobbee, G. van Poppel, and H. M. G. Princen LDL Oxidation and Extent of Coronary Atherosclerosis Arterioscler Thromb Vasc Biol, February 1, 1998; 18(2): 193 - 199. [Abstract] [Full Text] [PDF]
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M. E. Tornwall, J. Virtamo, J. K. Haukka, A. Aro, D. Albanes, B. K. Edwards, and J. K. Huttunen Effect of {alpha}-Tocopherol (Vitamin E) and ß-Carotene Supplementation on the Incidence of Intermittent Claudication in Male Smokers Arterioscler Thromb Vasc Biol, December 1, 1997; 17(12): 3475 - 3480. [Abstract] [Full Text]
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I. R. Brude, C. A. Drevon, I. Hjermann, I. Seljeflot, S. Lund-Katz, K. Saarem, B. Sandstad, K. Solvoll, B. Halvorsen, H. Arnesen, et al. Peroxidation of LDL From Combined-Hyperlipidemic Male Smokers Supplied With ¯-3 Fatty Acids and Antioxidants Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2576 - 2588. [Abstract] [Full Text]
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G. Davi, P. Alessandrini, A. Mezzetti, G. Minotti, T. Bucciarelli, F. Costantini, F. Cipollone, G. B. Bon, G. Ciabattoni, and C. Patrono In Vivo Formation of 8-Epi-Prostaglandin F2{alpha} Is Increased in Hypercholesterolemia Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 3230 - 3235. [Abstract] [Full Text]
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M. Seccia, E. Albano, and G. Bellomo Suitability of chemical in vitro models to investigate LDL oxidation: study with different initiating conditions in native and {alpha}-tocopherol-supplemented LDL Clin. Chem., August 1, 1997; 43(8): 1436 - 1441. [Abstract] [Full Text] [PDF]
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H. E. Brussaard, J. A. G. Leuven, C. Kluft, H. M. J. Krans, W. v. Duyvenvoorde, R. Buytenhek, A. v. d. Laarse, and H. M.G. Princen Effect of 17ß-Estradiol on Plasma Lipids and LDL Oxidation in Postmenopausal Women With Type II Diabetes Mellitus Arterioscler Thromb Vasc Biol, February 1, 1997; 17(2): 324 - 330. [Abstract] [Full Text]
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M. Seccia, E. Albano, E. Maggi, and G. Bellomo Circulating Autoantibodies Recognizing Peroxidase-Oxidized Low Density Lipoprotein: Evidence for New Antigenic Epitopes Formed In Vivo Independently From Lipid Peroxidation Arterioscler Thromb Vasc Biol, January 1, 1997; 17(1): 134 - 140. [Abstract] [Full Text]
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M.-F. Hau, A. H.M. Smelt, A. J.G.H. Bindels, E. J.G. Sijbrands, A. Van der Laarse, W. Onkenhout, W. van Duyvenvoorde, and H. M.G. Princen Effects of Fish Oil on Oxidation Resistance of VLDL in Hypertriglyceridemic Patients Arterioscler Thromb Vasc Biol, September 1, 1996; 16(9): 1197 - 1202. [Abstract] [Full Text]
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 J. M. Rapola, J. Virtamo, J. K. Haukka, O. P. Heinonen, D. Albanes, P. R. Taylor, and J. K. Huttunen Effect of Vitamin E and Beta Carotene on the Incidence of Angina Pectoris: A Randomized, Double-blind, Controlled Trial JAMA, March 6, 1996; 275(9): 693 - 698. [Abstract] [PDF]
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H. N. Hodis, W. J. Mack, L. LaBree, L. Cashin-Hemphill, A. Sevanian, R. Johnson, and S. P. Azen Serial Coronary Angiographic Evidence That Antioxidant Vitamin Intake Reduces Progression of Coronary Artery Atherosclerosis JAMA, June 21, 1995; 273(23): 1849 - 1854. [Abstract] [PDF]
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