1222 Am J C/in Nuir 1991;53:1222-9.. Printed in USA. © 1991 American Society for Clinical Nutrition
Effects of antioxidant supplementation on platelet function:a randomized pair-matched, placebo-controlled, double-blindtrial in men with low antioxidant status�3
Jukka T Salonen, Riitta Salonen, Kari Seppanen, Soili Rinta-Kiikka, Maija Kuukka,Heikki Korpela, Georg Alfihan, Marjatta Kantola, and Wolfgang Schalch
ABSTRACT We investigated the effect on platelet function
ofsupplementing men with low antioxidant status with 600 mg
ascorbic acid, 300 mg a-tocopherol, 27 mg /3-carotene, and 75
�tg selenium in yeast daily. Eighty men were randomly assigned
in pairs (matched for smoking, baseline antioxidant status, and
time and day ofentry) by use ofa double-blind design to receive
supplement or placebo for 5 mo. Compared with 39 control
subjects, 39 antioxidant-supplemented men experienced the fob-
bowing net reductions during the double-blind period: 20% (P
= 0.012) in serum lipid peroxides, 24% (P = 0.035) in ADP-
induced platelet aggregation, 42% (P = 0.040) in the rate of ATP
release during aggregation, 5 1% (P = 0.018) in serum (platelet-
produced) thromboxane B2 , and 29% (P = 0.024) in plasma /3-
thromboglobulin concentration. The data support our hypothesis
that antioxidant supplementation of men with low antioxidantstatus and high fat intake reduces lipid peroxidation, the capacity
of platelets to aggregate and to produce thromboxane A2 , andin vivo platelet activation. Am J Clin Nutr 199 l;53:
1222-9.
KEY WORDS Antioxidants, clinical trials, lipid peroxida-tion, platelet aggregability
Introduction
One prospective population study (1) and other case-controlstudies (2-4) observed an association between low selenium sta-
tus and ischemic heart disease (IHD). Cross-population studies
showed an inverse relationship between plasma vitamin C (5),
E, and A (6) concentrations and coronary mortality, but thesefindings were not confirmed in prospective within-population
studies (7-9). Although not all studies supported the relationship
between low antioxidant status and IHD, the information aboutthe potential mechanisms whereby antioxidant deficiency could
increase the risk of IHD is essential (10).
Prostacyclin synthesis in the arterial endothelium is, in cell
cultures, irreversibly inhibited by lipid peroxides (1 1). These are
generated either enzymatically by lipoxygenases and cycbooxy-
genase or by the influence of free radicals on cell-membrane
lipids. Intracellular lipid-peroxide concentration is regulated by
glutathione peroxidase, superoxide dismutase, and catabase en-
zymes as well as by the chain-breaking antioxidants a-tocopherol,
ascorbic acid, and fl-carotene. Thus, theoretically, a deficiency
in these antioxidants leads to reduced prostacyclin synthesis (12),
which can promote platelet aggregability. In an in vitro study,
ascorbic acid stimulated prostaglandin 12 (PGI2) synthesis by
endothelial cells whereas a-tocopherol and selenium, an integral
part ofgbutathione peroxidase, inhibited the platelet productionof thromboxane A2 (13).
There is clinical evidence suggesting that administration ofvitamin E and calcium could prevent postoperative throm-
boembolism (14). Marked platelet hyperaggregability was ob-
served in children with vitamin E deficiency, with complete re-
versal after vitamin E supplementation (15). Previous clinical
trials of the effects of ct-tocopherob (16-21) and ascorbic acid(22, 23) on platelet function have, however, produced inconsis-
tent results. Indicators of platelet activation in vivo have notbeen measured in any of the previous studies. We observed
markedly enhanced platelet aggregability at low serum selenium
concentrations in two independent, random population samples
of middle-aged Eastern Finnish men (24, 25). This inverse as-
sociation persisted even after other major determinants of plateletaggregability were allowed for.
The purpose of the present study was to test the hypothesis
that antioxidant supplementation of men with low antioxidant
status and relatively high intake of saturated fatty acids will re-
duce serum lipid peroxides and platelet activity as expressed byplatelet aggregability, serum thromboxane B� concentration, and
plasma concentrations of platelet-specific proteins.
Subjects and methods
Study subjects
The subjects ofthis trial were screened in a survey ofa random
population sample of men aged 54 y from eastern Finland at
I From the Departments ofCommunity Health and General Practice,and Chemistry and the Research Institute of Public Health, University
of Kuopio, Kuopio, Finland; the Kuopio Research Institute of ExerciseMedicine, Kuopio, Finland; the Department of Biochemistry, The Na-tional Public Health Institute of Finland, Helsinki, Finland; and theDepartment of Human Nutrition and Health, F Hoffman-La RocheLtd, Basle, Switzerland.
2 Supported by the Finnish Academy (grant for the KIHD study), the
University of Kuopio, and F Hoffman-La Roche Ltd.3 Address reprint requests to JT Salonen, University of Kuopio, P0
Box 6, 7021 1 Kuopio, Finland.
Received May 17, 1990.
Accepted for publication August 19, 1990.
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ANTIOXIDANTS AND PLATELET FUNCTION 1223
examination in the Kuopio Ischemic Heart Disease Risk Factor
Study (KIHD) (24-27). A total of 1 327 men was screened be-
tween March 1984 and June 1987. Plasma a-tocopherob was
not used as an inclusion criterion, but all subjects had relatively
low plasma a-tocopherol values (22. 1 ± 6.4 �tmol/L, I ± SD;
range 9.1-39.9 �mol/L) at the beginning ofthe study. Of these,
176 men with plasma ascorbic acid < 41.5 �tmol/L and serum
selenium < 1 .7 �tmol/L were invited to a reassessment of an-
tioxidant status in June and July 1987. Blood samples were ob-
tamed from 150 ofthese men; 26 did not show up, the majority
because of deaths and emmigration. Of the 150 men, 1 12 were
eligible for the study. Twelve men refused to participate in the
trial and 20 were excluded because ofillness or medications that
could have affected platelet function. The remaining 80 men
were entered into the trial. None ofthese men took antioxidants,
vitamin supplements, or prostaglandin-inhibiting drugs (eg,
acetosalicylic acid) before the trial. One subject randomly as-
signed to antioxidant supplementation discontinued the trial af-
ter 2 1 d of supplementation. The pairwise control was also ex-
cluded from the study population. Seventy-eight subjects com-
pleted the trial and underwent all assessments.
The mean baseline daily dietary intake offats was 122 g (40%
of total energy intake). There were no statistically significant
differences between the supplemented and the placebo men in
the changes in smoking, alcohol consumption, physical activity,
or any of the 60 nutrients assessed.
The study protocol was approved by the ethical committee
of the University of Kuopio, Kuopio, Finland.
Study design and intervention
The subjects were stratified by smoking status. There were 54
nonsmokers and 26 smokers. Subjects were paired in these strata
by their baseline values ofplasma ascorbic acid and a-tocopherol
and serum selenium. The members ofthe 40 pairs were randomly
assigned, one to antioxidant supplementation and the other to
placebo. To avoid confounding effects of seasonal variability,
both members ofeach pair were entered in the trial on the same
day and time. Two pairs were entered on each Monday and
Friday between August 1987 and January 1988. The purpose
ofthe extended subject entry over a period of6 mo was to balance
out the seasonal variation in vitamin intakes.
Each subject started the trial with a 1-mo placebo period.
Baseline refers to the end ofthis 1-mo lead-in period. The subjects
were instructed to keep diet, physical activity, and the amount
ofsmoking constant during the trial and not to use any vitamin
or mineral supplements. Four-day dietary recording was carried
out at the end of the trial as described in detail previously (27).
The blood antioxidant status was assessed at the beginning and
at the end of the lead-in period. After the 1-mo lead-in the sub-
jects receiving supplementation were treated with the antioxidant
preparation for 5 mo and those receiving placebo were given
visually identical placebo capsules for 5 mo.
The dosage was three capsules daily in both groups. An an-
tioxidant capsule contained 200 mg ascorbic acid, 100 mg a-
tocopherol, 9 mg /3-carotene, and 25 jzg selenium in selenium-
enriched yeast. The filling material was peroxide-free arachis oil.
Antioxidant status was reassessed at the end of the trial.
Weight, serum lipids, serum lipid peroxides, blood glutathione
peroxidase, platelet aggregability, serum thromboxane B2, and
platelet-specific proteins were measured at the end of the bead-
in period and at the end of the trial.
An 8-wk bioavailability study was carried out in 10 volunteers
who were not subjects ofthe present trial. The same dosing was
used as was planned for the trial. In this pilot study, mean plasma
ascorbic acid rose from 63.6 to 79.5 .tmol/L (25% increase), a-
tocopherob from 24.4 to 47. 1 zmol/L (93% increase), and /3-
carotene from 0.7 to 3.5 �mol/L (400% increase). The concen-
trations ofplasma ascorbic acid and a-tocopherol reached a pla-
teau in 1 wk, whereas plasma /3-carotene rose linearly for 3 wk
and more slowly for another 2 wk.
Blood collection
The subjects were asked not to take any antiinflammatory
analgesics during the 2 wk before blood sampling and to abstain
from alcohol for 1 wk before blood sampling. The subjects were
also asked to fast and not to smoke for 12 h before blood sam-
pling. Blood sampling took place after the subjects had been
supine for 30 mm. Blood was collected without a tourniquet by
a flawless venipuncture. Terumo Venoject VT-050X88 vacuum
tubes (Terumo Corporation, Tokyo, Japan) and Terumo 18 G
needles (Terumo Europe, Leuven, Belgium) were used in drawing
blood samples. The tubes contained 1 vol of 3. 1% trisodium
citrate as the anticoagulant to 9 vol of blood.
Biochemical determinations
Plasma concentrations of a-tocopherob and /3-carotene were
determined with HPLC as described by Vuilleumier et al (28)
and plasma ascorbic acid was determined by using a fluorometric
method (29). Serum selenium concentrations were determined
with the graphic-furnace atomic-absorption spectrophotometric
technique, Zeeman background correction, and pyrolyticabby
coated graphite tubes with a platform (Zeeman 5000 atomic
absorbtion spectrophotometer, HGA 500, Perkin-Elmer, Nor-
walk, CT) (1).
The main serum lipoprotein fractions (very-low-density,
(VLDL)bow-density (LDL)and high-density (HDL) lipoproteins)
were separated within 3 d of blood sampling by a combination
of ultracentrifugation and precipitation (30). The cholesterol
content (mmol/L) of all lipoprotein fractions was determined
enzymatically (cholesterol CHOD-pap method, Boehringer
Mannheim, Mannheim, FRG).
Serum lipid peroxides were measured fluorometrically by using
the thiobarbituric acid (TBA) reaction (31). In this method TBA-reacting substances other than lipid peroxides were eliminated
by precipitating lipid peroxides along with serum proteins in the
phosphotungstic acid-sulfuric acid system. The determinants
were done from deep-frozen (at -70 #{176}C)serum samples within
24 mo of blood sampling.
Blood glutathione peroxidase activity was determined from
prediluted (1:9) frozen (at -70 #{176}C)whole-blood samples with
an automated modification ofthe method described by Levander
et al (32) within 3 mo of blood sampling. Catalase activity of
red blood cells was inhibited with Drabkins’ solution (33).
For measuring platelet aggregation adenosine 5’-diphosphatesodium salt (ADP) and adenosine 5’-triphosphate disodium salt(ATP; Sigma Chemical Company, St Louis) were dissolved in9 g NaC1 in 1 L distilled water. Silicon-treated SMI capillary
tubes and an SMI micropipettor (Scientific Manufacturing In-
dustries, Miami) were used for adding ADP and ATP to the
reaction mixture. All plasma samples were handled with plastic
Pasteur pipettes (Orioba Oy Probab, Helsinki, Finland). Coulter
thrombocyte control CTC-3 (Coubter Electronics Ltd, Luton,
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1224 SALONEN ET AL
TABLE 1
Baseline and posttrial values and change during the trial in the plasma antioxidative vitamins, serum selenium, lipids, and indicators of lipid
peroxidation in the 39 p airs of supplem ented and place bo-treated mena
Net percent P forSupplemented Placebo change in the
supplementedmen
intrapairdifference
in changeBaseline Posttrial Change Baseline Posttrial Change
Plasma ascorbic acid
(�mol/L) 28.39 ± 2.32 75.52 ± 2.50 47.13 ± 3.S2t 31.22 ± 2.27 27.25 ± 2.89 -3.97 ± 3.18 +274 ± 52 <0.001Plasma a-tocopherol
(�mol/L) 23.22 ± 0.95 47.37 ± 0.87 24.15 ± l.S5t 20.90 ± 0.74 18.58 ± 0.79 -2.32 ± l.08f +73 ± 8 <0.001a-Tocopherol:(VLDL
+ LDL cholesterol)(�mol/mmol) 5.38 ± 0.16 10.96 ± 0.34 5.58 ± 0.28t 5.15 ± 0.18 5.18 ± 0.23 0.03 ± 0.18 +101 ± 9 <0.001
Plasma fl-carotene
(�mol/L) 0.57 ± 0.05 3.71 ± 0.37 3.14 ± 0.35t 0.67 ± 0.05 0.50 ± 0.05 -0.17 ± 0.03t +656 ± 75 <0.001Serum selenium
(�tmol/L) 1.58 ± 0.03 1.80 ± 0.03 0.22 ± 0.03t 1.60 ± 0.03 1.51 ± 0.03 0.09 ± 0.02t +19.5 ± 2.5 <0.001
Serum total cholesterol(mmol/L) 5.6 ± 0.16 5.5 ± 0.15 -0.1 ± 0.10 5.5 ± 0.15 5.3 ± 0.15 -0.2 ± 0.08 - NS
Serum LDL cholesterol(mmol/L) 3.8 ± 0.15 3.7 ± 0.13 -0.1 ± 0.1 1 3.7 ± 0.14 3.5 ± 0.13 -0.2 ± 0.08 - NS
Serum HDL
cholesterol (mmol/L) 1.28 ± 0.05 1.21 ± 0.06 -0.07 ± 0.05 1.33 ± 0.04 1.31 ± 0.06 -0.02 ± 0.06 - NSBody weight (kg) 80.72 ± 2.05 81.51 ± 2.13 0.80 ± 0.33f 81.29 ± 1.58 81.69 ± 1.64 0.40 ± 0.31 - NS
Serum lipid peroxidesLI
thrombocytes) 1.74±0.12 1.31 ±0.08 -0.43±0.lOt 1.46±0.08 1.36±0.07 -0.10±0.08 -19.9±7.2 0.012Blood glutathione
peroxidase(mU/g Hb) 1.64 ± 0.04 1.73 ± 0.05 0.09 ± 0.03� 1.62 ± 0.05 1.59 ± 0.05 -0.03 ± 0.05 +7.3 ± 3.6 0.032
a � � SEM. VLDL, very-low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein.
tf� Significantly different from zero: tP < 0.001, fP < 0.05, §P < 0.01.
UK) was used as a control when platelets in plasma samples
were counted.
The blood tubes were mixed well by gentle inversion and werekept at room temperature for 30 mm. The blood samples wereagain mixed gently and centrifuged at +20 #{176}Cfor 10 mm at 250x g. Platelet-rich plasma (PRP) was collected to a polypropylene
tube. For platelet-poor plasma (PPP) preparation the same blood
tubes were centrifuged once more at room temperature for 10
mm at 4000 X g and PPP was handled in the same way as PRP.
Thrombocytes in PRP and PPP were counted with a Throm-
bocounter C (Coulter Electronics Ltd).Thrombocyte aggregation in samples of PRP was measured
as a function of time by the Whole Blood Aggro-Meter Model
500 (Chronobog Corporation, Havertown, PA). Changes in both
optical density, as measured with changes in electrical impedance
(mV), and ATP secretion (luminescence) were recorded simul-
taneously. The measurement of aggregation was described in
detail previously (34). Luciferase-luciferin reagent (100 �tL;
Chronolog Corporation) was added to the sample of PRP (1.0
mL) and the PPP sample was adjusted with 100 �tL 9 g NaCb/
L to the same basic optical density. The aggregating agent, 5 �tL1.0 mmol ADP/L, was injected into the PRP samples to start
the reaction. Final concentration ofADP was 4.52 �tmob/L. Each
subject’s samples before and after the double-blind period were
always handled with the same dose ofaggregating stimulus. After
a 10-mm run the luminescence channel was calibrated by adding
4 �iL 2.004 mmol ATP/L into the PRP samples (final ATP con-
centration 7.26 zmol/L). The measurement of platelet aggreg-
ability was carried out within 3 h of blood drawing.
For analysis ofthromboxane B2 in serum (produced by plate-
lets during in vitro incubation), 1 mL blood was taken and al-
bowed to clot at 37 #{176}Cfor 30 mm. After centrifugation at 500
x g for 15 mm at 4 #{176}C,the serum was frozen at -20 #{176}Cuntil
assayed. The determinations were carried out after 20-26 mo
ofstorage. The extraction ofthromboxane B2 was performed by
Powell’s method (35). Thromboxane B2 concentration in the
extract was determined by using reverse-phase HPLC. In our
laboratory this method gives > 50% lower values of serum
thromboxane B2 than do the commercially available radioim-
munoassay (RIA) kits and thus may be more specific.Plasma /3-thromboglobulin (/3-TG) was determined by a com-
mercial RIA (Amersham, Amersham, UK). The method depends
on competition between sample fl-TG and ‘251-labeled /3-TG for
a limited number of binding sites on a fl-TG-specific antibody.
Plasma samples (25 ML), standard (25 .tL), ‘251-labeled /3-TG
(100 giL; radioactivity 7.4 MBqJL), and anti-fl-TG rabbit serum
(100 �.tL) were pipetted into the polystyrene tubes. The reaction
solutions were mixed and incubated for 1 h at room temperature.
After incubation 250 �iL saturated ammomum sulphate solution
were added into the tubes. The tubes were mixed and centrifuged
10 mm at 1 500 X g. Supernatants were removed and radioac-
tivity in precipitates was measured by a MiniGamma �y counter
(LKB Wallac, Turku, Finland). All tests were run in duplicate
and the results were calculated by using the spline function (36).
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ANTIOXIDANTS AND PLATELET FUNCTION 1225
TABLE 2Baseline and posttrial values and change during the trial in the indicators of in vitro and in vivo platelet function in 39 pairs of supplemented and
placebo-treated mena
Supplemented Placebo
Net percent
change in the
supplemented
men
P for
intrapair
difference
in changeBaseline Posttrial Change Baseline Posttrial Change
Extent (amplitude) of
secondary aggregationphase (mV) 19.8 ± 1.5 15.6 ± 1.8 -4.2 ± 1St 16.5 ± 1.8 16.9 ± 2.2 0.5 ± 2.2 -23.7 ± 13.3 0.035
Velocity (slope) ofsecondary aggregationphase(mV/s) 0.177±0.016 0.130±0.021 -0.048±0.Ol6t 0.143±0.017 0.132±0.019 -0.011 ±0.019 -20.9± 14.0 0.028
Maximum ATP release(�imol/L) 3.10 ± 0.38 2.22 ± 0.33 -0.88 ± 0.40� 2.36 ± 0.30 2.49 ± 0.42 0.13 ± 0.38 -32.6 ± 17.9 0.040
Velocity (slope) of ATP
release
(�mol. L’ .s’) 0.053 ± 0.009 0.034 ± 0.007 -0.020 ± 0.009� 0.038 ± 0.006 0.040 ± 0.007 0.002 ± 0.007 -41.5 ± 21.5 0.034
Serum thromboxane B2
Platelet-count adjustedI
thrombocytes) 121 ± 13 76 ± 18 -45 ± 21t 138 ± 18 155 ± 18 17 ± 26 -51.1 ± 27.2 0.018
Unadjusted (nmol/L) 287 ± 32 152 ± 27 -135 ± 361 372 ± 43 407 ± 42 36 ± 56 -59.6 ± 23.2 0.003
Plasma �3-thromboglobulin
Platelet-count adjusted(�ig/10”thrombocytes) 27.0 ± 3.1 19.5 ± 1.8 -7.5 ± 3.2t 20.5 ± 1.9 20.9 ± 4.0 0.3 ± 4.4 -29.1 ± 20.2 0.024
Unadjusted (�igIL) 64.3 ± 8.5 43.9 ± 4.4 -20.4 ± 7.8t 55.6 ± 4.3 54.6 ± 1 1.3 -1.0 ± 12.2 -30.2 ± 22.5 0.048
Plasma platelet factor 4
Platelet-count adjusted
(�g/l0”
thrombocytes) 6.97 ± 2.7 5.03 ± 0.7 -1.93 ± 2.7 3.79 ± 0.4 4.85 ± 0.9 1.06 ± 1.0 -43.0 ± 41.3 0.132
Unadjusted (�tgIL) 18.2 ± 8.2 1 1.6 ± 1.7 -6.6 ± 8.1 10.1 ± 0.9 12.4 ± 2.3 2.3 ± 2.6 -48.9 ± 46.7 0.134
aI± SEM.
ff1 Significantly different from zero: tP < 0.01, tP < 0.05, §P < 0.001.
Plasma platelet factor 4 (PF4) was measured by using an RIA
(Abbott Laboratories, Wiesbaden-Delkenheim, FRG). The
method is a competitive RIA in which nonradioactive PF4 in
plasma competes with a constant amount of ‘25I-PF4 reagent
solution (125 giL, radioactivity 13.0 MBCIJL). Anti-PF4 goat
serum (125 jzL) was pipetted into the polystyrene tubes. Tube
contents were mixed and the tubes were incubated at room tem-
perature for 2 h. After incubation 500 �tL ammonium sulphatesolution (73% saturated) was added into the tubes. Tube contents
were mixed and then allowed to stand 10 mm before centrifu-
gation. All tubes except total-count tubes were centrifuged at
room temperature at 1 500 X g for 20 mm. Supernatants were
removed by decanting and radioactivity in precipitates was
measured by a ‘y counter. All tests were run in duplicate and
the results were calculated by using the spline function (36).
Determinations of plasma /3-TO and PF4 from deep-frozen
(at -70 #{176}C)plasma samples were carried out within 3 mo of
the end of the trial.
Statistical methods
The association ofantioxidant status with indicators of platelet
activation at the baseline was tested with two-way SPSS-X anal-
ysis ofcovariance (37). Because subjects were matched pairwise,
statistical analysis was based on the t test for paired samples.
The a priori power to detect a difference of 5 mV (‘-25%) in
the ADP-induced platelet aggregation between the supplemented
and the placebo subjects in the change from end of bead in to
end of trial was > 0.8 at one-tailed a of 0.05, assuming an SD
of 10 mV for this platelet-aggregation variable (based on KIHD
data, ref 24). The net change was computed by subtracting the
percentage change in the control subject from the percentage
change in the corresponding paired supplemented subject. The
statistical significance levels for differences in change between
the supplemented and the placebo-treated men are based on
one-sided tests. The matching of subject pairs with regard to
smoking was done to enable the comparison of the treatment
effects between smokers and nonsmokers, even with suboptimal
study power.
Serum thromboxane B2 and plasma /3-TO and PF4 concen-
trations were adjusted for blood platelet count by computing
ratios. Both adjusted and unadjusted concentrations are pre-
sented for main results.
The statistical significance of linear trends in changes in in-
dicators of platelet activation over quartiles of changes in the
supplemented vitamins and selenium was tested with one-way
analysis of variance. The pairing ofsubjects was broken for this
analysis.
Results
To analyze the cross-sectional association of antioxidant vi-
tamin and selenium status with in vivo platelet activation, the
mean values of serum thromboxane B2 and plasma /3-TO con-
centrations at entry to the double-blind period were computed
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1226 SALONEN ET AL
end of the washout period. Plasma ascorbic acid concentration change in the placebo group (Table 2).
TABLE 3
Baseline values and change during the trial in the antioxidative vitamins and serum lipid peroxides, and indications of platelet activation in the
supplemented and placebo-treated men, by smoking statusa
Nonsmokers (2 6 pairs) Smokers (13 pairs)
Net change Net change
Supplemented Placebo Percent Pt Supplemented Placebo Percent Pt
Plasma ascorbic acidBaseline (�mol/L) 31.2 ± 3.1 31.5 ± 2.6 22.8 ± 2.7 30.7 ± 4.5Change (%) 273 ± 7 lf - 1 ± 13 274 ± 72 0.001 256 ± 44j - 18 ± 14 274 ± 46 <0.00 1
Plasma a-tocopherol: (VLDL
+ LDL cholesterol)Baseline (�tmo1/L) 5.60 ± 0.19 5.20 ± 0.22 4.99 ± 0.27 5.06 ± 0.30Change (%) 102 ± 9� 2 ± 5 100 ± 10 <0.001 105 ± 18t 1 ± 3 104 ± 19 <0.001
Plasma fl-caroteneBaseline (�mol/L) 0.60 ± 0.07 0.77 ± 0.07 0.50 ± 0.09 0.48 ± 0.08Change (%) 668 ± 82� -25 ± 5� 693 ± 82 <0.001 560 ± 159t -23 ± 8� 583 ± 160 0.002
Serum seleniumBaseline (�imol/L) 1.59 ± 0.04 1.59 ± 0.04 1.57 ± 0.05 1.62 ± 0.06Change (%) 1 1 ± 3j -6 ± 2� 17 ± 3 <0.001 19 ± 2f -6 ± 2� 24 ± 3 <0.001
Serum lipid peroxidesBaseline (zmol/l0�
thrombocytes) 1.75 ± 0.18 1.48 ± 0.10 1.74 ± 0.10 1.43 ± 0.13Change(%) -19±8t -9±7 -11 ± 11 0.157 -35±8� -2±7 -35± 11 0.006
Serum thromboxane B2Baseline (�imol/l0�
thrombocytes) 105 ± 15 132 ± 22 151 ± 23 150 ± 34Change(%) -20±26 5± 19 -25±32 0.188 -60± l6� 24±39 -84±42 0.019
Plasma fl-thromboglobulinBaseline(�g/10� thrombocytes) 25.1 ± 2.9 21.6 ± 2.0 30.7 ± 7.4 18.6 ± 3.9Change(%) -20± 14 15±29 -35±32 0.040 -40±2111 -28±22 -23±30 0.178
aj± SEM.
t One tailed, derived from paired t tests. All P values were consistent (at the same significance level) with those from Wilcoxon matched-pairssigned-rank tests.
t�II Significantly different from zero: �P < 0.001, §P < 0.01, lIP < 0.05.
for all subjects, grouped by plasma vitamin and selenium con-
centrations (broken down at medians), with an additional
breakdown by smoking status (no vs yes) in two-way covariance
analysis. Both serum thromboxane B2 (by 46%) and plasma /3-
TO (by 12%) were nonsignificantly higher in smokers than innonsmokers. When adjustments were made for body mass index
(expressed in kg/m2), history of or presence of current IHD (no
vs yes), serum LDL cholesterol (mmol), and coffee consumption
(g/wk) as covariates and for smoking status as a binary factor,
the mean serum thromboxane B2 concentration was higher in
the lower half of plasma ascorbic acid distribution (385 vs 271
nmol/L, two-tailed P = 0.039). There were similar nonsignificant
trends for the ratio ofplasma a-tocopherob to cholesterol (VLDL
plus LDL), /3-carotene, and serum selenium. Identically adjusted
plasma /3-TO concentration was higher (75.4 �g/L) in the bower
half than in the upper half(53.7 �g/L) of the ascorbic acid dis-
tribution (two-tailed P = 0.041) in a two-way covariance analysis.
This difference was greater in smokers than in nonsmokers (P
= 0.030 for interaction).
The baseline and posttriab means ofthe antioxidative vitamin
and selenium concentrations, serum lipoproteins, serum lipid
peroxides, and blood glutathione peroxidase are presented in
Table 1. The supplemented and the placebo-treated subjects had
comparable plasma vitamin and selenium concentrations at the
rose in the supplemented subjects by 166% (range -4% to
+ 1 358%), a-tocopherol rose by 104% (range 28-2 16%), /3-car-
otene rose by 549% (range 11-1938%), and serum selenium
concentration rose by 13% (range - 13% to +57%) (Table 1). In
the placebo-treated subjects neither plasma ascorbic acid nor
the ratio ofplasma a-tocopherob to cholesterol (VLDL plus LDL)
changed significantly whereas plasma /3-carotene decreased by
26%, plasma a-tocopherob decreased by 1 1%, and serum sebe-
nium decreased by 6%.
There were no statistically significant changes in the serum
total-, LDL-, or HDL-cholesterob concentrations in either
the supplemented or the placebo-treated men during the
supplementation (Table 1). Mean body weight increased by 1%
in the supplemented subjects. Platelet-count-adjusted serum
lipid peroxides were reduced by 25% in the supplemented
and by 7% in the placebo-treated subjects. Blood glutathi-
one peroxidase increased by 6% in the supplemented subjects
and did not change significantly in the placebo-treated men
(Table 1).
The extent of ADP-induced platelet aggregation was reduced
during the antioxidant supplementation by 21% (P < 0.01) (Ta-
ble 2) whereas there was practically no change in the placebogroup. The average velocity ofADP-induced platelet aggregation
declined 27% (P < 0.01) in the supplemented subjects, with no
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ANTIOXIDANTS AND PLATELET FUNCTION 1227
TABLE 4
Change of serum lipid peroxides, serum thromboxane B2, and plasma fl-thrombogbobulin in concentration quartiles (I-IV) ofchange in vitamins,selenium, and lipid peroxidesa
Quartile of I II III IV rtP for
linear trend
�mo//1O” thrombocytes
Serum /ipid peroxide change
Plasma ascorbic acid change
Plasma a-tocopherol change
Plasma fl-carotene change
Serum selenium changeSerum lipid peroxide change
0.01 ± 0.1 1
-0.26 ± 0.09
-0. 18 ± 0. 10
-0.17 ± 0.10-
-0.42 ± 0. 17 -0.22 ± 0.09
0.07 ± 0. 10 -0.37 ± 0. 10
-0.09 ± 0.09 -0.43 ± 0. 19
-0.01 ± 0.12 -0.54 ± 0.15- -
-0.43 ± 0. 13-0.50 ± 0. 18
-0.35 ± 0. 1 1
-0.33 ± 0.12-
-0.28-0.3 1
-0.06
-0.27
-
0.0290.035
0.102
0.003
-
Serum thromboxane B2 changePlasma ascorbic acid changePlasma a-tocopherol change
Plasma fl-carotenechange
Serum selenium changeSerum lipid peroxide change
3 1 ± 4375 ± 35-1 ± 34
25 ± 38
63 ± 22
2 1 ± 2 1 -7 1 ± 2 1
-4 1 ± 3 1 -69 ± 28
15 ± 40 -7 ± 35
-17 ± 34 -35 ± 24
30 ± 29 -8 ± 29
-29 ± 39
-20 ± 30
-62 ± 17
-17 ± 36
-8 ± 29
-0.28
-0.24
-0.23
-0.18
-0.09
0.025
0.002
0.041
0.084
0.330
Plasma fl-thromboglobulin changePlasma ascorbic acid changePlasma ct-tocopherol change
Plasma fl-carotene change
Serum selenium changeSerum lipid peroxide change
0.6 ± 5.9-4. 1 ± 1.8
-8.4 ± 3.3
6.0 ± 8.1-12.7 ± 4.8
- 1 .9 ± 7.0 - 1.8 ± 4. 14.6 ± 8.6 -9.5 ± 5.0
3. 1 ± 8.7 -8.3 ± 3.0
-3.5 ± 4.7 -12.8 ± 4.7
-6.2 ± 2.3 1.1 ± 6.6
- 1 1.6 ± 4. 1-5.6 ± 4.0
-0.9 ± 4.0
-3.5 ± 2.44.7 ± 7.0
-0. 18-0. 14
0.06
-0.150.24
0.071
0.228
0.3360.0610.008
ai+SEM
t Simple coefficient of correlation between changes in antioxidants and changes in lipid peroxides, serum thromboxane B2 , and plasma fl-throm-boglobulin.
The maximum ATP release, as measured by the maximal
concentration during ADP induction, decreased by 28% (P
< 0.05) and the average velocity of ATP release decreased by
38% (P < 0.05) in the supplemented men whereas no changes
occurred in the placebo group (Table 2). The differences in
changes in all four indicators of in vitro platelet aggregability
between the supplemented and placebo subjects were statistically
significant (P < 0.05).
Platelet-count-adjusted serum thromboxane B2 concentra-
tions were reduced by 37% P < 0.05; unadjusted by 47%, (P
< 0.001) in the supplemented men and did not change signifi-
cantly in the placebo-treated men. The difference in thrombox-
ane B2 change between the supplemented and placebo-treated
men was statistically significant (P = 0.018 for adjusted and P= 0.003 for unadjusted). The plasma concentrations of both
platelet-specific proteins, which indicate the platelet activity in
vivo, decreased during supplementation: platelet-count-adjusted
/3-TO by 28% (P < 0.05; unadjusted by 32%, (P < 0.01) andPF4 by 28%. The latter change did not reach statistical signifi-
cance because of the large variability of baseline values among
the supplemented men (Table 2). The difference in change be-
tween the supplemented and placebo-treated men was statisti-
cally significant (P < 0.05) for /3-TO but not for PF4.
In the baseline measurements for one supplemented subject
and for none of the control subjects, the ratio of plasma /3-TO
to PF4 was < 2, indicating a possibility of cx vivo platelet ac-
tivation. In the end-of-trial measurement this was the case for
three supplemented and three control subjects. For 32 pairs the
ratio was > 2 in both assessments. In this subgroup the mean
thromboxane B2 fell in the supplemented subjects from 276 ± 32
(1 ± SEM) to 1 64 ± 32 nmol/L (by 40.6%; P < 0.0 1 for change)
and increased in the control subjects from 403 ± 49 to 411
± 49 nmol/L, giving a net reduction of 43.6% (P = 0.032). In
the same subgroup plasma /3-TO was reduced among the sup-
plemented from 60.6 ± 6.5 to 45.5 ± 4.3 �sg/L (by 26.9%; P
< 0.05 for change) and was increased among the control subjects
from 56.8 ± 4.9 to 59.9 ± 13.1 �sg/L, the net reduction being
32.0% (P = 0.071).
The reduction in platelet-count-adjusted serum thromboxane
B2 in the supplemented smokers was 60% (P < 0.01) and in the
supplemented nonsmokers, 20% (Table 3). The net reduction
ofserum thromboxane B2 was 84% (P = 0.019) in smokers and
25% (P = 0. 188) in nonsmokers. Platelet-count-adjusted plasma
/3-TO concentration decreased among the supplemented menby 40% (P < 0.05) in smokers and by 20% in nonsmokers. The
net reduction of plasma /3-TO was 23% (NS) in smokers and
35% (NS) in nonsmokers (Table 3).
To analyze the contributions of changes in different antioxi-
dants to changes in platelet activation, the distributions of
changes in platelet-count-adjusted serum lipid peroxides, serum
thromboxane B2 , and plasma /3-TO concentrations were com-
puted in quartiles ofchanges in the supplemented vitamins and
selenium in all 78 subjects who completed the trial (Table 4).A great increase in plasma ascorbic acid, a-tocopherol, and serum
selenium was related (P < 0.05) to a large reduction in serum
lipid peroxides. Similarly, the change in plasma ascorbic acid,
a-tocopherol, and /3-carotene (P < 0.05 for each) and, though
nonsignificantly, serum selenium was inversely associated with
the change in serum thromboxane B2 . The changes in plasmaascorbic acid and serum selenium had a nonsignificant associ-
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1228 SALONEN ET AL
ation with the change in plasma /3-TO (Table 4). The greater
the reduction in serum lipid peroxides, the greater was the de-
crease in plasma fl-TO (P = 0.008 for linear trend for adjusted
and P = 0.019 for unadjusted), as shown in Figure 1. The lipidperoxide change had no significant association with the change
in serum thromboxane B2.
Discussion
It is well-established that increased concentrations of platelet-
specific proteins in plasma reflect enhanced platelet stimulation
in vivo (38-40), except possibly when there is deficient metab-
olism of these proteins in certain disease conditions. Several
studies suggested that /3-TO binds to endothelial cells and blocks
prostacyclin formation (4 1 ). A reduction in plasma concentra-
tions of/3-TG should thus indicate a reduction ofplatelet activity
in vivo.
The assessment ofin vivo platelet activity is tedious and com-
plicated because the platelets tend to get activated during blood
sampling. Because of its shorter half-life FF4 is proportionately
more elevated in ex vivo platelet activation than is fl-TO (39).
Thus, the ratio of these platelet-specific proteins can be used to
rule out spurious platelet activation. The exclusion of pairs of
subjects with possible ex vivo platelet activation did not influence
the results ofthis study decisively. Because plasma concentrations
ofplatelet products and peroxides are dependent on the platelet
count, we presented the main findings with an adjustment for
the platelet count. This adjustment had, however, a minor effect
on the results.
Serum thromboxane B2, as measured in this study, mainly
describes the production of thromboxane A2 by the platelets
during the 30-mm in vitro incubation ofblood at 37 #{176}C.About
one thousandth ofthe thromboxane B2 measured originates from
the circulating blood. For this reason we have also used the term
platelet-produced thromboxane for the same measurement (34).
This assessment of serum thromboxane B2 is an indicator of the
capacity of platelets to produce thromboxane A2.
As an index of lipid peroxidation, serum lipid peroxide con-
centration assayed by the TBA method was used because of its
sensitivity (3 1 ). This method has been criticized for lack of spec-
ificity (42) but this problem is eliminated or found to have a
negligible effect when Yagi’s method is used (3 1, 43). Moreover,
there is a good correlation between the results obtained by Yagi’s
method and those achieved with HPLC (44).
The lower lipid-peroxide concentrations in the end-of-trial
samples ofthe placebo-treated group as compared with the base-
line samples suggest that some lipid peroxidation took place
during the storage of serum samples at -70 #{176}C.If the higher
antioxidant concentrations of the supplemented subjects inhib-
ited this, some of the observed net reduction of lipid peroxides
during the trial would have been due to this ex vivo inhibition
of lipid peroxidation.
The findings of the present study disagree with those of a
recent small but well-controlled trial by Stampfer et al (2 1), who
observed no significant effects on either in vitro platelet aggreg-
ability or serum thromboxane B2 concentration in 20 healthy
university-hospital staff members who were supplemented with
727 mg vitamin E/d for 5 wk. In addition to study power and
measurement method, the most likely explanation for the dis-
crepancy is the difference in the baseline antioxidant status and
dietary fat intake between the two study populations. Five weeks
QUARTILE OF LIPID PEROXIDE CHANGE
FIG 1. Mean change in platelet-count-adjusted plasma fi-thrombo-
globulin Lg,/” thrombocytes) in the quartiles of platelet-count-adjusted
serum lipid peroxide change. r = 0.24, P = 0.008.
may also be too short a period to establish lipid peroxidation-
mediated changes in platelet function.
We also used our data to analyze the cross-sectional associ-
ations between antioxidants and platelet activity and the rela-
tionship between changes in antioxidants and changes in mdi-
cators of platelet activity. The large range of changes in plasma
antioxidant concentrations between subjects enabled the batter
analysis. Both nonexperimental analyses supported the main
finding. Considering the small sample size and the conservative
statistical approach taken (45), the presence of the associations
between antioxidant changes and change in lipid peroxides on
one hand and the associations of lipid peroxide change with
change in plasma fl-TO on the other are in accordance with the
hypothesis that antioxidants reduce platelet activity through the
elimination of circulating lipid peroxides.
This study demonstrates that long-term supplementation witha combination of ascorbic acid, a-tocopherol, fl-carotene, and
selenium of men with low antioxidative vitamin status and rel-
atively high intake of saturated fatty acids reduces serum lipid
peroxide content, the activation of platelets in vivo, and the
aggregabibity and thromboxane production of the platelets in
vitro. Antioxidant supplementation had no effect on the cho-
lesterol concentrations in the main lipoprotein fractions. Al-
though the sample size was insufficient for a formal analysis
comparing smokers with nonsmokers, the data suggest that the
effect of antioxidant supplementation may be greater in smokers
than in nonsmokers. These observations provide additional in-
formation of possible mechanisms through which low antioxi-
dant status can increase the risk of IHD and promote the pro-
gression of atherosclerosis. U
We are deeply grateful to Hannele Kastarinen, public health nurse,
for subject management.
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