Influence of vitamin E administration on platelet functions in hormonal contraceptive users

CONTRACEPTION

INFLUENCE

OF VITAMIN E ADMINISTRATION ON PLATELET 11: HORMONAL CONTRACEPTIVE USERS

FUNCTIONS

Serge Renaud, Maryvonne Ciavatti, Laurence Perrot, Francois Berthezene, Daniel Dargent ano Pierre Condamin INSERM Unit 63, Bran (France), INSERM Unit 297 and Department of Gynecology and Obstetrics, Hopital E. Herriot, Lyon (France)

ABSTMCT Platelet functions (aggregation, clotting activity), platelet iipid biosynthesis, stcrol composition and phospholipid fatty acids were studied in relation to plasma lipids and fatty acids as well as dietary habits at day 5 and 21 of the menstruai cycle in 30 women having used hormonal contraceptive for several years. The women were after they had taken for studied again on day 21 of the second cycle two months 200 mg vitamin E per day in addition to the contraceptive. At day 21 (after 3 weeks oi hormonal contraceptive) as compared to day increase in the ciotting activity of 5, there was a signiricant platelets and the response to ADP-induced aggregation concomitant with vitamin E administration, a decrease in plasma vitamin C. After platelet activity at day 21 was markedly decreased, being similar to the activity at day 5, with a significant increase in the level of vitamin E in plasma bun platelets. The high response of platelets tc aggregation in women using contraceptives does not seem to be associated with the intake of saturated fat but rather with that of 18:2. The aggregation to throrobin was mostly related to lanosterol biosynthesis and the aggregation to ADP to platelet cholesterol, but not to Plasma lipids or apoproteins. The present piiot study suggests that the platelet hyperaccility of long-term hormonal contraceptive users might be dependent upon a low level of platelet a-tocopherol which can be rapidly overcome by giving a supplement ot vitamin E.

LNTRODUCTION Our previous studies have shown that hormonal contraceptive, mostly the oestrogen moiety (l), administered to female rats (2) and aiso to women (3), increases the platelet susceptibility to aggregation. The enhanced response of platelets was associated both in women and animals with an increase in the platelet lipid biosynthesis especially in the fraction (2,3). lanosterol-dihydrolanosterol Morecver, vitamin E supplementation could markedly reduce in female rats the effect of estrogen on platelet aggregation and lipid biosynthesis (4). Finally, a recent unexpected result was the observation that the untoward effect of hormonal contraceptive on platelets was cbserved only in polyunsaturated fat-fed animais (5). Thus the main purpose of the present study in women was to determine whether: Reprint Lepine,

requests to : S. Renaud, INSERM Unit Case 18, 69675 Bran Cedex, France.

63,

22 avenue

du Loyen

Submitted for publication April 1, 1987 Accepted for publication September 4, 1987

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CONTRACEPTION

a) Vitamin E administration is able to antagonize the platelet noxious effects of hormonal contraception; and b) Diet would influence the platelet reactivity in oral contraceptive users. MATERIAL The project has been INSERM (Institut National

AND METHODS

approved by the Ethical Committee of the de la Sante et de la Recherche Medicale).

Subjects They were 30 healthy women with a mean age of 38 years (range 2942) and long-time contraceptive users, mean 8.5 years (range 0.5-16). The hormonal contraceptives used by the subjects were low ethinylestradiol (30-40 ug)-progestin (levo-norgestrel 0.15-0.20 mg: 18 subjects; norethisterone acetate 1-2 mg: 4 subjects; desogestrel 0.15 mg: 6 subjects). In addition, 2 subjects were taking a pill containing 50 ng ethinylestradiol and 2 mg cyproterone acetate. None or the subjects had any additional medication in the 10 days preceding Seven subjects were smoking more than 10 cigarettes per venesection. day but none oi them had smoked in the mcrning before blood sampling. Previous studies in man have shown that the effect of smoking on piatelets lasted less than one hour (6). Dietary

survey

At each venescccion performed in the laboratory, the women were interviewed by a dietitian, according to the 24-h recall technique. The data reported in Figure 3 are the pooled results of the 3 Calculation of nutrients was performed by- computer with interviews. (7) used in previous studies (8, the help of a food table composition 9). blood

samples

Blood was taken on the 5th (range 4-6) and Zlst day (range 19-23) of the menstrual cycle, and then after 3 weeks of continuous hormonal During the subsequent two cycles, the contraceptive administration. 200 mg subjects were taking, in addition to the oral contraceptive, per day of Vitamin E (Ephynal) at the same time as the contraceptive, usualiy in the evening. Then venesection was done on the 21st day of the second cycle of vitamin E administration. Ninety-two ml of venous blood were taken in the morning in the fasting state. After eliminating the first few drops of blood the foilowing samples were collected: 1. 10 ml, on ice with sodium citrate (0.13 M) (l/9) (adjusted to pH = 7.4 with citric acid) as the anticoagulant, for vitamin E and lipid HDL-cholesterol, (total cholesterol, triglycerides, apoproteins and fatty acids) (centrifugation 1000rC, 20 min at 4°C) determinations. 2. 2 ml for the Stypven time with citrate as anticoagulant stored at 25°C (centrifugation 700xG, 6 min). (PRP) also with 3. 20 ml for aggregation tests in platelet rich-plasma citrate and stored at 25°C. Crntrifugation 130 x G, 10 nin for the PKP; 1000xG, 18 min for the platelet poor plasma-(PPP) as in previous studies (8).

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CONTRACEPTION b. 60 ml for platelet lipid biosynthesis and biochemical determinations with ACD (l/5) (3) at room temperature. The blood was centrifuged (ZOOxG, 20 min) to obtain the PRP, acidified and centrifuged again (700xG, 25 min) to obtain a platelet pellet which was gently resuspended in 5 ml of Tyrode gelatin solution as described elsewhere (3). The platelets were then counted (thrombocoulter, Coultronics) and Tyrode added to obtain log platelets per ml. 3x10' platelets were incubated for lipid biosynthesis evaluation and the remaining platelets were washed twice in Tyrode gelatin without calcium but containing 0.5 mM EGTA (ethylene glycol-bis (fiaminoethyl ether) N,N,N'N'-tetraacetic acid) for biochemical determinations. Coagulation and aggregation tests Tests were performed in duplicate : 1. The Stypven time in PRP which evaluates essentially the clotting activity of platelets (lo), was performed in a recording coaguloaggregometer (8) by adding 0.1 ml of 0.02 M CaClz solution containing Russell's viper venom (l/100,000, Sigma Chemical Co., St. Louis, MO) to 0.1 mi of PKP diluted with 0.1 ml of NaCl (0.95 %). 2. Platelet aggregation on PRP with a platelet count adjusted to 400,00G/~l.,by dilution of PRP with PPP as in previous studies (6, 8). The aggregating agents used were : a. Thrombin (human lyophilized, Sigma Chemical Co., St.Louis, MO) final concentration in PRP 0.04 and 0.1 NIH Unit/ml. b. ADP (from Cquine Muscle, Grade I sodium salt, Sigma Chemical Co.) final concentrntion in PRP, 0.92 x lO-6 M. The extent of aggregation was evaluated as the maximal deflection in cm. Incorporation and lipid fractionation Platelets (3~10~) in 3 ml of Tyrode were incubated for 9@ min at 37'C with 60 01 of 12 tii sodium (14C)-acetate (15 pCi) in aqueous solution. After incubation, platelets were centrifuged, washed, extracted as previously (3). The extract was evaporated under nitrogen and dissolved in 100 JJ~ CHCls/CHsOH (2:1, v/v). Total radioactivity was determined on an aliquot (10 ~1) of the extract and lipid fractions separated by TLC (1,3). The radioactive spots were located by autoradiography (Kodirex, films 2) and identified as previously described (3). Platelet phospholipids and sterols Platelet lipids were extracted by the Folch technique: 1 nL of platelet suspension was extracted by 25 ml chloroform-methanol (2/l) to which was added 150 clgBHT and 10 pg of epicoprostanol as internal standard for sterols determinations. The tube was vigorously stirred for 1 min on a Vortex. After filtration, 5 ml of 0.73 % kaC1 were added and the mixture mixed for 20 seconds. After eliminating the aqueous phase, the chloroform extract was evaporated to dryness under nitrogen. Sterols were dissolved in 4 ml acetone while precipitated phospholipids were washed several times with acetone and dissolved in chloroform-methanol. Total phospholipids were separated by TLC in one dimension on

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CONTRACEPTION silica gel 60 F 254 aluminium foils (Merck, E., Darnstadt, Germany), with the following solvent: petroleum ether-diethyl ether-acetic acid, 9ci:30:% (v/v/v). After methylation, the fatty acids were analysed by GLC with a Hewlett-Packard 5840 A on i2-foot glass columns packed with 8% Supelco 2340 on 100/120 chromosorb W-AW and temperature programming (135-230°C, 1’/min starting at 10 minj. Sterols contained in acetone extract were transferred into d small conic tube and evaporated to dryness unotr nitrogen. loooctane was added (40 ul/lO’ platelets) and 1 ul injected (on into a Carlo Erba (HKGC 5166) GLC with flame ionisation column) detector ar,c fused silica capillary column SE 52 crossbond, 25mx0.32mm ID, 0.35 PC: film. Hydrogen was useu a8 t,arrier gas and the temperature programming was as ioll.ows: hold 1 min at 9U”C, then lO’/min Lo 240°C, l’/min to EHO”C, hold 15 min. Dctrctor temperature was 300°C and ilow rate 1.5 ml/mil:. Plasma

lipids

total lipic iatty acids, after extraction by the Folch Piasr~a meth!,lated by ZFa/CHsOH reagent (Merck) and technique, were purirrtd by ‘TIC as previously uc:,s.r-ihtd (9). They were d~~termined b) plaLeiet phospholipid fatty GLC under t.he same condi: ions as for acids. Plasma Lipid determindticns were pcirormed by enzymatic techniqur6, for tcthl and with the following lzits: PAP ICO (EioMerieux, France) for triglycerides. Apoproterns HUL-cholrstr: ~1; L\ 100 (UioP!erieus) were measured by rsdioimmurrozssay (i 1,12). Statistical

anal*

The results are expressed
vi.tamin E administration, comparison As shown in Figure 1, before of the platelet function test:; at the 5th and 21st day of the cycle in all the tests at day 21. i.e. a signiiicant shows an incr-edse a higher platelets to t irx and response of shortened Stypven signiricant only for the secondary aggregation to ADP. At agl;regation, decrease. in the level of the same time, there v&s a significant vitamin E in pldsma. E with the contr‘*c.eptive, Concomitant administration l;f viLamin significantly prolunged rhe Stypven time at day >i, dnd reduced the response of aggregaLion to thrombin and X:P, signiiicant only for the that at day 21, rhe secondary aggregation to ADP. It can be noted platelet reactivity of womzn with vitamin E is not any different from of viiamin the reactivity at day 5, Also tc be noted is that the level alightly above the normal level I: in plasma 11ad increased to a level at day S. Similar results are shown in Figure 2, but concern only the 10 subjects with the highest platelet reactiviLy. It can be seen that in difference the ploteiet reactivity these women there is more in between cay 21 and day 5. Cancomltant &dministrdtion of vit.anin F ccmpletely normalized the hyperactjvity observed at day 21.

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CONTRACEPTION

0

wtthout

m

with

wtamin

50_

‘P< “P< “‘P< .. . -

05 02 01 .

,D

STYP-CT

(WC)

E

Coagulation

THR (cm)

ADP (P) (cm) Aggregation

ADP (S) (Cm)

VIT

E

(vg/ml) Plasma

Fig. 1. Influence of vitamin E (200 mg/day) on the combined effect of the menstrual cycle and hormonal contraceptive on platelet functions. Tests were performed on the 5th (5) and 21st (21) day of the cycle. Stypven clotting time (STYP-CT); Aggregation to thrombin (THR) (0.1 NIB Unit); and hnP, primary (P) and secondary (S). Significance, paired t-test.

r

W,th ‘P< 05 “P < 02 “‘P < 01

,f

50

20

STYP~CT

HR ml

’ (P) m) ?gatIo”

A

p 6)

VIT E

Fig.2. Influence of vitamin E on platelet functions in the 10 subjects with the highest platelet reactivity. Abbreviations as in Fig. 1. In Table I are shokn the values of several other blood parameters determined at the 3 periods. None is significantly different at any of the periods except the level of alpha-tocopherol in platelets, which was increased after vitamin E administration.

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CONTRACEPTION

To evaluate whether platelet aggregation might be related to the intake of nutrients, the subjects at day 21 were classified in tertile with regard to their response to the secondary platelet aggregation as shown in Figure 3. The primary response to aggregation induced by ADP as well as by thrombin is also parallel to ADP-S. The intake of saturated and of monounsaturated fat is certainly not higher in By contrast, the tertile 3 with the highest -response to aggregation. intake of l&:2 and especiaily the PiS ratio (Figure 3), are significantly more elevated in tertile 3 as compared to 1. Platelet cholesterol is also enhanced in tertile 3.

Table I. Blood 30 women using

parameters determined oral contraceptives

Biosynthesis cpm/109 Plat

Plasma

lipids

at the 3 different

periods

in the

Sterols ug/109 Plat

(mg/dl)

Total Chol Lano

Chol HDL Trig AI

AII

B

Taco

Lano

Chol

lipids

5

21

21E

12925 139

199

166

45

77

124

34

79

0.25

0.17

67

? 818 111

220

254

I2

"4

15

r2

14

I.01

A.02

x2

13050 113

308

152

43

83

118

30

74

0.23

0.17

69

~878 116 1147

~6

?3

-10

~6

12

14

1.02

T.02

+3

134

185

155

45

79

123

30

74

#.$2

0.15

75

~1152 ~17

14200

~24

14

+2

~7

26

-1

13

I.03

1.02

14

Mean z S.E. 5,?i,21E = day of the menstrual cycle, E = after vitamin E administration. Chol = cholesterol Plat = platelets Trig = triglycerides HDL = HDL-cholesterol AI, AII, B = apoproteins Taco = alpha-tocopheroi *p <.Ol, 21E "S 21.

Lano = lanosterol

In Figure 4 are shown, divided in tertile as in Figure 3, cholesterol and lanosterol biosynthesis in addition to several plasma lipid fractions. The only parameter which runs parallel to the aggregation (Figure 3)is the biosynthesis of lanosterol. Iu confirmation of the results obtained from the dietary evaluation, there is habit apparently not a higher level of saturated and monounsaturated fatty acids in the plasma total lipids of tertile 3. By contrast, there is a slight, non-significant increase in 18:2 in tertile 3. Concerning the platelet phospholipid fatty acids determined at day 5 and 21, and also day 21 after vitamin E (results not shown but available on request), there was no significant difference in any of the 3 periods, the fatty acids between not between tertile when classified as in Figs. 3 and 4.

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CONTRACEPTION

(!a/1

:m)

pIat)

;i

1

T

5 T

! 100

50

THR

ADP-S ADP-P AG GI REGATION

MONO DIET

SAT

P/S

Cholest PLAT

Fig. 3. Platelet aggregation and fat intake in 30 women using hormonal contraceptives, classified in tertile with regard to secondary aggregation to ADP (ADP-S). Sat = 14:0 + 16:0 + 18:O Kono = 16:l + 18:l Cholest plat = platelet chclesterol Aggregation to thrombin (THR) (0.04 NIH Unit) Other abbreviations as in Figure 1. Significance unpaired t-test.

mc

Icpm 111 p plat)

500

200

1 1

SAT

Chol

HONO

BIOSYNTHESIS

Fig. using with Chol Sat Mono

-.

18.2

PLASMA

Shol

HDL

LIPIDS

4. Platelet lipid biosynthesis and plasma lipids in 30 women hormonal contraceptive, classified in tertile as in Figure 3, regard to ADP-S values. = cholesterol Lano = lanosterol = 14:0 + 16:0 + 18:0 = 16:l + 18:l HDL = HDL-cholesterol

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CONTRACEPTION

In Figure 5 are shown the results of platelet aggregation at day 5 and day 21 after vitamin E administration during two cycles. The subjects have been classified in the same tertile as in Figures 3 and 4. The results are closely parallel, especially at day 5, with those of day 21 (Figure 3). except that they are much lower.

Mean

t S.E Dav 21 E

Day 5

r

ADP-P

THR

ADP-S THR AGGREGATION

IDP-P

ADP-S

'riatelet aggregation at day 5 and 21E (after vitamin E) in Fig. 5. subjects classified in tertile according to the ADP-S value at day 21. Aggregation to thrombin (0.04 NIH Unit).

In Table I1 are reported some ccrrelation coefficients between various blood parameters, on the pooled results obtained at day 5, Ll Table 11 Correlation coefficients in 30 women _------* contraceptives at days 5 and 21 of their menstrual vitamin E administration and at uay Ll after vitamin E

using oral cycle before

Aggregation THK

ADP-P

Plat

NIP-S

Chol

HDL

Taco

.k-L

Aggr

THR

Plat

Cholesteroi Lanost

PltlS

.35

.z

.f-G

.06

.13

-.14

-.09

-.lJ4

.lO

.19

*** .41

-.03

-.08

.07

.14 *x* .35

.2;

Vit E

-.19

-.04

-.o:

Apo AI

-.12

-.I1

-.I1

**I: .36 X*x .48

-.2$

xxx .51

.32

-.Ui

.iY

*** .70

.05

-.07

Apo AI1 Apo B *p<.05

354

Bios

-.OO *x* .69

xx*

.32

-.I8 -.19 **p<.Ll1

-.16 -.2?

***p<.ooi

.Ol

?I*

n = 90

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CONTRACEPTION and 21E. The results are closely similar to those obtained at day 21 alone, but are more significant. There is a significant positive association, in individuals as well as on a tertile basis (Figure 3) between thrombin and ADP-induced aggregation. Thrombin aggregation was mostly related to the lanosterol biosynthesis and ADP aggregation to cholesterol in platelets. The level of vitamin E in plasma was closely related to the levei of plasma cholesterol, as was platelet vitamin E (a-tocopherol) to platelet cholesterol. Plasma total cholestero! was significantly associated with apoproteins AI, AI1 and mainly R.

DISCUSSION The results of the present pilot study indicate that vitamin E at a moderate dosage (200 mg/day) (probably the most efficient as suggested by preliminary studies) is able to completely normalize platelet functions exacerbated by 21 days of contact with the hormones (endogenous and exogenous). Nevertheless, preliminary studies suggest that without the hormonal contraceptive, it is rather a prolongation of the clotting time (13) and a slight decrease in the susceptibility of platelets to aggregation which is observed at day 21. Thus, although it has to be clearly demonstrated in further studies, the hyperactivity of plateiets at day 21 of the menstrual cycle observed here seems to result mostly from the hormonal contraceptive. Perhaps the most encouraging result is that vitamin E was especially efficient atcounteractingthe platelet noxious effect of the hormonal contraceptive, in subjects with the highest platelet reactivity. Also of interest is that the 2 subjects receiving a pill with a higher ethinylestradiol level (50 up) had their platelet functions (especially primary and secondary aggregation to ADP) decreased by more than 50%. The present results in women reproduce those obtained in female rats in which vitamin E administration inhibited most of the enhanced plateiet response to aggregation induced by ethinylestradiol (4) and the concomitant increase in the platelet ianosterol biosynthesis (4). Here also in women, the lanosterol biosynthesis was decreased by vitamin E, but the result did not reach statistical siguificance. As to the level of vitamin E in plasma in the women studied here (12.1 at day 5 and 11.2 (ug/mi) at day 21), it is comparable to the one found in 26 female students aged LO-23 years (10.9 ug/ml) at different days of their cycle (14) and higher than u-tocopherol (9.9 pg/ml) in 49 normal meu
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also depend on the pill itself since one (Varnoline) used here by a few subjects contained a small amount of vitamin E, apparently sufficient to prevent the lowering in plasma, but not in platelets. The level in carefully washed plateiets was not significantly lower at day 21 than at day 5 although it was significantly increased after vitamin E administration. However the main interest is perhaps the level of a-tocopherol per se in the platelets of the hormonal E administration the level of contraceptive users. Before vitamin u-tocopherol was 0.24 ug/lO' platelets. In the study mentioned above in 49 men (is), the level found by HPLC was 0.35 ng/lOg platelets. The discrepancy could have been due to differences in the technique and sex of the subjects, Nevertheless, at about the same time that we performed the sterol analysis by GLC in the women of the present was determined by the same technique in platelets study, u-tocopherol from 40 women not using hormonal contraceptives (mean age 39 years), but from a different French region (Moselle) before and one year after diet modification (a study not yet published but similar to the one reported recently in men (9)). Before diet modification, the dietary P/S ratio was 0.47 and the level of a-tocopherol (mean ? S.E.) 0.33 I 0.02 ng/lO' platelets. After diet modification (P/S ratio 0.91) a-tocopherol was 0.45 + 0.05 us/log platelets. In the present study, in women taking a hormonal contraceptive, the P/S ratio was 0.40. as Thus, although the women studied in Moselle cannot be considered that in controls for the present study, it seems the perfect contraceptive users and depending on the diet, there is a sizeable a result which has to be confirmed decrease in platelet u-tocopherol, interest is that a by studies with adequate controls. Of additional similar decrease of u-tocopherol in platelets has been reported in type I diabetes (19) although the plasma level of vitamin E was (15), it seems that "platelet vitamin E normal. As already emphasized than could be a better indicator of vitamin E nutritional status especially in hormonal contraceptive users. Thus, plasma tocophrrol", for vitamin E it seems also that there is "an increased requirement during oral contraceptive therapy" (20). Concerning the influence of the dietary habits, it seems that as in fat diet that hormonal female rats (5), it is not on a saturated the response of platelets to aggregation, contraceptives potrntiate but rather on a diet rich in linolric acid. Nevertheless, the present as indications which have to be results can only be considered confirmed by more extensive studies. Also as in animals (2) and previous studies in women (3), the response to thrombin aggregation was closely related to the lanosterol Although lanosterol in platelets was determined in the biosynthesis. present study by capillary column GLC, its level was not increased at day 21 of the menstrual cycle, and was not related to platelet The lack of association might be simply due to the aggregation. technique used, not sensitive enough to determine with the precision needed tiny amounts of lanosterol in platelets. The fact that the platelet response at day 21 is enhanced by the contraceptive but runs parallel to the response at day 5 suggests that from the platelet response before the administration of the contraceptive one could somehow predict the response after its administration. Although those results have to be confirmed by further investigations, they suggest that the study of platelet aggregation before taking the after pill might be of interest to predict possible hyperactivities

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the administration of the pill. This seems of interest to detect the predisposition to thrombosis since platelet aggregation (21) as well as platelet clotting activity (2.2) have been shown to be closely related to the thrombotic tendency in animals. In addition, preliminary results in an ongoing prospective study performed by the British Medical Research Council in Wales indicate that the response of platelets to aggregation by ADP and thrombin is significantly higher in prevalent cases of coronary heart disease (23). Therefore, the protective effect of vitamin E on platelets in hormonal contraceptive users if confirmed, might have direct implications for human health especially since several studies claim that the administration of vitamin E prevents thromboembolic diseases in man (24). ACKNOWLEDGEMENTS This work was supported in part by Grants from INSERM (Reseau Clinique) and from "Groupe Lipide et Nutrition". The authors wish to thank M.C. Attie, C. Benoit, M. Fiers and C. ThCvenon for their skilful technical assistance and J.L. Martin (INSERM U. 265) for statistical evaluation of the results.

REFERENCES 1. Ciavatti, M and Renaud, S. Modification by oral contraceptives in rat of lsC-acetate incorporation into platelet lipids. Hormon Metab Res 11, 441-444 (1979) 2. Ciavatti, M, Michel, G and Renaud, S. Biosynthesis of cholesterol and cholesterol precursors in platelets of female rats treated with oral contraceptives. Biochem Biophys Acta 620, 297-307 (1980) 3. Ciavatti, M. Davenas, E, Blache, D, bonnier, A and Renaud, S. Biosynthesis of platelet lipids in relation to aggregation in women using oral contraceptives. Contraception 25, 629-638 (1982) 4. Ciavatti, M, Davenas, E, Blache, D and Renaud, S. Vitamin E prevents the platelet abnormalities induced by estrogen in rat. Contraception 30, 279-287 (1984) 5. Ciavatti, M, Blache, D and Renaud, S. Dietary fats modulate hormonal contraceptive induced changes in platelet aggregation, composition and lipid biosynthesis in female rats. Nutr Res (in press) (1987) 6. Ranaud, S, Blache, D, Dumont, E and Wiessendanger, T. Platelet function after cigarette smoking in relation to nicotine and carbon monoxide. Clin Pharmacol Therapeut 36, 389-395 (1984). 7. Renaud, S and Attie, M.C. La table de composition des aliments. Astra-Calve, Paris, France, 1986 8. henaud, S, Dumont, E, Goasey, F., Suplisson, A and Thevenon, C. Platelet functions in relation to dietary fats in farmers from two regions of France. Thromb Haemost 40, 518-531 (1979) 9. Renaud, S, Godsey, F, Dumont, E, Thevenon, C, Ortchanian, E and Martin, JL. Influence of long-term diet modification on platelet function and composition in Moselle farmers. Amer .I Clin Nutr 43: 136-150 (1986)

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10. Renaud, activity

S, Gautheron, P and Rosenstein, H. Platelet factor 3 in washed platelets. Thromb. Diath. Haemorrh. 30, 557-566

11. Karlin, (lY73) IB, Juhn, DJ, Scanu, AM. and Rubenstein, AH. Measurement of serum apolipoprotein B by radioimmunoassay. Europ J Clin Invest 8: 19-26 (1978) 12. Perrot, I, and Berthezene, F. Dosage radioimmulogique des apolipoproteines AI et AII. Lyon Medical 246, 441-445 (1481) 13. Recaud, S. The recalcification plasma clotting time. A valuable general clotting test in man and rats. Canad .I. Physiol Pharmacol 47, 689-6Y3 (1969) 14. Wittitig, LA and Lee, L. Dietary levels of vitamin E and polyunsaturated fatty acids ano plasma vitamin E. Amer .I Clin Nutr 28, 571-576 (1975) Krezowski, t&i 15. Vatassery, CT, and Eckfeldt, JH. Vitamin E concentration in human blood plasma and platelets. Amer J Clin Nutr 37, 1020-1024 (1983) 16. Aftergood, L, Alexander, AR and Alfin-Slater, RB. Effect of oral lipoproteins, and contraceptives on plasma cholesterol u-tocopherol levels in young women. Nutr Kept Inter 11, 295-304 (1975) 17. Tangeney, CC arid Driskell JA. Vitamin E, status of young women on 49'1-512 oral cotltraceptives. Contraception 17, combined-type (lY7bj i8. Horwitt, AK, Harvey, CC and Dahm, 01. .Jr. Relationship between levels of blood lipids, vitamins C, A, and E, serum copper and urinary excretions of tryptophan metabolitrs in compounds, women taking or&l contraceptive therapy. Amer J Clin Nutr 28, 403-41: (1975) S, O'Dorisio, TM and Panganamala. RV. 19. Karpen, CW, Cataland, Interrelation of platelet vitamin E and thrumboxane synthesis in Type 1 diabetes mellitus. Diabetes 33, 239-243 (1984) and Alfin-Slater, RB. Grel contraceptive-o20. Aftergood, L tocopherol interrelationships. Lipids 9, 91-96 (1973) 21. Renaud, S, Kuba, K, Goulet, C and Allard, C. Relationship between of platelets and platelet aggregation in fatty-acid composition to thrombosis. Circulat Res 26, 553-564 rat and man. Erlation (1970) induced by hyperli22. Renaud, S and Lecompte, F. hypercoagulability pemia in rat, rabbit and man. Role of platelet factor 3. Circulat Res 27, 1003-1011 (1970). function and 23. ‘iarnell, JWG, Elwood, PC and Renaud, S. Platelet ischaemic heart disease in the Caerphilly study: preliminary results. Biblthca Nutr. Dieta 40, 19-27, 1987. disease 24. Kanofsty, JD and Kanofsty PB. Prevention oi thromboembolic by vitamin E. New Engl J Pled 305, 173-174 (1981).

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