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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1939-1946

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1939-1946.)
© 1997 American Heart Association, Inc.

Articles

Folic Acid Deficiency Enhances Oral Contraceptive-Induced Platelet Hyperactivity

Philippe Durand; Michel Prost; ; Denis Blache

From INSERM CJF 93-10, Laboratoire de Biochimie des Lipoprotéines, Faculté de Médecine, Université de Bourgogne, Dijon, France (P.D., D.B.); and CEDRA, Centre Européen de Recherches et Analyses, Couternon, France (M.P.).

Correspondence to Denis Blache, PhD, INSERM CJF 93-10, Laboratoire de Biochimie des Lipoprotéines, Université de Bourgogne, 7, boul. Jeanne d'Arc, 21033 Dijon, France. E-mail dblache{at}satie.u-bourgogne.fr


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
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Abstract In previous studies conducted in female rats and in women, oral contraceptives (OC) were found to induce a platelet hyperactivity that was related to an oxidative stress. Because cases of megaloblastic anemia have been reported to occur in women taking OC, these treatments are suspected of depleting folate stores. In the study presented herein, which was conducted in rats, we sought to determine the influence of dietary folic acid deficiency (FD) on the thrombogenicity of OC. Animals were fed for 6 weeks with either a folic acid-deficient diet (250 µg/kg folic acid) or a control diet (750 µg/kg). One-half of the animals in each group were treated with OC (ethinyl estradiol plus lynestrenol). FD and OC individually potentiated platelet aggregation in response to thrombin and ADP and the release and metabolism of arachidonic acid, in particular, the biosynthesis of thromboxane. These platelet activities were further enhanced in animals given both the folic acid-deficient diet and the OC treatment. In addition, FD enhanced the pro-oxidant state in OC-treated rats characterized by (1) a fall in platelet and plasma n-3 fatty acids, (2) an increase in plasma lipid peroxidation products such as conjugated dienes, lipid peroxides, and thiobarbituric reactive substances, (3) a rise in ex vivo erythrocyte susceptibility to free radicals. Moreover, we found that OC treatment led to a reduction of plasma and erythrocyte folate concentrations associated with a moderate hyperhomocysteinemia. Under our experimental conditions, we did not find significant synergistic effects between OC and FD. We propose that, although the untoward effects associated with the OC treatment may not primarily be dependent on FD, the folic acid deficiency magnified OC-induced oxidative stress, which resulted in platelet hyperactivity by elevating the pro-oxidant homocysteine plasma concentration. Despite the limitations of this animal model, the data of the present study suggest that in addition to cigarette smoking, inadequate folic acid intake might predispose those taking OC to vascular thrombosis.


Key Words: oral contraceptives • homocysteine • folic acid • oxidative stress • platelet function


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
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Epidemiologic studies of atherosclerotic patients have shown that mild to moderate hyperhomocysteinemia is an independent risk factor for vascular occlusion.1 2 3 4 Plasma folate concentration was found to be inversely correlated with plasma homocysteine3 4 5 and related to the incidence of occlusive diseases.4 Moreover, supplementation with folic acid has been found to normalize hyperhomocysteinemia.2 3 6 With regard to thrombosis, we have recently shown in animal studies that FD can potentiate platelet activation. This could be related to lipid peroxidation induced by hyperhomocysteinemia.7 However, further studies are required to document, in humans, the efficacy of folic acid supplementation on atherothrombosis reduction.

An increased risk of thromboembolic diseases, myocardial infarction, and thrombotic stroke has been formally demonstrated in women taking OC, although the risk tended to diminish with the use of low doses of hormonal steroids.8 9 During the past 25 years, various studies have attempted to find a basis for the thrombogenicity of OC. Beside the impact of OC on lipid and lipoprotein metabolism, a shift toward a hypercoagulable state has been reported.10 11 12 An increase in the plasma level of various clotting factors, especially fibrinogen and factor VII, a decrease in the plasma anticoagulant factors antithrombin III and protein C, and an altered fibrinolytic system have been emphasized. In keeping with these prothrombotic effects, platelet hyperactivity has been shown to occur in women and in rats taking OC.13 14 In a recent study conducted in OC-treated female rats, we found that such an alteration might be related to the occurrence of an oxidative stress, which enhanced platelet thromboxane synthesis.15

OC and other drugs have been shown to change the requirements for folic acid.16 17 Several investigations have shown decreased serum and red blood cell folate concentrations in those taking OC, and cases of megaloblastic anemia related to folate deficiency have been reported.16 18 Although there are theoretic and experimental indications that the increased cardiovascular risk in those taking OC might be mediated by hyperhomocysteinemia,19 the influence of folates on OC-induced prothrombotic effects has not yet been investigated.

The aim of the present study was to determine in a female rat model whether FD could enhance an OC-induced prethrombotic state in a female rat model and whether this state was related to impaired folate metabolism. We report that the hyperhomocysteinemia associated with the dietary FD amplified the platelet hyperactivity caused by OC intake and that the OC-induced FD might be secondary to an oxidative stress.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
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Animals and Diets
Female Sprague-Dawley rats (Charles River), weighing 210 to 230 g, were housed in groups of three rats in wire-bottomed, stainless steel cages to minimize coprophagy, in a well ventilated room maintained at 22°C, on a 12-hour light-dark cycle. They were pair-fed daily with 20 g of food per rat and were permitted free access to water. The standard semi-synthetic diet used was composed (purchased from USB, in weight %) of vitamin-free casein (15), vitamin mixture without folic acid (1), salt mixture (1.8), sawdust (10), starch (22), glucose (20), fructose (9), butter (8), vegetalin (1.2), rapeseed oil (7), and sunflower oil (5). The dietary fat consisted of (by weight) 53.8% saturated fatty acids, 26.6% monounsaturated fatty acids, and 19.6% polyunsaturated fatty acids. To evaluate the influence of FD on the thrombogenicity of OC, rats fed with a folic acid-deficient diet were compared with rats fed with a control diet (groups: FD and CTL, respectively) and in each group, one-half of the animals were treated with OC (groups: FD-OC and CTL-OC, respectively). The control diet contained 750 µg/kg folic acid, whereas the deficient diet only contained 250 µg/kg folic acid, which represented 7.5 µg and 2.5 µg of folic acid for 100 g of body weight per day, respectively. These two diets also contained 0.5 weight % of succinylsulfathiazole (Fluka) to avoid bacterial proliferation and subsequent folate neoproduction.20 Hormonal treatment was performed by mixing the estrogen and progestogen with the dietary fats. The preparation supplied 10 µg of ethinyl estradiol and 50 µg of lynestrenol per 100 g of body weight per day. Because of a difference in the sensitivity of the rat model, it was necessary to increase the estrogen dose to approximately 100 times compared with that for women. Analyses were performed after 6 weeks of feeding.

Platelet Preparation and Aggregation
Platelets were prepared from blood drawn from rats fasted overnight into syringes containing 1 volume of anticoagulant (38 mmol/L citric acid; 75 mmol/L sodium citrate; 136 mmol/L glucose) for 3 volumes of blood. Aggregation experiments were performed as previously described.15 21 Briefly washed platelets were adjusted to a count of 0.35x109/mL in a Ca2+-free Tyrode's buffer (pH 7.4), and 0.5 mL of the platelet suspension was dispensed in cuvettes and placed in a turbidimetric coaguloaggregometer. After a 1-minute incubation with Ca2+ (final concentration, 0.3 mmol/L), platelets were stimulated with 0.04 IU/mL thrombin or 0.7 µmol/L ADP (Sigma).

Platelet Arachidonic Acid Metabolism
Platelet labeling and extraction of arachidonate metabolites were performed as previously described.15 21 Briefly, washed rat platelets (2x109) were labeled with 3.75 µCi of [1-14C]arachidonate (55 mCi/mmol, Amersham). After washing in Tyrode's buffer, an aliquot of cell suspension was used for determination of arachidonate incorporation in platelet phospholipids, and radiolabeled platelets were stimulated for 2 minutes with thrombin (final concentration, 0.2 IU/mL) in the presence of 0.5 mmol/L Ca2+. Arachidonate metabolites were directly extracted by ethylacetate after acidification and were analyzed by thin layer chromatography in CHCl3/CH3OH/CH3COOH/H2O (90:8:1:0.8 by volume). The radioactive spots detected by autoradiography were then assessed for radioactivity. The synthesis of thromboxane A2 in thrombin-stimulated platelets was also determined by measuring its stable hydrolysis product, thromboxane B2, by RIA (Amersham) as described.22

Fatty Acid Analysis
Plasma and platelet samples were stored at -20°C under argon for fatty acid analysis. Plasma and platelet lipids were extracted according to Folch et al,23 and the extracts were transmethylated with BF3/CH3OH according to Morrison and Smith.24 The fatty acid methyl esters were separated by capillary gas liquid chromatography with temperature programming using a Di 200 chromatograph (Delsi, France) equipped with a SP2340 column (Supelco) as described.15

Analyses of Lipid Peroxidation Products and Erythrocyte Susceptibility to Oxidation
Plasma lipid peroxidation was measured as thiobarbituric acid reactive substances and expressed in malondialdehyde equivalents as described.22 25 Conjugated dienes were evaluated at 234 nm on plasma lipid extracts solubilized in heptane.26 Lipid peroxides were measured at 365 nm by a iodometric method.27 The erythrocyte susceptibility to oxidative stress was evaluated in vitro by submitting a 15% hematocrit suspension of erythrocytes washed in 154 mmol/L NaCl to free radicals produced from the thermal decomposition of 100 mmol/L 2,2'-azo-bis (2-amidinopropane) HCl.15 28 The time course of hemolysis was monitored by spectrophotometry at 405 nm of the released hemoglobin and fitted by computer analysis (Prism, GraphPad Software, San Diego, Calif). The 50% hemolysis time (HT50% in minutes) is referred to as the erythrocyte susceptibility to free radicals.

Folate and Aminothiol Assay
Blood and plasma samples mixed with 0.2% sodium ascorbate were stored at -80°C for folate analysis. Total folates were measured using a radioimmunoassay (Simultrac kit, Becton Dickinson Immunodiagnotics, Grenoble, France). Total plasma homocysteine, cysteine, and glutathione concentrations were determined by high-performance liquid chromatography performed in isocratic conditions with a Beckman Gold system equipped with a reverse-phase column (C18 ODS, 150x4.66 mm, Beckman) and a fluorescence detector using N-acetylcysteine as the internal standard.29

Statistical Analysis
Data are expressed as mean±standard deviation (SD) from n=3 to 9 rats per group. The main effects of OC treatment and of the FD or their interactions (OCxFD) were determined using two-way analysis of variance (ANOVA) performed with the Statistical Analysis System developed by the SAS Institute.30 When the interactions were found to be statistically significant, the significance of the main effects of OC and FD was not taken into consideration. After ANOVA, significant differences (P<0.05) between means were analyzed by multiple comparisons using the Honestly Significant Difference test of Tukey.


*    Results
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 *Results
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Weekly body weight measurements during the 6 weeks of feeding showed that OC-treated rats gained less weight than controls, irrespective of the folic acid content of the diet. The difference between all OC-treated animals and controls reached 15%. No difference in body weight gains were observed between animals fed the folic acid-deficient and the control diet.

Folate Concentrations
The effectiveness of the reduced folic acid intake on the folate status was confirmed by the highly significant decrease in both plasma and erythrocyte folate levels (Table 1Down). As expected, OC treatment significantly reduced the plasma and the erythrocyte folate concentrations. Although OC tended to decrease the folate concentrations of the folic acid-deficient group further than those of the control group (-30% versus -16%, for plasma and -23% versus -18% for erythrocytes, respectively), no significant interaction between the dietary folic acid supply and OC treatment was evidenced by two-way ANOVA analyses (Table 1Down).


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  		Table 1. OC Effects on Folate Status in Relation to Dietary Folic Acid

Platelet Aggregation
Similarly, OC and FD both potentiated platelet aggregation in response to ADP and thrombin (Fig 1ADown and 1BDown, respectively). In addition, dietary deficiency of folic acid significantly enhanced OC-mediated platelet hyperaggregation (FD-OC versus CTL-OC rats). Moreover, the increase in ADP-evoked platelet activity after OC treatment was significantly dependent on the dietary folic acid content. Indeed, we observed a higher percent increase in the platelet aggregation of deficient animals than in controls (+49% versus +21%). With regard to the platelet response to thrombin, the OC-mediated increase in platelet aggregation was not significantly dependent on the folic acid supply (Fig. 1Down).



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  		Figure 1. ADP-induced (A) or thrombin-induced (B) platelet aggregation in oral contraceptive-treated rats receiving a FD or a CTL diet. Washed platelets, incubated with 0.3 mmol/L Ca2+, were stimulated with 0.7 µmol/L ADP or 0.04 IU/mL thrombin. Results (mean±SD, n=6) represent the amplitude of the recorded aggregation curve in millimeters. Bars with different symbols are significantly different (P<0.05) as determined by the Tukey Honestly Significant Difference method after two-way ANOVA. Two-way ANOVA has revealed an interaction between the OC treatment and the folic acid deficiency (OCxFD) for ADP-evoked platelet aggregation (P=0.0001). No significant OCxFD interaction but significant main effects (P<0.0001) were found for thrombin-induced platelet aggregation with both OC and FD.

Platelet Arachidonate Incorporation and Metabolism
OC and FD had dissociated effects on [1-14C]arachidonate incorporation into washed platelet phospholipids (Table 2Down). A decrease in the arachidonate incorporation was found after OC treatment in both control and folic acid-deficient rats. Conversely, FD induced a rise in this incorporation in both control and OC-treated rats. However, the OC-induced decrease in arachidonate incorporation was significantly dependent on the folic acid supply. A 54% decrease in incorporated arachidonate was observed in the deficient group compared with the 35% decrease observed in the control group.


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  		Table 2. Arachidonic Acid Metabolism in Thrombin-Stimulated Platelets

The thrombin-induced release of arachidonic acid from platelet phospholipids showed that both treatment with OC and FD decreased the radioactivity remaining in platelet phospholipids and increased the platelet-free arachidonic acid and its conversion into cyclo-oxygenase and lipoxygenase products (Table 2Up). The OC-induced elevation of arachidonate release and metabolism was amplified by FD but was independent of the folic acid content of the diet except for the 12-HETE increment, which was reduced in the deficient animals compared with controls. The RIA results of the thrombin-evoked platelet thromboxane synthesis (Fig 2Down) confirm the above data. Platelets of OC-treated animals produced significantly more TXB2 than those of controls, independently of the folic acid content of the diet, whereas FD enhanced the increased thromboxane synthesis brought about by OC intake.



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  		Figure 2. The thrombin-induced thromboxane B2 secretion by platelets from OC rats according to the folic acid supply. Washed platelets were stimulated for 5 minutes at 37°C with 0.06 IU/mL thrombin. Thromboxane B2 was then assayed in the incubation medium using a RIA and results were expressed as pmol/108 platelet (plt) for 5 minutes (n=3). Bars with different symbols are significantly different (P<0.05) as determined by the Tukey Honestly Significant Difference method. Two-way ANOVA revealed significant main effects (P<0.0001) for both OC treatment and FD without interaction between OC and FD.

Plasma and Platelet Fatty Acid Composition
OC and FD had similar effects on the percent composition of most plasma polyunsaturated fatty acids (Table 3Down). OC strongly decreased the plasma unsaturation index in both control and deficient rats by affecting mainly arachidonic acid (20:4 n-6) and the long chain n-3 PUFA, eicosapentaenoic (20:5), docosapentaenoic (22:5), and docosahexaenoic acids (22:6). Despite an increase in adrenic acid (22:4 n-6), FD reduced the plasma unsaturation index by diminishing circulating long chain n-3 PUFA and arachidonate and, thus, enhanced the OC-induced changes. However, the disappearance of these PUFA after OC treatment tended to be lower in folate-deficient rats than in controls. Significant interactions between the FD and OC effects were found for plasma 20:4 n-6 and 20:5 and 22:6 n-3.


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  		Table 3. Plasma Fatty Acid Composition of OC-Treated Rats Fed a FD Diet

OC treatment drastically increased the circulating levels of linoleate (18:2 n-6), {alpha}-linolenate (18:3 n-3), monounsaturated fatty acids (palmitoleic (16:1 n-7), oleic (18:1 n-9), and cis-11-eicosenoic acids (20:1 n-9) and the saturated fatty acids, myristic (14:0) and palmitic acids (16:0), although the relative content of stearic acid (18:0) was decreased. Whereas FD did not significantly affect the plasma composition of total saturated and monounsaturated fatty acids, it increased the proportions of 18:2 n-6 and 18:3 n-3, thus enhancing the effects of OC. Interaction tests indicated that the OC-induced rises in these PUFA were independent of the folic acid supply.

Modifications of the platelet fatty acid pattern were different from those of plasma (Table 4Down). OC and FD similarly affected the cell fatty acids. Despite the decrease in palmitoleic induced by OC, platelet saturated and monounsaturated fatty acids were not modified, whereas the PUFA profile was changed. A marked elevation in arachidonate associated with a marked reduction in the platelet eicosapentaenoate content was observed in all OC-treated rats compared with controls. In contrast to plasma, these changes contributed to a strong decrease in the platelet 20:5/20:4 ratio, and this also held true for the n-3/n-6 PUFA ratio. This redistribution between n-3 and n-6 fatty acids was markedly enhanced after FD. No significant interaction was found between FD and OC on the platelet content in arachidonate and total n-3 fatty acids.


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  		Table 4. Platelet Fatty Acid Composition of OC-Treated Rats Fed a FD Diet

Lipid Peroxidation Products
The plasma-conjugated dienes, lipid peroxides, and TBARS were increased after OC treatment and after folate deficiency (Table 5Down). FD amplified the increase in conjugated dienes and TBARS induced by OC (FD-OC versus CTL-OC rats). In addition, the highest lipid peroxide concentration was observed in OC-treated rats fed with the folic acid-deficient diet. Statistical analyses revealed that the rise in the peroxidation products was significantly independent of the dietary folic acid.


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  		Table 5. Plasma Peroxidation Products of OC-Treated Rats Given a FD Diet

Erythrocyte Susceptibility to Free Radicals
The susceptibility to free radical-induced oxidation of erythrocytes from the control and OC-treated animals fed the control or the folic acid-deficient diet was investigated in vitro. Fig 3Down shows that OC shortened the HT50% of erythrocytes of control rats (CTL-OC versus CTL: 71.47±2.85 minutes versus 98.35±4.27 minutes, respectively). FD also significantly shortened the HT50% of red blood cells from control rats (FD versus CTL: 76.20±4.45 minutes versus 98.35±4.27 minutes, respectively), and it further amplified the OC-induced elevation in erythrocyte susceptibility to free radicals (FD-OC versus CTL-OC: 64.35±3.76 minutes versus 71.47±2.85 minutes, respectively).



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  		Figure 3. Susceptibility to free radicals of washed erythrocytes from OC-treated rats in relation to dietary FD. The time course of erythrocytes hemolysis was plotted (in percent) and fitted by computer analysis using a sigmoid. Results are expressed as 50% hemolysis time in minutes (n=6). Bars with different symbols are significantly different (P<0.05) as determined by the Tukey Honestly Significant Difference method after two-way ANOVA, which revealed an interaction between OC and FD (P=0.0001).

Homocysteine and Other Aminothiol Concentrations
FD markedly increased total homocysteine and glutathione concentrations in plasma (Table 6Down). In accordance with the overall reduction of both plasma and erythrocyte folate concentrations, OC increased plasma homocysteine. Despite a trend toward a higher increment in homocysteinemia in deficient rats than in controls, no interaction between OC and FD was observed. In addition, our results revealed that OC significantly enhanced total plasma cysteine independently of FD.


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  		Table 6. OC Effects on Plasma Concentrations of Total Aminothiols in Relation to Dietary Folic Acid


*    Discussion
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 *Discussion
down arrowReferences
 
In a previous report, we suggested that platelet hyperactivity, possibly through stimulated free radical-induced arachidonate acid metabolism, might be involved in the known thrombogenic risk of OC intake.15 We were also the first to show that dietary FD potentiated platelet activation.7 The present study was designed to determine whether FD could amplify OC effects and whether an impaired folate metabolism could contribute to the thrombogenicity of OC. We found that feeding rats with a folic acid-deficient diet enhanced OC-induced platelet hyperactivity. Although a rat model has been used with a high dose of estrogen that may not be comparable with that for women, we also observed that OC treatment led to a loss of folates associated with moderate hyperhomocysteinemia. We further suggested that involvement of an impaired folate metabolism in the prothrombotic effects caused by OC might be secondary to the OC-induced oxidative stress, which would initially affect platelet activity.

An effective FD was established by diminishing the intake of folic acid (Table 1Up). This deficiency potentiated platelet aggregation, as previously reported.7 As expected, it further enhanced the OC-induced increase in platelet aggregation in response to ADP and thrombin (Fig 1Up). The mechanism underlying platelet hyperaggregation was then explored as regards platelet eicosanoid synthesis. Stimulation of the arachidonic acid metabolism resulting in increased platelet thromboxane production might be relevant to the potentiating action of FD. Indeed, we found that FD enhanced the increased release and metabolism of membrane-incorporated, radiolabeled arachidonate in OC-treated rats and, in particular, it raised production of TXB2, the stable metabolite of the powerful proaggregating agent TXA2 (Table 2Up). Consistent with a higher thromboxane-dependent platelet aggregation, the amplified rise in thromboxane synthesis induced by FD was confirmed, using a radioimmunoassay of platelet-derived TXB2 (Fig 2Up). In addition, the major change in fatty acid composition of OC-treated rats potentiated by FD was a fall in the plasma and platelet n-3/n-6 fatty acid ratio (Tables 3Up and 4Up). The amplified decrease in the platelet 20:5/20:4 ratio would lead to an increase in the production of TXA2.31 Alternatively, the accentuated plasma increase in linoleate and decrease in arachidonate could impair the vascular production of the potent platelet antagonist prostacyclin (PGI2). This fatty acid redistribution occured in favor of an unbalanced eicosanoid synthesis resulting in platelet hyperactivity.

We also confirmed that FD increased lipid peroxidation and, in parallel, decreased cellular antioxidant defense.7 The observation that the disappearance of n-3 fatty acids, which are highly susceptible to free radical attack, was more pronounced in the plasma and platelets of OC-treated rats given a folic acid-deficient diet than in the animals receiving a control diet suggested that FD increased OC-initiated lipid peroxidation, which previously has been reported to be involved in platelet hyperactivity.15 This idea was reinforced by the finding of the important OC-induced increase in cell-oxygenated products (12-HETE) derived from the enzymatic oxidation of arachidonate to hydroperoxide (12-HPETE; Table 2Up) and in the plasma lipid peroxidation products, TBARS and conjugated dienes (Table 5Up). The significant enhancement of erythrocyte susceptibility to in vitro oxidation observed in OC-treated rats fed the folic acid-deficient diet compared with rats on the control diet also indicated that FD worsened OC-induced impairment of the antioxidant defense system (Fig 3Up). Altogether, these results showed that FD and OC similarly potentiated the platelet hyperactivity related to oxidative stress resulting in the stimulation of arachidonate metabolism and that FD had an additive effect on OC-induced platelet hyperactivity.

The amplification of OC-induced platelet activation by FD could be partly explained by an elevation of plasma homocysteine (Table 6Up), of which the pro-oxidant activities have been often underlined.32 33 34 35 In agreement with this proposed mechanism, studies have reported prothrombotic effects of hyperhomocysteinemia related to its pro-oxidant action. Specifically, an enhanced thromboxane synthesis partly reflecting in vivo platelet activation, which was reduced by antioxidant administration, has been reported in homocystinuric patients.36 An inhibition of PGI2 synthesis by endothelial cells has been found after in vitro endothelial cell incubation with homocysteine. This was probably caused by the generation of hydrogen peroxide.37 In addition, Stamler et al38 suggested that exposure of endothelial cells to homocysteine would impair nitric oxide-mediated inhibition of platelet aggregation. Nitric oxide was also shown, by forming nitroso-thiols, to prevent homocysteine from generating hydrogen peroxide.38 Nevertheless, we could not exclude indirect effects of homocysteine such as an influence on in vivo methylation capacity. FD is known to decrease hepatic S-adenosylmethionine synthesis,32 39 which represents the physiologic methyl donor for the N-methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). In keeping with an altered methylation capacity, we found a 40% increase in liver PE/PC ratio 2 hours after intraperitoneal injection of radiolabeled ethanolamine (data not shown). Although the N-methylation pathway is quantitatively a minor pathway of PC biosynthesis in extrahepatic cells, this pathway may play a role in cell signal transduction by remodeling membrane phospholipid organization and/or membrane bilayer assymetry and, hence, might potentially influence platelet activity.40 Consequently, we propose that FD could worsen the prethrombotic state in OC-treated rats partly by enhancing the circulating concentration of the pro-oxidant homocysteine.

The OC-mediated folate loss and moderate hyperhomocysteinemia reported in our study were associated with an oxidative stress that resulted in platelet hyperaggregation. This finding fits well with the elevated plasma total homocysteine described in OC users with an incidence of vascular occlusion.41 42 Conversely and despite a trend toward low blood folates, no significant elevation in homocysteinemia has been reported in healthy young women taking OC compared with age-matched controls.43 However, under our experimental conditions, statistical analyses of interactions have unexpectedly revealed no synergistic effect between FD and OC, except for ADP-evoked platelet aggregation. Thus, although the huge FD-induced hyperhomocysteinemia could mask the FD-dependent OC action, it might be that the oxidative stress-related OC effects are not a direct consequence of folic acid deficiency. Indeed, an increased lipid biosynthesis, mainly cholesterol and its precursor lanosterol, which was inhibited by a vitamin E supplement, has been reported to be involved in OC-induced platelet hyperactivity.14 We did confirm this enhanced lipid biosynthesis, but under the conditions of our study, this pathway was not affected by folic acid deficiency in platelets of control and OC-treated rats (data not shown).

We, thereby, proposed that the primary action of OC would be to induce an oxidative stress that would generate oxygenated radical species leading directly to modification of cell membranes and lipoproteins and, thereafter, increasing platelet activity as has been demonstrated previously.15 26 44 In favor of this hypothesis, a generation of free radicals by redox cycling of estrogens has been described.45 In addition, we reported previously that the appearance of lipid hydroperoxides in plasma would be the initial event stimulating platelets after OC.25 Furthermore, we showed that vitamin E supplementation in vitro and in vivo normalized the platelet hyperaggregation associated with the stimulation of thromboxane synthesis caused by OC intake.14 15 The OC-induced oxidative stress might secondarily lead to folate depletion that would amplify the untoward effects of OC. Consistent with this suggestion, reactive oxygen species may cleave in vitro N5-methyltetrahydrofolate, the main circulating folate derivatives serving as a methyl donor in cellular homocysteine remethylation to methionine.46

In summary, our data show that by increasing the oxidative stress that stimulated thromboxane synthesis, FD enhanced markedly the OC-induced platelet hyperactivity. In addition, our study documented the decreased folate status and the increased plasma concentration of the pro-oxidant homocysteine in response to OC treatment. This is consistent with a recent report that demonstrated that hyperhomocysteinemia was an additional risk factor for vascular occlusion in women taking OC.42 Thus, although some limitations of our animal model should be taken into account (especially, the high dose of estrogen used) before considering the human situation, it is conceivable that in addition to age and smoking, dietary FD could predispose women who take OC to vascular thrombosis.


*    Selected Abbreviations and Acronyms
 
CTL = control
FD = folic acid deficiency
OC = oral contraceptives
PUFA = polyunsaturated fatty acids
TBARS = thiobarbituric acid reactive substances
TX = thromboxane


*    Acknowledgments
 
This work was partly supported by the Institut National de la Santé et de la recherche Médicale (INSERM), the Conseil Régional de Bourgogne, and the Université de Bourgogne. The financial support from SPIRAL R.D. to Ph. Durand was greatly appreciated. The authors thank Dr J. Davignon and L. J. Fortin (IRCM, Montreal, Canada) for analyses of aminothiol compounds, Dr S. Lussier-Cacan (IRCM) for kindly editing the manuscript, and P. Brunet-Lecompte for expert advice in statistical analyses.

Received February 22, 1996; accepted January 28, 1997.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med.. 1991;324:1149-1155.[Abstract]
  2. Malinow MR. Hyperhomocyst(e)inemia: a common and easily reversible risk factor for occlusive atherosclerosis. Circulation. 1990;81:2004-2006.[Free Full Text]
  3. Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA.. 1992;268:877-881.[Abstract]
  4. Pancharuniti N, Lewis CA, Sauberlich HE, Perkins LL, Go RCP, Alvarez JO, Macaluso M, Acton RT, Copeland RB, Cousins AL, Gore TB, Cornwell PE, Roseman JM. Plasma homocyst(e)ine, folate, and vitamin B-12 concentrations and risk for early-onset coronary artery disease. Am J Clin Nutr.. 1994;59:940-948.[Abstract/Free Full Text]
  5. Selhub J, Jacques PF, Wilson PWF, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA.. 1993;270:2693-2698.[Abstract]
  6. Ubbink JB, Vermaak WJH, Van der Merwe A, Becker PJ. Vitamin B-12, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia. Am J Clin Nutr.. 1993;57:47-53.[Abstract/Free Full Text]
  7. Durand P, Prost M, Blache D. Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids. Atherosclerosis. 1996;121:231-243.[Medline] [Order article via Infotrieve]
  8. Royal College of General Practitioners'. Oral contraception study: mortality among oral-contraceptive users. Lancet.. 1977;2:727-731.[Medline] [Order article via Infotrieve]
  9. Kaplan NM. Cardiovascular complications of oral contraceptives. Annu Rev Med.. 1978;29:31-40.[Medline] [Order article via Infotrieve]
  10. Meade TW. Risks and mechanisms of cardiovascular events in users of oral contraceptives. Am J Obstet Gynecol.. 1988;158:1646-1652.[Medline] [Order article via Infotrieve]
  11. Inauen W, Baumgartner HR, Haeberli A, Straub PW. Excessive deposition of fibrin, platelets and platelet thrombi on vascular subendothelium during contraceptive drug treatment. Thromb Haemost.. 1987;57:306-309.[Medline] [Order article via Infotrieve]
  12. Beller FK, Ebert C. Effects of oral contraceptives on blood coagulation: a review. Obstet Gynecol Surv.. 1985;40:425-436.[Medline] [Order article via Infotrieve]
  13. Ciavatti M, Davenas E, Blache D, Monnier A, Renaud S. Biosynthesis of platelet lipids in relation to aggregation in women using oral contraceptives. Contraception. 1982;25:629-638.[Medline] [Order article via Infotrieve]
  14. Ciavatti M, Davenas E, Blache D, Renaud S. Vitamin E prevents the platelet abnormalities induced by estrogen in rat. Contraception. 1984;30:279-287.[Medline] [Order article via Infotrieve]
  15. Durand P, Blache D. Enhanced platelet thromboxane synthesis and reduced macrophage-dependent fibrinolytic activity related to oxidative stress in oral contraceptive-treated female rats. Atherosclerosis. 1996;121:205-216.[Medline] [Order article via Infotrieve]
  16. Shojania AM. Oral contraceptives: effects on folate and vitamin B12 metabolism. Can Med Assoc J.. 1982;126:244-247.[Abstract]
  17. Ueland PM, Refsum H. Plasma homocysteine, a risk factor for vascular disease: plasma levels in health, disease, and drug therapy. J Lab Clin Med.. 1989;114:473-501.[Medline] [Order article via Infotrieve]
  18. Lindenbaum J, Whitehead N, Reyner F. Oral contraceptive hormones, folate metabolism, and the cervical epithelium. Am J Clin Nutr.. 1975;28:346-353.[Abstract/Free Full Text]
  19. McCully KS. Homocystine, atherosclerosis and thrombosis: implications for oral contraceptive users. Am J Clin Nutr.. 1975;28:542-549.[Free Full Text]
  20. Akesson B, Fehling C, Jagerstad M, Stenram U. Effect of experimental folate deficiency on lipid metabolism in liver and brain. Br J Nutr.. 1982;47:505-520.[Medline] [Order article via Infotrieve]
  21. Blache D, Ciavatti M. Rat platelet arachidonate metabolism in the presence of Ca2+, Sr2+ and Ba2+: studies using intact platelets and semi-purified phospholipase A2. Biochim Biophys Acta.. 1987;921:541-551.[Medline] [Order article via Infotrieve]
  22. Polette A, Blache D. Effect of vitamin E on acute iron load-potentiated aggregation, secretion, calcium uptake and thromboxane biosynthesis in rat platelets. Atherosclerosis. 1992;96:171-179.[Medline] [Order article via Infotrieve]
  23. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem.. 1957;226:497-509.[Free Full Text]
  24. Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethyl acetates from lipids with boronfluoride methanol. J Lipid Res.. 1964;5:600-608.[Abstract]
  25. Ciavatti M, Blache D, Renaud S. Hormonal contraceptive increases plasma lipid peroxides in female rats: relationship to platelet aggregation and lipid biosynthesis. Arteriosclerosis. 1989;9:84-89.[Abstract/Free Full Text]
  26. Blache D. Involvement of hydrogen and lipid peroxides in the acute tobacco smoking-induced platelet hyperactivity. Am J Physiol.. 1995;268:H679-H685.[Abstract/Free Full Text]
  27. El-Saadani M, Esterbauer H, El-Sayed M, Goher M, Nassar AY, Jürgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res.. 1989;30:627-630.[Abstract]
  28. Blache D, Prost M. Free radical attack: biological test for human resistance capability. In: Ponnamperuma C, Gehrke CW, eds. Proceedings of the IX College Park Colloquium on Chemical Evolution: A Lunar-Based Chemical Analysis Laboratory (LBCAL). Washington: NASA; 1992:82-98.
  29. Durand P, Fortin L-J, Lussier-Cacan S, Davignon J, Blache D. Hyperhomocysteinemia induced by folic acid deficiency and methionine load—applications of a modified HPLC method. Clin Chim Acta.. 1996;252:83-93.[Medline] [Order article via Infotrieve]
  30. SAS Institute Inc. SAS User's Guide, Version 6. Carry, NC: SAS Institute Inc.; 1989.
  31. Iritani N, Narita R. Changes of arachidonic acid and n-3 fatty acids of phospholipid classes in liver, plasma and platelets during dietary fat manipulation. Biochim Biophys Acta.. 1984;793:441-447.[Medline] [Order article via Infotrieve]
  32. Munday R. Toxicity of thiols and disulphides: involvement of free-radical species. Free Radic Biol Med.. 1989;7:659-673.[Medline] [Order article via Infotrieve]
  33. Starkebaum G, Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest.. 1986;77:1370-1376.
  34. Parthasarathy S. Oxidation of low-density lipoprotein by thiol compounds leads to its recognition by the acetyl LDL receptor. Biochim Biophys Acta.. 1987;917:337-340.[Medline] [Order article via Infotrieve]
  35. Heinecke JW, Kawamura M, Suzuki L, Chait A. Oxidation of low density lipoprotein by thiols: superoxide-dependent and -independent mechanisms. J Lipid Res.. 1993;34:2051-2061.[Abstract]
  36. Di Minno G, Davì G, Margaglione M, Cirillo F, Grandone E, Ciabattoni G, Catalano I, Strisciuglio P, Andria G, Patrono C, Mancini M. Abnormally high thromboxane biosynthesis in homozygous homocystinuria: evidence for platelet involvement and probucol-sensitive mechanism. J Clin Invest.. 1993;92:1400-1406.
  37. Panganamala RV, Karpen CW, Merola AJ. Peroxide mediated effects of homocysteine on arterial prostacyclin synthesis. Prostaglandins Leukot. Med.. 1986;22:349-356.[Medline] [Order article via Infotrieve]
  38. Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M, Singel D, Loscalzo J. Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest.. 1993;91:308-318.
  39. Miller JW, Nadeau MR, Smith J, Smith D, Selhub J. Folate-deficiency-induced homocysteinaemia in rats: disruption of S-adenosylmethionine's co-ordinate regulation of homocysteine metabolism. Biochem J.. 1994;298:415-419.
  40. Zwaal RFA, Comfurius P, Bevers EM. Mechanism and function of changes in membrane-phospholipid asymmetry in platelets and erythrocytes. Biochem Soc Trans.. 1993;21:248-253.[Medline] [Order article via Infotrieve]
  41. Wong PWK, Kang SS. Accelerated atherosclerosis. Am J Med.. 1991;84:1093-1094.
  42. Beaumont V, Malinow MR, Sexton G, Wilson D, Lemort N, Upson B, Beaumont JL. Hyperhomocyst(e)inemia, anti-estrogen antibodies and other risk factors for thrombosis in women on oral contraceptives. Atherosclerosis. 1992;94:147-152.[Medline] [Order article via Infotrieve]
  43. Brattström L, Israelsson B, Olsson A, Andersson A, Hultberg B. Plasma homocysteine in women on oral oestrogen-containing contraceptives and in men with oestrogen-treated protatic carcinoma. Scand J Clin Lab Invest.. 1992;52:283-287.[Medline] [Order article via Infotrieve]
  44. Ambrosio G, Golino P, Pascucci I, Rosolowsky M, Campbell WB, DeClerck F, Tritto I, Chiariello M. Modulation of platelet function by reactive oxygen metabolites. Am J Physiol.. 1994;267:H308-H318.[Abstract/Free Full Text]
  45. Liehr JG, Roy D. Free radical generation by redox cycling of estrogens. Free Radic Biol Med.. 1990;8:415-423.[Medline] [Order article via Infotrieve]
  46. Shaw S, Jayatilleke E, Herbert V, Colman N. Cleavage of folates during ethanol metabolism: role of acetaldehyde/xanthine oxidase-generated superoxide. Biochem J.. 1989;257:277-280.[Medline] [Order article via Infotrieve]



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