Vitamin E and omega-3 fatty acids: effectors of platelet responsiveness.

This overview focuses on recent evidence in support of vitamin E and omega-3 fatty acids as positive effectors of cardiovascular health through their ability to influence signaling processes in platelets. Special emphasis is placed on vitamin E actions independent of antioxidant activity. The sometimes discordant findings among observational studies, clinical trials, and in vitro cellular studies have been scrutinized for clues to explain possible mechanisms of actions and suggest strategies for future work to define appropriate intakes of these two nutrients.

Population doubling time, phosphatase activity, and hydrogen peroxide generation in Jurkat cells.

Differences in growth characteristics, phosphatase activity, and hydrogen peroxide generation in two clones of a T-cell leukemic line are described in this communication. Wurzburg cells had significantly shorter population doubling times compared with the parental Jurkat cells (16.6 +/- 2.0 h and 20.7 +/- 2.2 h, respectively; mean +/- SD, p < .0001, n = 20). In addition, total phosphatase activity was significantly decreased (p < .006) and hydrogen peroxide production was significantly increased (p < .002) in Wurzburg cells compared to Jurkat cells. That the cell line with the faster growth rate should have these latter two properties is entirely consistent with the positive effects of increased kinase activity and hydrogen peroxide on proliferative cellular responses in T cells. As originally described, Wurzburg cells were distinguished from Jurkat cells by their lack of CD45, a membrane protein tyrosine phosphatase, and their positive response to hydrogen peroxide-stimulation of NF-kappaB activation. We propose that these two clones, with their distinguishing characteristics, can be used to advantage in experiments designed to study the effects of antioxidants on signaling pathways that control cell life and death.

Dietary soy isoflavones inhibit activation of rat platelets.

Isoflavones (isoflavonoids) have been proposed to be the active compounds that contribute to decreased mortality from chronic diseases in populations that consume large amounts of soy products. Diets containing soy protein with and without isoflavones were fed to rats to determine if these compounds could exert in vivo effects on physiologic markers of platelet activation. Three methods were employed to monitor platelet activation: measurement of electronic mean platelet volume, which is an indicator of shape change; monitoring of collagen-induced production of reactive oxygen signals (hydrogen peroxide); and determination of increases in phosphorylation of protein tyrosine residues after collagen stimulation. Apparent volumes were significantly smaller for platelets from rats fed isoflavones, suggesting that these platelets were in a more disc-like, quiescent state compared with platelets from rats fed the isoflavone-reduced diet (means +/- SEM, 5.37 +/- 0.08 vs. 5.70 +/- 0.06 fL, n = 6/group, P < 0.008). Results from the other functional tests were consistent with this finding. Platelet production of hydrogen peroxide was found be significantly lower 1, 3, and 5 minutes after addition of collagen for rats fed isoflavones versus rats fed the isoflavone-reduced diet (n = 6/group, P < 0.004). Phosphorylated tyrosine residues in platelet proteins after stimulation also were shown to be significantly lower in the platelets exposed to dietary isoflavones (n = 5/group, P < 0.047). These combined results indicate that soy isoflavones can alter early-event signaling networks that result in less activated platelets and may partially explain the beneficial effects of dietary soy against human heart disease.

Design criteria: tests used to assess platelet function.

A brief overview of platelet function tests used in the past to assess relations between dietary fatty acids and thrombogenicity reveals problems that need to be recognized and addressed before planning future studies. Implementation of new strategies that integrate new technologies with measures of two or more markers of platelet activity may be more successful in predicting the thrombotic potential of dietary fatty acids.

Effect of oral vitamin B6 supplementation on in vitro platelet aggregation.

A randomized, double-blind study was conducted with 12 healthy adult males to determine the effects of oral pyridoxine supplementation on in vitro platelet aggregation. Following a 4-wk baseline period, half the subjects received 100 mg/day of pyridoxine X HCl while the remaining subjects received a placebo for 6 wk. In vitro platelet responses to ADP and collagen and the plasma pyridoxal 5'-phosphate (PLP) concentrations were measured at biweekly intervals. Plasma PLP concentrations increased significantly (p less than 0.001) for those receiving the vitamin B6 compared to baseline values or compared to those receiving the placebo. However, there was no significant effect of increased levels of plasma PLP on collagen-stimulated platelet aggregation and only a slight effect on ADP-stimulated aggregation. Acute administration of 100 mg pyridoxine X HCl failed to alter the in vitro response of platelets to either ADP or collagen. Reevaluation of conclusions based solely on in vitro studies suggesting the use of pyridoxine as an effective in vivo antithrombotic agent may be warranted.

Effect of dietary sucrose on the SHR/N-corpulent rat: a new model for insulin-independent diabetes.

A new congenic strain of genetically obese rat, SHR/N-corpulent (cp), was studied. Young male corpulent (cp/cp) and lean (cp/+ or +/+) rats approximately 5 wk of age were fed a diet containing 54% carbohydrate as either sucrose or cooked cornstarch for 9 wk. A phenotype effect was observed with body weight, fasting levels of serum insulin, triglyceride and total cholesterol, levels of serum insulin and glucose after an oral glucose load, and level of urine glucose (corpulent greater than lean), and with systolic blood pressure (corpulent less than lean). Only lean rats were hypertensive. Corpulent rats were hyperinsulinemic, hyperlipidemic, exhibited glycosuria, and were hyperglycemic after an oral glucose load. Lean rats were hyperinsulinemic, but normoglycemic. A diet effect (sucrose greater than starch) was observed with body weight, level of serum glucose after an oral glucose load, and urine volume in both corpulent and lean rats, and with levels of serum insulin and total urine glucose in corpulent rats. Corpulent rats fed sucrose had 20 to 40% higher levels of serum glucose and insulin after an oral glucose load, and twice the amount of total urine glucose, than did corpulent rats fed starch. The data demonstrate that corpulent rats have metabolic characteristics associated with insulin-independent diabetes in humans and that sucrose is more diabetogenic than starch. Manifestation of hyperglycemia in this model may be the result of superimposing obesity on an insulin-resistant genetic background.

Identification and quantification of prostaglandin E3 in renal medullary tissue of three strains of rats fed fish oil.

Three strains of rats were fed a fish oil diet to verify their ability to incorporate and convert dietary eicosapentaenoic acid (20:5 omega 3) into trienoic prostaglandins. Our results show that such conversion indeed occurs in kidney medullae homogenates. Specifically, the presence of prostaglandin E3 (PGE3) was established by gas chromatographic-mass spectrometric (GC-MS) analysis. That compound was conclusively identified by comparison of fragment ions and their relative intensities with those obtained from authentic PGE3. Further evidence was provided by studying the recovery of exogenously added PGE3. Further evidence was provided by studying the recovery of exogenously added PGE3. The crude ethyl acetate extracts of the medullary homogenates were methylated and cleaned up by liquid-gel chromatography with Lipidex-5000 prior to conversion to PGB3 for GC-MS analysis. The PGE3 was quantified by selected ion monitoring (SIM) with [3,3,4,4-2H4] PGE2 as internal standard. The levels of PGE3 were similar, about 3 ng/mg of wet tissue, in the 3 strains of rats. Identical in vivo conversion of the 2.0:5 omega 3 fatty acid to PGE3 could not be positively established by analysis of pooled urine specimens.

Production of prostaglandins in homogenates of kidney medullae and cortices of spontaneously hypertensive rats fed menhaden oil.

Menhaden oil (MO), whose polyunsaturated fatty acids consist mainly of (n-3) fatty acids, was fed to spontaneously hypertensive rats to determine the effect of (n-3) fatty acid on the in vitro production of prostaglandins produced from arachidonic acid (20:4[n-6]). Capacity to form PGE2 and PGF2 alpha was impaired in homogenates of kidney medullae and cortices from rats fed the MO diet compared to rats fed the control diet. The lower amounts of diene prostaglandins produced corresponded to the decrease in the amount of 20:4 (n-6) in the tissue. Possibly changes produced in tissue lipids by dietary fatty acids affect prostaglandin production by reducing the availability of substrate in tissue lipids.

Properties of platelet phospholipase A2.

Thrombin, within seconds after its addition, stimulated the release of 1-14C-20:4n-6 from the platelet phospholipids, phosphatidylcholine and phosphatidylinositol. Although inhibitors of prostaglandin cyclooxygenase did not inhibit this PLA2 activity, the anti-malarial drug, quinacrine, did. The location of the PLA2 that furnishes 20:4n-6 for the cyclooxygenase is not clear, but if it were in or near membranes containing the cyclooxygenase, phospholipids in the vicinity might serve as the source of 20:4n-6. How substrate specificity relates to the asymmetric distribution of the various phospholipid classes in membranes is yet to be determined.