Oxygen-radical-mediated lipid peroxidation and inhibition of ADP-induced platelet aggregation

The effects of lipid peroxidation on ADP-induced aggregation of washed rat platelets were examined using a oxygen-radical-generating system consisting of H2O2 and ferrous ion. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS). Incubation of the platelets with various concentrations of H2O2 (2-10 mM) in the presence of 10 microM Fe2+ resulted in a decrease of the aggregating capacity and an increase of TBARS value, depending on the concentrations of H2O2. Addition of catalase (0.1 mg/ml) to the incubation medium containing 10 microM Fe2+ and 10 mM H2O2 effectively protected the aggregating capacity, but superoxide dismutase (0.1 mg/ml) did not protect H2O2/Fe(2+)-induced inhibition of the platelet aggregation. The results of kinetic studies on the platelet aggregation with varying ADP and Ca2+ concentrations suggested that treatment of the platelets with H2O2/Fe2+ causes decreases in the binding affinities of ADP and Ca2+ for the platelets. On the basis of these results, change in the aggregating capacity of the platelets by treatment with H2O2/Fe2+ is discussed in relation to lipid peroxidation.     

Inhibition of human platelet aggregation and membrane lipid peroxidation by food spice, saffron

The inhibitory activity of saffron extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with platelet rich plasma (PRP) and platelet membranes respectively obtained from blood of healthy human volunteers. Human platelets were subjected to stimulation with a variety of agonists like ADP (61 μM), epinephrine (76 μM), collagen (11 μg/ml), calcium ionophore A 23187 (6 μM) and ristocetin (1.25 μg/ml) in the presence and absence of saffron extract with IC₅₀ being 0.66, 0.35, 0.86 and 0.59 mg respectively and no inhibition with ristocetin. The inhibitory effect was dose dependent with concentrations varying between 0.16 to 0.80 mg and time dependent at IC₅₀. A significant decrease was observed in malondialdehyde (MDA) formed, one of the end products of arachidonic acid metabolism and of serotonin released from dense granules of platelets at respective IC₅₀. Lipid peroxidation in platelet membranes induced by iron-ascorbic acid system was inhibited by saffron extract significantly with IC₅₀ of 0.33 mg. Hence, it may be said that aqueous extract of saffron may have component(s), which protect platelets from aggregation and lipid peroxidation. (Mol Cell Biochem 278: 59–63, 2005)     

Effects of palm oil and transesterified palm oil on chylomicron and VLDL triacylglycerol structures and postprandial lipid response

The effects of positional distribution of triacylglycerol (TAG) fatty acids to TAG structures in chylomicrons and VLDL, and to postprandial lipemia, were studied in 10 healthy premenopausal women using a 6-h oral fat load test and a randomized, double-blind cross-over design. Molecular level information of TAG regioisomerism was obtained with a tandem mass spectrometric method. The positional distribution of fatty acids in chylomicron TAGs was similar to the respective dietary fat; 79% of the analyzed regioisomers in palm oil and 84% of the analyzed regioisomers in transesterified oil were found in chylomicron TAGs 3 h after the oral fat loads. VLDL TAGs were equal after the two fat loads in all but one regioisomer. Similarities in the fatty acid compositions of chylomicron TAGs suggest that palmitic acid was absorbed equally from both test fats. The proportion of palmitoleic acid in the chylomicrons was increased. Fat with palmitic acid predominantly in the sn-1 and sn-3 positions caused a larger incremental area of total TAGs in plasma and reduced plasma insulin values at the beginning of the postprandial response (0-90 min) compared with fat with palmitic acid randomly distributed. The relationship between TAG molecular structures in dietary fats and in lipoproteins provides new means for understanding the effects of fatty acid positional distribution on human lipid metabolism.     

Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum.

1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively.     

Effects of dietary polyunsaturated fatty acid on platelet aggregation in rats

In the present study, we changed the fatty acid profile in blood and platelet membranes by dietary manipulation, and examined the effect on platelet aggregation in rats. Fifty-five rats were divided into five groups and fed for 56 days with 1% cholesterol and different types of fatty acid-rich diets: basal, lard, lard + fish oil, soybean oil, and soybean oil + fish oil. a decrease in serum arachidonic acid (20:4, omega-6, AA) and an increase in serum eicosapentaenoic acid (20:5, omega-3, EPA) were found in all experimental dietary groups fed with refined fish oil. Similar changes in the polyunsaturated fatty acids were also found in the platelet membrane phospholipids. Platelet aggregation, quantitated by the slope and height of the aggregation curve induced by different concentrations of ADP in a platelet aggregometer, was inhibited in all groups fed with refined fish oil. This inhibition of platelet aggregation may be related to an increase in the ratio of EPA and AA in the platelet membrane phospholipids after dietary manipulation. The differences in the platelet aggregation and thromboxane B2 (TXB2) concentration between the lard and the lard + fish oil groups were more profound than that between the soybean oil and the soybean oil + fish oil group. However, the malondialdehyde (MDA) concentration revealed no significant differences between the five groups.     

Effect of dietary oils on lipid peroxidation and on antioxidant parameters of rat plasma and lipoprotein fractions.

In order to investigate the influence of fatty acid pattern and antioxidants other than vitamin E on lipid peroxidation and antioxidant levels of plasma very low density and low density lipoproteins (VLDL + LDL), the effects of three diets (equalized for vitamin E) containing soybean oil, olive oil, or an oleate-rich mixture of triglycerides (triolein) were studied in rats. A significantly lower concentration of thiobarbituric acid-reactive substances (TBA-RS) in plasma and lipoproteins was found after the olive oil diet (soybean oil, 3.7 +/- 0.4 nmol/ml; triolein, 2.1 +/- 0.5 nmol/ml; olive oil, 1.5 +/- 0.3 nmol/ml, in plasma) (soybean oil, 0.99 +/- 0.16 nmol/ml; triolein, 0.96 +/- 0.13 nmol/ml; olive oil, 0.38 +/- 0.12 nmol/ml, in the VLDL + LDL fraction). Furthermore, the results from in vitro copper-induced lipid peroxidation, expressed in terms of conjugated dienes, lipid hydroperoxides, and TBA-RS content, showed that VLDL + LDL particles from olive olive oil-fed rats were remarkably resistant to oxidative modification. The results suggest that the fatty acid unsaturation of dietary oils is not the only determining factor of the antioxidant capacity of lipoproteins in this animal model. The maximal protection observed after the olive oil diet may be explained by the presence of other unidentified antioxidants in addition to vitamin E, derived from oil intake. Therefore, the optimal balance between the content of unsaturated fatty acids and natural antioxidants in dietary oils appears to be of major importance.     

Effects of protein kinase C activation on human platelet cyclic AMP metabolism

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).     

Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).     

Effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase in trained mice.

The purpose of this study was to investigate the effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase (SOD) in exercised mice. In the first part of the study, 48 male weanling mice were randomly divided into three groups. They were fed a zinc-deficient diet containing 1.6 mg/kg zinc or were pair-fed or fed ad libitum a zinc-adequate diet supplemented with 50 mg/kg zinc. Half of each group received an exercise training program that consisted of swimming for 60 min per day in deionized water. The diets and exercise program persisted for 6 weeks. In the second part of the study, 64 mice were fed zinc-deficient diets for 6 weeks, and then one group was fed the zinc-deficient diet for an additional 3 weeks, and the other three groups were fed diets supplemented with 5, 50, and 500 mg/kg zinc, respectively. Half of each group also received the exercise program. Both blood and liver samples were examined. Free radicals in liver were directly detected by electron spin resonance techniques and the extent of lipid peroxidation was indicated by malonic dialdehyde (MDA). Both CuZn-SOD and Mn-SOD were measured. The results showed that exercise training increased the metabolism of zinc, and zinc deficiency induced an increased free radical generation and lipid peroxidation and a decreased hepatic CuZn-SOD activity in exercised mice. Furthermore, although exercise training had no effect on the level of free radicals in zinc-adequate mice, it could increase the hepatic mitochondrial MDA formation further in zinc-deficient animals and zinc deficiency would eliminate the exercise-induced increase in SOD activities which existed in zinc-adequate mice. A total of 50 mg/kg zinc supplemented in the diet was adequate to correct the zinc-deficient status in exercised mice while 5 mg/kg zinc had a satisfactory effect on the recovery of only sedentary zinc-deficient mice. However, 500 mg/kg zinc had a harmful effect on both sedentary and exercised zinc-deficient animals.     

Effects of piretanide on plasma fibrinolytic activity,platelet aggregation and platelet factor-4 release in man

Piretanide, 4-phenoxy-3-(pyrrolidinyl)-5-sulphamoyl benzoic acid, apart from being an efficient diuretic, enhances endogenous plasma fibrinolytic activity after a single dose of 6 mg administered by oral route. After ingestion of the drug, acceleration of fibrinolytic acitivity became manifest within 1 h, reached its peak in 3 h and was associated with a fall in fibrinogen and diminished urokinase excretion. Piretanide did not cause lysis of fibrinin vitro. Primary platelet aggregation, induced by adenosine-diphosphate, was inhibited by piretanide. Inin vitro experiments piretanide led to effective inhibition of adenosine-diphosphate-induced platelet aggregation with complete inhibition at 5 mM concentration. Piretanide led to a highly significant decrease of platelet factor-4 release.     

Effect of exercise on plasma cyclic AMP

The purpose of this study was to confirm the relationship between cyclic AMP(cAMP) level in plasma and changes of hormones concentrations in blood, during and after physical exercise. The results were as follows: At rest, plasma cAMP were 23.1 p mole/ml on the average and decreased after glucose loading. The level in plasma increased in proportion to the intensity of exercises. Under the 50% condition of the maximal intensity, cAMP level in plasma was about 40 p mole/ml and the contents of both thyroxine and growth hormone in serum clearly increased. And, under the 70% of the maximal, the contents of both adrenaline and noradrenaline in serum as well as that of cAMP in plasma increased. Plasma cAMP level also increased by prolongation of exercise (ca 45 p mole/ml). And when exercise lasted over 1.5 hrs, plasma glucagon level began to rise. The effect of carbohydrate load to lower the levels of plasma cAMP were also found during physical exercise. These results suggested that the cAMP level in plasma was affected, not only by the some regulating factors of glycolytic activities such as adrenaline and glucagon, but also by the production of thyroxine and growth hormone at the onset of exercise.     

The effects of plasma from IgA nephropathy patients on vascular prostacyclin and platelet cyclic AMP production

The effects of plasma from 10 IgA nephropathy patients and from ten controls were studied on vascular prostacyclin (PGI2) production, the cyclic AMP (cAMP) level and the aggregation of normal platelets. The ability of the plasma to support PGI2-like activity (PSA) was significantly lower in the group of patients (18.0 +/- 13.3%) than in the controls (52.6 +/- 12.9%). The concentration of 6-keto-PGF1 alpha in the supernatant of the vascular tissue was also lower following incubation with patient plasma than with control plasma (p less than 0.001). The reduced PGI2 released by the patient plasma led to a significantly lower platelet cAMP than that following the control plasma (p less than 0.01). There was a significantly positive correlation between the 6-keto-PGF1 alpha and the plasma PSA, and also between both the plasma PSA and 6-keto-PGF1 alpha concentrations and the platelet cAMP level. These findings suggest that a vascular PGI2 defect may cause a reduced cAMP production and an uninhibited aggregation of platelets, which might play a role in the pathogenesis of IgA nephropathy.     

Effects of cigarette smoking on cyclic AMP, cyclic GMP, dopamine beta-hydroxylase and nicotine in human plasma and urine during water diuresis.

Effects of cigarette smoking on dopamine β-hydroxylase activity (DBH), cyclic AMP, cyclic GMP and nicotine in plasma and urine during water diuresis were examined in 19 healthy men. After smoking, plasma DBH decreased and plasma cyclic AMP increased significantly, followed by the increases in urinary cyclic AMP and nicotine excretion. No significant change in both plasma and urinary cGMP was observed.     

Effects of adrenergic blockers on platelet aggregation.

The action of orciprenaline, tolazoline, propanolol and inpea on platelet aggregation induced by ADP epinephrine and norepinephrine was studied in vitro in human platelet-rich plasma. Orciprenaline did not significantly affect aggregation induced by ADP. Tolazoline inhibits the aggregation induced by epinephrine and norepinephrine more intensely than the beta-blockers. Inpea blocks the platelet aggregation induced by epinephrine and norepinephrine to a greater extent than propanolol at similar concentrations. The beta-blockers inhibit platelet aggregation non-specifically.     

Effects of deferrioxamine on iron-catalyzed lipid peroxidation.

The kinetics of iron binding by deferrioxamine B mesylate and the ramifications of this process upon iron-catalyzed lipid peroxidation were assessed. The relative rates of Fe(III) binding by deferrioxamine varied for the chelators tested as follows: ADP greater than AMP greater than citrate greater than histidine greater than EDTA. The addition of a fivefold molar excess of deferrioxamine to that of Fe(III) did not result in complete binding (within 10 min) for any of the Fe(III) chelates tested except ADP:Fe(III). The rates of Fe(III) binding by deferrioxamine were greater at lower pH and when the competing chelator concentration was high in relationship to iron. The relatively slow binding of Fe(III) by deferrioxamine also affected lipid peroxidation, an iron-dependent process. The addition of deferrioxamine to an ascorbate- and ADP:Fe(III)-dependent lipid peroxidation system resulted in a time-dependent inhibition or stimulation of malondialdehyde formation (i.e., lipid peroxidation), depending on the ratio of deferrioxamine to iron. Converse to Fe(III), the rates of Fe(II) binding by deferrioxamine from the chelators tested above were rapid and complete (within 1 min), and resulted in the oxidation of Fe(II) to Fe(III). Lipid peroxidation dependent on Fe(II) autoxidation was stimulated by the addition of deferrioxamine. Malondialdehyde formation in this system was inhibited by the addition of catalase, and a similar extent of lipid peroxidation was achieved by substituting hydrogen peroxide for deferrioxamine. Collectively, these results suggest that the kinetics of Fe(III) binding by deferrioxamine is a slow, variable process, whereas Fe(II) binding is considerably faster. The binding of either valence of iron by deferrioxamine may result in variable effects on iron-catalyzed processes, such as lipid peroxidation, either via slow binding of Fe(III) or the rapid binding of Fe(II) with concomitant Fe(II) oxidation.     

Effects of dietary fibre on serum lipid levels and fecal bile acid excretion.

Epidemiologic studies have suggested that dietary fibre protects humans against coronary heart disease, but interpretation of the data is confounded by coexisting differences in both dietary and environmental variables. The hypocholesterolemic action of dietary fibre varies: in general mucilaginous fibres such as pectin and oat bran are more effective than particulate fibres such as wheat bran. Although the mechanism of action of mucilaginous fibres is not completely understood, there is evidence that they induce small increases in the fecal excretion of bile acids and neutral steroids that are not fully compensated for by de novo cholesterol synthesis.     

Effects of the lipid peroxidation product 4-hydroxynonenal on the aggregation of human platelets

The stimulation by ADP or arachidonic acid of the aggregation of human platelets in plasma was inhibited by 4-hydroxynonenal (HNE). This reduction of aggregation was time related, and was increased by prolonged preincubation of the platelets with the aldehyde. HNE was more potent than its homologue 4-hydroxypentenal (HPE). HNE was less active in decreasing the aggregation induced by calcium ionophore A23187 or collagen in comparison with ADP. HNE was inactive against aggregation of platelet-rich plasma (PRP) stimulated by thrombin whereas it potently inhibited the aggregation of washed platelets in response to both thrombin and collagen. Platelets were found to degrade HNE, and mechanisms additional to covalent binding to glutathione are indicated by the results obtained. The aldehydes, including HNE, generated by platelets originated principally from arachidonic acid metabolism.