Impact of a weight loss and fitness intervention on exercise-associated plasma oxylipin patterns in obese, insulin-resistant, sedentary women.

Very little is known about how metabolic health status, insulin resistance or metabolic challenges modulate the endocannabinoid (eCB) or polyunsaturated fatty acid (PUFA)-derived oxylipin (OxL) lipid classes. To address these questions, plasma eCB and OxL concentrations were determined at rest, 10 and 20 min during an acute exercise bout (30 min total, ~45% of preintervention V̇O2peak , ~63 W), and following 20 min recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled diet conditions. We hypothesized that increased fitness and insulin sensitivity following a ~14-week training and weight loss intervention would lead to significant changes in lipid signatures using an identical acute exercise protocol to preintervention. In the first 10 min of exercise, concentrations of a suite of OxL diols and hydroxyeicosatetraenoic acid (HETE) metabolites dropped significantly. There was no increase in 12,13-DiHOME, previously reported to increase with exercise and proposed to activate muscle fatty acid uptake and tissue metabolism. Following weight loss intervention, exercise-associated reductions were more pronounced for several linoleate and alpha-linolenate metabolites including DiHOMEs, DiHODEs, KODEs, and EpODEs, and fasting concentrations of 9,10-DiHODE, 12,13-DiHODE, and 9,10-DiHOME were reduced. These findings suggest that improved metabolic health modifies soluble epoxide hydrolase, cytochrome P450 epoxygenase (CYP), and lipoxygenase (LOX) systems. Acute exercise led to reductions for most eCB metabolites, with no evidence for concentration increases even at recovery. It is proposed that during submaximal aerobic exercise, nonoxidative fates of long-chain saturated, monounsaturated, and PUFAs are attenuated in tissues that are important contributors to the blood OxL and eCB pools.

Elevated levels of 15-lipoxygenase-1 contribute to the abnormal phenotypes of osteoblasts in human osteoarthritis.

AIMS: 15-lipoxygenase-1 (15-LOX-1) plays a vital role in aggravating the inflammatory response in various pathological processes, including osteoarthritis (OA). Abnormal osteoblast phenotypes including elevated runt-related transcription factor 2 (RUNX2), collagen type 1 alpha 1 (COL1), and osteocalcin (OCN) lead to osteosclerosis of the subchondral bone, which eventually causes OA. However, the pathogenesis of OA is poorly defined, and it is unclear if 15-LOX-1 induces osteoblast abnormal phenotypes in OA. Therefore, this study aimed to determine the roles of 15-LOX-1 on the abnormal phenotypes present in osteoblasts of the subchondral bone in OA. MAIN METHODS: The expression levels of 15-LOX-1 were measured by Immunohistochemistry, qRT-PCR and western blotting from the OA subchondral bone osteoblasts. To further investigate the roles of 15-LOX-1 in abnormal phenotypes of osteoblasts and its mechanisms in OA, 15-LOX-1 siRNA or overexpressing lv-15-lox-1 were transfected into osteoblasts, respectively. The effects of 15-LOX-1 on abnormal phenotypes of osteoblasts in OA were assessed by qRT-PCR, and western blotting. We also examined the role of 15-LOX-1-inhibited autophagy in OA osteoblasts by qRT-PCR, and western blotting, transmission electron microscopy. KEY FINDINGS: The expression levels of 15-LOX-1 along with osteoblast phenotype markers such as RUNX2, COL1, and OCN were significantly increased in OA subchondral bone. Furthermore, 15-LOX-1 inhibited autophagy significantly upregulated the expression levels of RUNX2, COL1 and OCN through activated mTORC1. Similarly, treatment with autophagy inhibitors alleviated osteoblast abnormal phenotypes of osteoblasts in OA. SIGNIFICANCE: In conclusion, our results suggested that the expression of 15-LOX-1 on osteoblasts from the subchondral bone increased in OA. 15-LOX-1 inhibited autophagy by activated mTORC1, which in turn upregulated the markers of abnormal osteoblast phenotypes RUNX2, COL1, and OCN.

Maximal exercise and plasma cytochrome P450 and lipoxygenase mediators: a lipidomics study.

Epoxides derived from arachidonic acid (AA) are released during exercise and may contribute to vasodilation. However, exercise may also affect circulating levels of other epoxides derived from cytochromes P450 (CYP) monooxygenase and lipoxygenase (LOX) pathways, many of whose exhibit cardiovascular activity in vitro. The effects of exercise on their levels have not been documented. We tested the hypothesis that acute, maximal exercise would influence the plasma concentrations of these vasoactive substances. We measured plasma CYP and LOX mediators derived from both the n - 3 and n - 6 fatty acid (FA) classes in healthy volunteers before, during and after short-term exhaustive exercise. Lipid mediators were profiled by means of LC-MS/MS tandem mass spectrometry. A maximal Bruce treadmill test was performed to voluntary exhaustion. Exhaustive exercise increased the circulating levels of epoxyoctadecenoic (12,13-EpOME), dihydroxyeicosatrienoic (5,6-DHET), dihydroxyeicosatetraenoic acids (5,6-DiHETE, 17,18-DiHETE), but had no effect on the majority of CYP and LOX metabolites. Although our calculations of diol/epoxide ratios revealed preferred hydrolysis of epoxyeicosatrienoic acids (EEQs) into their diols (DiHETEs), this hydrolysis was resistant to maximal exercise. Our study is the first documentation that bioactive endogenous n - 3 and n - 6 CYP lipid mediators are released by short-term exhaustive exercise in humans. In particular, the CYP epoxy-metabolite status, 12,13-EpOME/DiHOME, 5,6-EET/DHET, 5,6-EEQ/DiHETE and 17,18-EEQ/DiHETE may contribute to the cardiovascular response during maximal exercise.

Impaired Production and Diurnal Regulation of Vascular RvDn-3 DPA Increase Systemic Inflammation and Cardiovascular Disease.

RATIONALE: Diurnal mechanisms are central to regulating host responses. Recent studies uncovered a novel family of mediators termed as specialized proresolving mediators that terminate inflammation without interfering with the immune response. OBJECTIVE: Herein, we investigated the diurnal regulation of specialized proresolving mediators in humans and their role in controlling peripheral blood leukocyte and platelet activation. METHODS AND RESULTS: Using lipid mediator profiling and healthy volunteers, we found that plasma concentrations of n-3 docosapentaenoic acid-derived D-series resolvins (RvDn-3 DPA) were regulated in a diurnal manner. The production and regulation of these mediators was markedly altered in patients at risk of myocardial infarct. These changes were associated with decreased 5-lipoxygenase expression and activity, as well as increased systemic adenosine concentrations. We also found a significant negative correlation between plasma RvDn-3 DPA and markers of platelet, monocyte, and neutrophil activation, including CD63 and CD11b. Incubation of RvDn-3 DPA with peripheral blood from healthy volunteers and patients with cardiovascular disease significantly and dose-dependently decreased platelet and leukocyte activation. Furthermore, administration of RvD5n-3 DPA to ApoE-/- (apolipoprotein E deficient) mice significantly reduced platelet-leukocyte aggregates, vascular thromboxane B2 concentrations, and aortic lesions. CONCLUSIONS: These results demonstrate that peripheral blood RvDn-3 DPA are diurnally regulated in humans, and dysregulation in the production of these mediators may lead to cardiovascular disease.

Serial Metabolome Changes in a Prospective Cohort of Subjects with Influenza Viral Infection and Comparison with Dengue Fever.

Influenza virus infection (IVI) and dengue virus infection (DVI) are major public health threats. Between IVI and DVI, clinical symptoms can be overlapping yet infection-specific, but host metabolome changes are not well-described. Untargeted metabolomics and targeted oxylipinomic analyses were performed on sera serially collected at three phases of infection from a prospective cohort study of adult subjects with either H3N2 influenza infection or dengue fever. Untargeted metabolomics identified 26 differential metabolites, and major perturbed pathways included purine metabolism, fatty acid biosynthesis and ß-oxidation, tryptophan metabolism, phospholipid catabolism, and steroid hormone pathway. Alterations in eight oxylipins were associated with the early symptomatic phase of H3N2 flu infection, were mostly arachidonic acid-derived, and were enriched in the lipoxygenase pathway. There was significant overlap in metabolome profiles in both infections. However, differences specific to IVI and DVI were observed. DVI specifically attenuated metabolites including serotonin, bile acids and biliverdin. Additionally, metabolome changes were more persistent in IVI in which metabolites such as hypoxanthine, inosine, and xanthine of the purine metabolism pathway remained significantly elevated at 21-27 days after fever onset. This study revealed the dynamic metabolome changes in IVI subjects and provided biochemical insights on host physiological similarities and differences between IVI and DVI.

The n-3 Polyunsaturated Fatty Acids Supplementation Improved the Cognitive Function in the Chinese Elderly with Mild Cognitive Impairment: A Double-Blind Randomized Controlled Trial.

OBJECTIVE: Intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) may protect against mild cognitive impairment (MCI). However, there is still a lack of the n-3 PUFAs intervention in the elderly with MCI in China. The aim of the present study was to investigate the effect of n-3 PUFA supplementation on cognitive function in the Chinese elderly with MCI. METHODS: Eighty six MCI individuals aged 60 years or older were randomly assigned to receive either n-3 PUFAs (480 mg DHA and 720 mg EPA per day, n = 44) or placebo (olive oil, n = 42) capsules. The changes of cognitive functions were assessed using Basic Cognitive Aptitude Tests (BCAT). RESULTS: The mean age of participants was 71 years old, and 59% of the participants were men. n-3 PUFA supplementation was associated with improved total BCAT scores, perceptual speed, space imagery efficiency, and working memory (p < 0.01), but not with mental arithmetic efficiency or recognition memory (p > 0.05). Subgroup analysis by sex showed that n-3 PUFAs significantly improved perceptual speed (p = 0.001), space imagery efficiency (p = 0.013), working memory (p = 0.018), and total BCAT scores (p = 0.000) in males. However, in females, the significant beneficial effects can only be observed in perceptual speed (p = 0.027), space imagery efficiency (p = 0.006), and total BCAT scores (p = 0.015)-not working memory (p = 0.113). CONCLUSION: n-3 PUFAs can improve cognitive function in people with MCI. Further studies with different fish oil dosages, longer intervention periods, and larger sample sizes should be investigated before definite recommendations can be made.

Influence of pomegranate seed oil and bitter melon aqueous extract on polyunsaturated fatty acids and their lipoxygenase metabolites concentration in serum of rats.

Competition with polyunsaturated fatty acids (PUFA) and an impact on eicosanoid biosynthesis may be one of mechanisms of conjugated linolenic acids (CLnA) action. The aim of this study was to investigate the influence of diet supplementation with pomegranate seed oil, containing punicic acid (PA)-one of CLnA isomers, and an aqueous extract of dried bitter melon fruits, administered separately or together, on PUFA and their lipoxygenase metabolites' concentration in serum of rats. Percentage share of fatty acids was diversified in relation to applied supplementation. PA was only detected in serum of pomegranate seed oil supplemented group, where it was about 1%. Cis-9, trans-11 conjugated linoleic acid (rumenic acid, RA) level tended to increase in group supplemented simultaneously with both dietary supplements whereas its highest share in total fatty acids pool was detected in group receiving solely bitter melon dried fruits aqueous extract. This indicates that consumption of bitter melon tea significantly increased RA content in fatty acids pool in serum. However, pomegranate seed oil elevated procarcinogenic 12-hydroxyeicosatetraenoic acid concentration. Taking into account that pomegranate seed oil and bitter melon dried fruits are dietary supplements accessible worldwide and willingly consumed, the biological significance of this phenomenon should be further investigated. We presume, that there may be a need for some precautions concerning the simultaneous use of these products.

Inhibition of the soluble epoxide hydrolase promotes albuminuria in mice with progressive renal disease.

Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.

Peroxynitrite signaling in human erythrocytes: synergistic role of hemoglobin oxidation and band 3 tyrosine phosphorylation.

Peroxynitrite crosses the red blood cell (RBC) membrane and reacts with hemoglobin (Hb) producing mainly metHb, which is reduced back to ferrousHb by NADH- and NADPH-dependent reductases. Peroxynitrite also induces band 3 (B3) tyrosine phosphorylation, a signaling pathway believed to activate glucose metabolism. This study was aimed to decipher the relationship between these two peroxynitrite-dependent processes. Peroxynitrite induced a burst of the hexose monophosphate shunt (HMS), revealed by NMR studies, and a burst of the glycolytic pathway, measured by lactate production. The HMS plays a prominent role in membrane signaling, as demonstrated by B3 phosphotyrosine inhibition by the glycolytic pathway inhibitor 2-deoxy-glucose (2DG) and activation by dehydroepiandrosterone (DHEA), an inhibitor of HMS. Peroxynitrite-induced B3 tyrosine phosphorylation was paralleled by the inhibition of membrane-associated phosphotyrosine phosphatase (PTP) activity, which was protected by 2DG but not DHEA. Interestingly, heme poisoning with CO inhibited peroxynitrite-dependent Hb oxidation and lactate production but did not affect PTP down regulation. These results suggest two distinct and concurrent effects of peroxynitrite: one mediated by Hb which, likely in its oxidized state, binds more strongly to B3, and another mediated by PTP-dependent B3 phosphorylation. Both effects are directed towards a surge in glucose utilization.

Development of novel peptide inhibitor of Lipoxygenase based on biochemical and BIAcore evidences.

Lipoxygenase (LOX) are enzymes implicated in a broad range of inflammatory diseases, cancer, asthma and atherosclerosis. These diverse biological properties lead to the interesting target for the inhibition of this metabolic pathway of LOX. The drugs available in the market against LOX reported to have various side effects. To develop potent and selective therapeutic agents against LOX, it is essential to have the knowledge of its active site. Due to the lack of structural data of human LOX, researchers are using soybean LOX (sLOX) because of their availability and similarities in the active site structure. Based on the crystal structure of sLOX-3 and its complex with known inhibitors, we have designed a tripeptide, FWY which strongly inhibits sLOX-3 activity. The inhibition by peptide has been tested with purified sLOX-3 and with LOX present in blood serum of breast cancer patients in the presence of substrate linoleic acid and arachidonic acid respectively. The dissociation constant (K(D)) of the peptide with sLOX-3 as determined by Surface Plasmon Resonance (SPR) was 3.59x10(-9) M. The kinetic constant (K(i)) and IC(50), as determined biochemical methods were 7.41x10(-8) M and 0.15x10(-6) M respectively.

Isomers of trans fatty acids modify the activity of platelet 12-P lipoxygenase and cyclooxygenase/thromboxane synthase.

OBJECTIVES: Trans isomers of fatty acids (TFA) have been implicated in the initiation and progression of atherosclerosis. Previous studies have shown that trans-unsaturated fatty acids like their cis-unsaturated counterparts exert a modifying effect on platelet aggregation. The aim of this work was to determine the influence of TFA on the aforementioned enzymes: lipoxygenase and cyclooxygenase/thromboxane synthase. METHODS: Two C18 cis-trans fatty acid pairs: oleic/elaidic and linoleic/linolelaidic were chosen. Fasting blood was sampled from 30 healthy volunteers without any lipid abnormalities and not on medication for at least 7 days prior to sampling. Platelet-rich plasma (PRP) was prepared and the platelet count was adjusted to 3 x 10(8) cells x ml(-1). Fatty acids in hexane were added to a final concentration of 7 microM and evaporated to dryness under nitrogen, 500 microl of PRP was pipetted into each tube, thoroughly mixed and incubated at 37 degrees C for 60 min. 12-P LOX activity was measured with a spectrophotometric method and expressed per mg protein. Cyclooxygenase/thromboxane synthase activity was determined by TXB(2) production with an ELISA-based assay. Statistics were done with the Kruskal-Wallis test and Statistica software package. RESULTS: We have found that the activity of 12-P LOX was suppressed by all cis-trans fatty acids used by us. Cyclooxygenase/thromboxane synthase activity was significantly inhibited by polyunsaturated fatty acids only. Linolelaidic acid was more potent in comparison to its monounsaturated (elaidic acid) counterpart. CONCLUSIONS: We believe that the effects of fatty acids are demonstrated at the membrane level where fatty acids may produce a perturbation in specific lipid domains. TFA are able to interact with the platelet membrane and transmembrane proteins just as the cis isomers. By interacting with proteins exposed on the cytoplasmic membrane, TFA may modify the activity of receptors and other membrane proteins. In this way, changes on the membrane surface are propagated into the cell affecting the activity of cytoplasmic enzymes like 12-P LOX and cyclooxygenase/thromboxane synthase.

Diabetic patients without vascular complications display enhanced basal platelet activation and decreased antioxidant status.

Vascular complications are the leading causes of morbidity and mortality in diabetic patients. The contribution of platelets to thromboembolic complications is well documented, but their involvement in the initiation of the atherosclerotic process is of rising interest. Thus, the aim of the present study was to evaluate basal arachidonic acid metabolism in relation to the redox status of platelets in both type 1 and type 2 diabetic patients, in the absence of vascular complications, as compared with respective control subjects. For the first time, we show that basal thromboxane B(2), the stable catabolite of thromboxane A(2), significantly increased in resting platelets from both type 1 and type 2 diabetic patients (58 and 88%, respectively), whereas platelet malondialdehyde level was only higher in platelets from type 2 diabetic subjects (67%). On the other hand, both vitamin E levels and cytosolic glutathione peroxidase activities were significantly lower in platelets from diabetic patients as compared with respective control subjects. We conclude that platelet hyperactivation was detectable in well-controlled diabetic patients without complications. This abnormality was associated with increased oxidative stress and impaired antioxidant defense in particular in type 2 diabetic patients. These alterations contribute to the increased risk for occurrence of vascular diseases in such patients.

Effects of non-steroidal anti-inflammatory drugs on cyclo-oxygenase and lipoxygenase activity in whole blood from aspirin-sensitive asthmatics vs healthy donors.

1. Cyclo-oxygenase (COX) and lipoxygenase (LO) share a common substrate, arachidonic acid. Aspirin and related drugs inhibit COX activity. In a subset of patients with asthma aspirin induces clinical symptoms associated with increased levels of certain LO products, a phenomenon known as aspirin-sensitive asthma. The pharmacological pathways regulating such responses are not known. 2. Here COX-1 and LO activity were measured respectively by the formation of thromboxane B(2) (TXB(2)) or leukotrienes (LT) C(4), D(4) and E(4) in whole blood stimulated with A23187. COX-2 activity was measured by the formation of prostaglandin E(2) (PGE(2)) in blood stimulated with lipopolysaccharide (LPS) for 18 h. 3. No differences in the levels of COX-1, COX-2 or LO products or the potency of drugs were found in blood from aspirin sensitive vs aspirin tolerant patients. Aspirin, indomethacin and nimesulide inhibited COX-1 activity, without altering LO activity. Indomethacin, nimesulide and the COX-2 selective inhibitor DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] inhibited COX-2 activity. NO-aspirin, like aspirin inhibited COX-1 activity in blood from both groups. However, NO-aspirin also reduced LO activity in the blood from both patient groups. Sodium salicylate was an ineffective inhibitor of COX-1, COX-2 or LO activity in blood from both aspirin-sensitive and tolerant patients. 4. Thus, when COX activity in the blood of aspirin-sensitive asthmatics is blocked there is no associated increase in LO products. Moreover, NO-aspirin, unlike other NSAIDs tested, inhibited LO activity in the blood from both aspirin sensitive and aspirin tolerant individuals. This suggests that NO-aspirin may be better tolerated than aspirin by aspirin-sensitive asthmatics.

Improved high-performance liquid chromatographic method for the combined analysis of phospholipase, lipoxygenase and cyclooxygenase activities.

We report herein an improved method for the high-performance liquid chromatographic separation and analysis of eicosanoids formed during the stimulation of human platelets in vitro with collagen. Since the products of interest, excepting arachidonic acid, contain hydroxyl groups (one to several), our method involves the conversion of the hydroxyl groups into acetates (pyridine/acetic anhydride) after derivatization with anthryl diazomethane (ADAM) rendering the compounds with much decreased polarity for separation on a reversed-phase column. This procedure is superior to that involving ADAM esters only, i.e. with free hydroxyl groups, as it leads to the excellent separation of the desired compounds from each other and from extraneous peaks observed due to the ADAM reagent and sharpens the peak of thromboxane. We have successfully applied the method to investigate the formation of thromboxane B2 and 12-hydroxyheptadecatrienoic acid (HHT) (products of cyclooxygenase and thromboxane A2 synthase), 12-hydroxyeicosatetraenoic acid (12-HETE, a 12-lipoxygenase product) and arachidonic acid (AA, product of phospholipase A2) formed during the in vitro aggregation of human platelets induced by collagen. A correlation between the inhibition of aggregation by aspirin and thromboxane/HHT formation was observed. All four compounds can be chromatographed in a single run. We employed prostaglandin B1 (PGB1) as internal reference standard to quantify the products. The method is useful to investigate selectivity of drugs which may affect either or all of these enzyme pathways at the same time.

Platelet arachidonate cascade of migraineurs in the interictal phase.

Morphological and functional alterations of platelets in migraineurs may be linked to the development of migraine. We examined the eicosanoid synthesis of platelets of untreated female migraineurs in a headache-free period and compared it to that of age- and blood group-matched healthy female volunteers. In the platelets of headache-free migraineurs significantly less amounts of anti-aggregatory prostaglandin D2 and prostacyclin, as well as of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (a potent endogenous inducer of endothelial prostacyclin production) were produced, while the synthesis of platelet aggregatory thromboxane did not differ when compared to that of healthy women. These results suggest that the platelet eicosanoids of migraineurs in the headache-free period might promote the development of cellular, vascular and neurological events inducing headache.

Prostaglandin H synthase and lipoxygenase mediated activation of xenobiotics in platelets.

To investigate the involvement of prostaglandin H synthase (PHS) and lipoxygenase (LPO) in the activation of xenobiotics in platelets, platelet sonicates were preincubated with alpha-naphthol. Protein covalent binding of alpha-naphthol was measured following addition of arachidonic acid. Protein covalent binding was increased in a dose-dependent manner until it plateaued at 500 microM arachidonic acid. Pretreatment by two inhibitors of PHS, aspirin and indomethacin, resulted in a dose-dependent inhibition of alpha-naphthol-induced covalent binding, confirming PHS involvement. In addition, pretreatment by a LPO inhibitor, nordihydroguaiaretic acid (NDGA), also prevented covalent binding substantially, showing that LPO may be an alternative pathway for xenobiotic activation in platelets. Furthermore, combined treatment of aspirin and NDGA almost abolished the increase of alpha-naphthol-induced covalent binding, suggesting that PHS and LPO are both major pathways for xenobiotic activation in platelets.

The effect of substance P on the arachidonate cascade of rat platelets.

The role of substance P (SP) in neurogenic inflammation is well known. Through neurokinin receptors, SP activates cells, including the arachidonate cascade of platelets. Our in vitro experiments were carried out to determine the effect of SP on the arachidonate cascade of rat platelets. The platelets were labelled with 14C-arachidonic acid, and the 14C-eicosanoids were then separated by means of overpressure thin-layer chromatography or high-performance liquid chromatography and were quantitatively determined. SP (10(-9) and 10(-8)) mol/L significantly increased the rate of the arachidonate cascade. The lipoxygenase pathway of platelets was stimulated by SP, which can result in the activation of protein kinase C mediated intracellular events. The cyclooxygenase system was inhibited by 10(-12) mol/L, and stimulated by 10(-9) mol/L SP. In our experiments SP in the physiological range of plasma concentration (10(-12) mol/L) decreased the synthesis of vasoconstrictor arachidonate metabolites (TxA2 and PGF2 alpha). These data suggest that in physiologic conditions the arachidonate cascade of platelets may play role in the vasodilator effect of SP. The formation of thromboxane in rat platelets was stimulated by higher concentration of SP (10(-9) mol/L), and therefore the SP-induced cytotoxicity against parasites might be mediated by the stimulation of thromboxane A2 synthesis.

Platelet lipoxygenase in spontaneously hypertensive rats.

We have previously reported that the nonselective lipoxygenase inhibitor phenidone is a potent hypotensive agent in the spontaneously hypertensive rat (SHR). In the present study, we examined the relationship between production of platelet 12-hydroxyeicosatetraenoic acid (12-HETE) and intra-arterial blood pressure in SHR and Wistar-Kyoto rats (WKY) using both a cross-sectional analysis and an acute pharmacological intervention. Basal generation rate of 12-HETE by platelets collected from the SHR was approximately 3.7-fold higher than in the WKY (0.86 +/- 0.24 versus 0.23 +/- 0.05 nmol/mL per 10 minutes, respectively; P < .01). Systolic arterial pressure was positively related to platelet 12-HETE formation rate when the entire rat population was considered (r = .70, P < .001). The specific 12-lipoxygenase inhibitor cinnamyl-3,4-dihydroxycyanocinnamate induced lowering of both arterial blood pressure and platelet 12-lipoxygenase activity in SHR. At 15 mg/kg, cinnamyl-3,4-dihydroxycyanocinnamate elicited a marked hypotensive effect in SHR but not in WKY. This reduction in arterial pressure was accompanied by an approximate 70% inhibition in platelet 12-HETE production rate. The return of high blood pressure to basal levels was associated with a significant rise in the production of platelet 12-HETE toward control values (baseline, 0.97 +/- 0.33 nmol/mL per 10 minutes; nadir of blood pressure, 0.19 +/- 0.03; resumption of basal pressure, 0.42 +/- 0.14). In contrast, captopril (15 mg/kg) induced a quantitatively similar decrease in blood pressure but had no effect on platelet 12-HETE generation rate. Thus, hypertension in SHR is linked to increased production rate of platelet 12-HETE. Acute blood pressure reduction attained during lipoxygenase inhibition but not by angiotensin converting enzyme inhibition leads to a concomitant reduction in the production of platelet 12-HETE. We speculate that since rat arterial tissue produces 12-HETE, increased 12-lipoxygenase activity in SHR may contribute to the maintenance of elevated arterial pressure in this strain.

Wines and grape juices as modulators of platelet aggregation in healthy human subjects.

To test the hypothesis that red wine, by virtue of its relatively high concentration of polyphenols, is more protective against atherosclerosis and coronary heart disease (CHD) than white wine, and that grape juice enriched in one of these, trans-resveratrol, may share some of these properties, studies were performed on 24 healthy males aged 26-45 years. Each consumed the following beverages for periods of 4 weeks: red wine, white wine, commercial grape juice and the same grape juice enriched with trans-resveratrol. Apart from the last beverage, 2 weeks abstinence was maintained before commencing the schedule. Blood was taken at the beginning and end of each schedule to determine plasma thromboxane B2 (TxB2) concentration and the IC50 (concentration required for 50% aggregation) for ADP and thrombin-induced platelet aggregation. White wine (P < 0.05) but not red wine increased the IC50 for ADP. Both wines increased the IC50 for thrombin (P < 0.02 and P < 0.001, respectively) and also lowered plasma TxB2 concentrations (P < 0.01 and P < 0.025, respectively). Neither grape juice altered ADP-induced aggregation or TxB2 concentrations, but the commercial juice lowered the IC50 for thrombin (P < 0.001) whereas the resveratrol-enriched juice caused a dramatic increase (P < 0.001). In vitro experiments demonstrated that the aggregation of fresh washed human platelets by ADP and thrombin was moderately reduced by both grape juices, strongly by red wine and not at all by white wine. The synthesis of TxB2 by platelets from labelled arachidonate was stimulated by commercial grape juice, slightly enhanced by resveratrol-enriched juice and strongly inhibited by red wine with white wine having little effect. Platelets from subjects consuming the commercial juice had a higher ratio of cyclo-oxygenase to lipoxygenase product formation and those consuming the resveratrol-enriched juice a lower ratio than during the control period. We conclude that trans-resveratrol can be absorbed from grape juice in biologically active quantities and in amounts that are likely to cause reduction in the risk of atherosclerosis. The failure of red wines (which have a 20-fold excess of polyphenols over white wines) to show any advantage suggests that, in vivo, ethanol is the dominant anti-aggregatory component in these beverages which are more potent than grape juices in preventing platelet aggregation in humans.

Nitric oxide-donor compounds inhibit lipoxygenase activity.

The nitric oxide (N0-releasing agents sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) inhibit dioxygenase activity of lipoxygenase in human platelets and human CHP100 neuroblastoma cells, leading the latter to necrosis. The effect of both NO-donors on the dioxygenase reaction was investigated by using soybean lipoxygenase type II (LOX-2) as a model for the mammalian enzyme. SNP and SNAP were competitive inhibitors of LOX-2, with inhibition constants of 525 microM and 710 microM, respectively. Both compounds inactivated LOX-2 by reducing the catalytic iron to the inactive Fe(II) form and counteracted the H2O2-mediated activation of the LOX-2 catalyzed dioxygenase reaction. Similarly, the co-oxidative and per-oxidative activities of LOX-2 were also inhibited by the NO-releasing agents. These findings suggest that the biological role played by NO can be mediated, at least in part, by the inactivation of lipoxygenase, a key-enzyme for the arachidonic acid metabolism in human cells.