Untargeted metabolomics reveals alterations in metabolites of lipid metabolism and immune pathways in the serum of rats after long-term oral administration of Amalaki rasayana.

Amalaki rasayana, a traditional preparation, is widely used by Ayurvedic physicians for the treatment of inflammatory conditions, cardiovascular diseases, and cancer. Metabolic alterations induced by Amalaki rasayana intervention are unknown. We investigated the modulations in serum metabolomic profiles in Wistar rats following long-term oral administration of Amalaki rasayana. Global metabolic profiling was performed of the serum of rats administered with either Amalaki rasayana (AR) or ghee + honey (GH) for 18 months and control animals which were left untreated. Amalaki rasayana components were confirmed from AR extract using HR-LCMS analysis. Significant reductions in prostaglandin J2, 11-dehydrothromboxane B2, and higher levels of reduced glutathione and glycitein metabolites were observed in the serum of AR administered rats compared to the control groups. Eleven different metabolites classified as phospholipids, glycerophospholipids, glucoside derivatives, organic acids, and glycosphingolipid were exclusively observed in the AR administered rats. Pathway analysis suggests that altered metabolites in AR administered rats are those associated with different biochemical pathways of arachidonic acid metabolism, fatty acid metabolism, leukotriene metabolism, G-protein mediated events, phospholipid metabolism, and the immune system. Targeted metabolomics confirmed the presence of gallic acid, ellagic acid, and arachidonic acid components in the AR extract. The known activities of these components can be correlated with the altered metabolic profile following long-term AR administration. AR also activates IGF1R-Akt-Foxo3 signaling axis in heart tissues of rats administered with AR. Our study identifies AR components that induce alterations in lipid metabolism and immune pathways in animals which consume AR for an extended period.

The impact of vitamin C on the relationship among inflammation, lipid peroxidation and platelet activation during analgesic nephropathy in rats.

BACKGROUND: Oxidative stress and inflammation are involved in the pathogenesis of paracetamol-induced renal damage. This study examines the relationship between 8-iso-prostaglandin F2α (8-iso-PGF2α) and platelet activation as well as the relative contribution of the pro-inflammatory markers interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α) in enhanced 8-iso-PGF2α biosynthesis, as a complementary onset during analgesic nephropathy induced by chronic treatment with paracetamol. The protective effects of vitamin C on the aforementioned settings are also investigated. METHODS: Analgesic nephropathy was induced in Wistar rats. Renal function markers and the activity of antioxidant enzymes were determined spectrophotometrically. Immunoassays were used to measure the pro-inflammatory markers and the markers of lipid peroxidation and platelet activation. RESULTS: The chronic treatment with paracetamol led to renal dysfunction, represented by the elevation of plasma urea and creatinine and the decline in the enzymatic antioxidant status, but did not cause a significant increase in TNF-α and IL-1ß. The paracetamol-induced lipid peroxidation and enhanced production of 8-iso-PGF2α was not sufficient to cause changes in platelet activation represented by the level of 11-dehydro thromboxane B2. CONCLUSIONS: Our results suggest that oxidative stress cannot circumvent the need of stimulation by circulatory cytokines in order to induce inflammatory response and changes in platelet activation during analgesic nephropathy. Vitamin C proved to be beneficial in restoring the renal function markers to normal, increasing the renal enzymatic antioxidant potential, inhibiting lipid peroxidation, and lowering cytokine production and 11-dehydro thromboxane B2 excretion. The observed effects of vitamin C offer support for its potential use as protective treatment in cases of chronic paracetamol overdose.

Anti Xa oral anticoagulants inhibit in vivo platelet activation by modulating glycoprotein VI shedding.

Anti Xa non-vitamin K oral anticoagulants (anti Xa NOACs) seem to possess antiplatelet effect in vitro, but it is unclear if this occurs also in vivo. Aim of the study was to compare the effect on platelet activation of two anti Xa NOACs, namely apixaban and rivaroxaban, to warfarin, and to investigate the potential underlying mechanism by evaluating soluble glycoprotein GPVI (sGPVI), a protein involved in platelet activation. We performed a cross-sectional including AF patients treated with warfarin (n=30), or apixaban 10mg/day (n=40), or rivaroxaban 20mg/day (n=40). Patients were balanced for sex, age and cardiovascular risk factors. Platelet activation by urinary excretion of 11-dehydro-thromboxane (Tx) B2 and soluble GPVI (sGPVI) were analysed at baseline and after 3 months of treatment. Baseline TxB2 value was 155.2±42.7ng/mg creatinine. The 3 months-variation of urinary excretion of TxB2 was -6.5% with warfarin (p=0.197), -29% with apixaban (p<0.001) and -31% with rivaroxaban (p<0.001). Use of anti Xa NOACs was independently associated to the variation of urinary TxB2 (B: -0.469, p<0.001), after adjustment for clinical characteristics; sGPVI was significantly lower in patients treated with NOACs at 3 months (p<0.001), while only a trend for the warfarin group (p=0.116) was observed. The variation of sGPVI was correlated with that of TxB2 in the NOACs group (Rs: 0.527, p<0.001). In 15 patients (5 per each group) platelet recruitment was significantly lowered at 3 months by NOACs (p<0.001), but not by warfarin. The study provides evidence that anti Xa NOACs significantly inhibit urinary TxB2 excretion compared to warfarin, suggesting that NOACs possess antiplatelet property.

The effect of flaxseed dose on circulating concentrations of alpha-linolenic acid and secoisolariciresinol diglucoside derived enterolignans in young, healthy adults.

PURPOSE: The primary endpoint was to determine the plasma concentration of alpha-linolenic acid (ALA), and its metabolites, following milled flaxseed consumption at four doses. Secondary outcomes focused on plasma enterolignan concentrations and the effects on tolerability, platelet aggregation, plasma lipids and urinary thromboxane levels. METHODS: Healthy, younger adults (n = 34; 18-49 years old) were randomized into four groups consuming one muffin daily for 30 days fortified with 10, 20, 30 or 40 g of milled flaxseed. Blood and urine were collected at baseline and 4 weeks. RESULTS: Plasma ALA concentrations increased with all flaxseed doses (P < 0.01), except the 20 g/day dose (P = 0.10), yet there was no significant dose-dependent response (P = 0.81). Only with the 30 g/day diet were n-3 polyunsaturated fatty acids (P = 0.007), and eicosapentaenoic acid (EPA) (P = 0.047) increased from baseline values. Docosapentaenoic acid and docosahexaenoic acid were not detected at any dose. Plasma total enterolignan concentrations significantly increased over time in all treatment groups, yet despite a dose-dependent tendency, no between-group differences were detected (P = 0.22). Flaxseed was well tolerated, even at the highest dose, as there were no reported adverse events, changes in cholesterol, platelet aggregation or urinary 11-dehydro-thromboxane B2. CONCLUSIONS: In healthy, younger adults, 10 g/day of milled flaxseed consumption is sufficient to significantly increase circulating ALA and total enterolignan concentrations; however, 30 g/day is required to convert ALA to EPA. Although all doses were well tolerated, 40 g/day is too low to attenuate cholesterol in this population.

Moderate oral supplementation with docosahexaenoic acid improves platelet function and oxidative stress in type 2 diabetic patients.

Platelets from patients with type 2 diabetes are characterised by hyperactivation and high level of oxidative stress. Docosahexaenoic acid (DHA) may have beneficial effects on platelet reactivity and redox status. We investigated whether moderate DHA supplementation, given as a triglyceride form, may correct platelet dysfunction and redox imbalance in patients with type 2 diabetes. We conducted a randomised, double-blind, placebo-controlled, two-period crossover trial (n=11 post-menopausal women with type 2 diabetes) to test the effects of 400 mg/day of DHA intake for two weeks on platelet aggregation, markers of arachidonic acid metabolism, lipid peroxidation status, and lipid composition. Each two week-period was separated from the other by a six-week washout. Daily moderate dose DHA supplementation resulted in reduced platelet aggregation induced by collagen (-46.5 %, p< 0.001), and decreased platelet thromboxane B2 (-35 %, p< 0.001), urinary 11-dehydro-thromboxane B2 (-13.2 %, p< 0.001) and F2-isoprostane levels (-19.6 %, p< 0.001) associated with a significant increase of plasma and platelet vitamin E concentrations (+20 % and +11.8 %, respectively, p< 0.001). The proportions of DHA increased both in plasma lipids and in platelet phospholipids. After placebo treatment, there was no effect on any parameters tested. Our findings support a significant beneficial effect of low intake of DHA on platelet function and a favourable role in reducing oxidative stress associated with diabetes.

Relation of fish oil supplementation to markers of atherothrombotic risk in patients with cardiovascular disease not receiving lipid-lowering therapy.

Fish oil supplementation (FOS) is known to have cardiovascular benefits. However, the effects of FOS on thrombosis are incompletely understood. We sought to determine if the use of FOS is associated with lower indices of atherothrombotic risk in patients with suspected coronary artery disease (sCAD). This is a subgroup analysis of consecutive patients with sCAD (n=600) enrolled in the Multi-Analyte, Thrombogenic, and Genetic Markers of Atherosclerosis study. Patients on FOS were compared with patients not on FOS. Lipid profile was determined by vertical density gradient ultracentrifugation (n=520), eicosapentaenoic acid+docosahexaenoic acid was measured by gas chromatography (n=437), and AtherOx testing was performed by immunoassay (n=343). Thromboelastography (n=419), ADP- and collagen-induced platelet aggregation (n=137), and urinary 11-dehydrothromboxane B2 levels (n=259) were performed immediately before elective coronary angiography. In the total population, FOS was associated with higher eicosapentaenoic acid+docosahexaenoic acid content (p<0.001), lower triglycerides (p=0.04), total very low-density lipoprotein cholesterol (p=0.002), intermediate-density lipoprotein cholesterol (p=0.02), and AtherOx levels (p=0.02) but not in patients on lipid-lowering therapy. Patients not on lipid-lowering therapy taking FOS had lower very low-density lipoprotein cholesterol, intermediate-density lipoprotein cholesterol, remnant lipoproteins, triglycerides, low-density lipoprotein cholesterol, AtherOx levels, collagen-induced platelet aggregation, thrombin-induced platelet-fibrin clot strength, and shear elasticity (p<0.03 for all). In clopidogrel-treated patients, there was no difference in ADP-induced aggregation between FOS groups. Patients on FOS had lower urinary 11-dehydrothromboxane B2 levels regardless of lipid-lowering therapy (p<0.04). In conclusion, the findings of this study support the potential benefit of FOS for atherothrombotic risk reduction in sCAD with the greatest benefit in patients not receiving lipid-lowering therapy. Future prospective studies to compare FOS with lipid-lowering therapy and to assess the independent effects of FOS on thrombogenicity are needed.

The intake of broccoli sprouts modulates the inflammatory and vascular prostanoids but not the oxidative stress-related isoprostanes in healthy humans.

Current evidence supports the positive association between the consumption of plant foods and health. In this work, we assessed the effect of consuming a half-serving (30 g) or one serving (60 g) of broccoli sprouts on the urinary concentrations of biomarkers of oxidative stress (isoprostanes) and inflammation (prostaglandins and thromboxanes). Twenty-four volunteers participated in the project. A quantitative determination of sulforaphane and its mercapturic derivatives, eicosanoids, and total vitamin C in urine was performed. The intake of broccoli sprouts produced an increase in the urinary concentrations of sulforaphane metabolites and vitamin C. Among the 13 eicosanoids analyzed, tetranor-PGEM and 11ß-PGF2α as well as 11-dehydro-TXB2 showed a significant decrease in their urinary concentrations after the ingestion of broccoli sprouts. Therefore, the consumption of broccoli sprouts modulated the excretion of biomarkers linked to inflammation and vascular reactions without exerting a significant influence on the oxidation of phospholipids in vivo.

Fatty acids and TxA(2) generation, in the absence of platelet-COX-1 activity.

BACKGROUND AND AIMS: Omega-3 fatty acids suppress Thromboxane A(2) (TxA(2)) generation via mechanisms independent to that of aspirin therapy. We sought to evaluate whether baseline omega-3 fatty acid levels influence arachidonic acid proven platelet-cyclooxygenase-1 (COX-1) independent TxA(2) generation (TxA(2) generation despite adequate aspirin use). METHODS AND RESULTS: Subjects with acute myocardial infarction, stable CVD or at high risk for CVD, on adequate aspirin therapy were included in this study. Adequate aspirin action was defined as complete inhibition of platelet-COX-1 activity as assessed by <10% change in light transmission aggregometry to ≥1 mmol/L arachidonic acid. TxA(2) production was measured via liquid chromatography-tandem mass spectrometry for the stable TxA(2) metabolite 11-dehydro-thromboxane B2 (UTxB2) in urine. The relationship between baseline fatty acids, demographics and UTxB(2) were evaluated. Baseline omega-3 fatty acid levels were not associated with UTxB(2) concentration. However, smoking was associated with UTxB(2) in this study. CONCLUSION: Baseline omega-3 fatty acid levels do not influence TxA(2) generation in patients with or at high risk for CVD receiving adequate aspirin therapy. The association of smoking and TxA(2) generation, in the absence of platelet COX-1 activity, among aspirin treated patients warrants further study.

Oxidative stress and platelet activation in subjects with moderate hyperhomocysteinaemia due to MTHFR 677 C→T polymorphism.

The methylenetetrahydrofolate reductase (MTHFR) 677 C→T polymorphism may be associated with elevated total homocysteine (tHcy) levels, an independent risk factor for cardiovascular disease. It was the study objective to evaluate in vivo lipid peroxidation and platelet activation in carriers of the MTHFR 677 C→T polymorphism and in non-carriers, in relation to tHcy and folate levels. A cross-sectional comparison of urinary 8-iso-prostaglandin (PG)F(2α) and 11-dehydro-thromboxane (TX)B(2) (markers of in vivo lipid peroxidation and platelet activation, respectively) was performed in 100 carriers and 100 non-carriers of the polymorphism. A methionine-loading test and folic acid supplementation were performed to investigate the causal relationship of the observed associations. Urinary 8-iso-PGF(2α) and 11-dehydro-TXB(2) were higher in carriers with hyperhomocysteinaemia than in those without hyperhomocysteinaemia (p<0.0001). Hyperhomocysteinaemic carriers had lower folate levels (p=0.0006), higher urinary 8-iso-PGF(2α) (p<0.0001) and 11-dehydro-TXB(2) (p<0.0001) than hyperhomocysteinaemic non-carriers. On multiple regression analysis, high tHcy (p<0.0001), low folate (p<0.04) and MTHFR 677 C→T polymorphism (p<0.001) independently predicted high rates of 8-iso-PGF(2α) excretion. Methionine loading increased plasma tHcy (p=0.002), and both urinary prostanoid metabolites (p=0.002). Folic acid supplementation was associated with decreased urinary 8-iso-PGF(2α) and 11-dehydro-TXB2 excretion (p<0.0003) in the hyperhomocysteinaemic group, but not in the control group, with substantial inter-individual variability related to baseline tHcy level and the extent of its reduction. In conclusion, hyperhomocysteinaemia due to the MTHFR 677 C→T polymorphism is associated with enhanced in vivo lipid peroxidation and platelet activation that are reversible, at least in part, following folic acid supplementation. An integrated biomarker approach may help identifying appropriate candidates for effective folate supplementation.

Dietary fish oil alters the lysophospholipid metabolomic profile and decreases urinary 11-dehydro thromboxane B2 concentration in healthy Beagles.

Increased concentrations of dietary fish oil and antioxidants have been shown previously to change circulating concentrations of individual fatty acids (FAs) and vitamin E. The purpose of this study was to further investigate the effects of vitamins E and C, in combination with dietary fish oil, on selected blood and urinary biomarkers. Fifty adult Beagle dogs (mean age 5.3 years, range 1.4-14.2 years) were randomized into five dietary treatment groups for 90 days. All foods were complete and balanced and met the nutrient profiles of AAFCO for adult dogs. For 60 days before study initiation, dogs consumed a pretrial food that contained 74 IU/kg vitaminE and 0mg/kg vitaminC. The five experimental foods were confirmed by analytical methods to contain ≥ 640 IU/kg vitaminE and 130 mg/kg vitaminC (as fed). Experimental foods ranged from low levels of EPA and DHA (pretrial food and lowest experimental food had 0.01% EPA and no detectable DHA) to the highest experimental food with 0.25% EPA and 0.17% DHA. Serum was analyzed for FAs, vitamin E, and cholesterol concentrations; urine was analyzed for 11-dehydro thromboxane B(2) (TXB(2)). Serum was also used for metabolomic analysis. FA intake ranged from 0.02 g/day EPA and 0.02 g/day DHA to 0.58 g/day EPA and 0.39 g/day DHA. Increasing dietary concentrations of EPA and DHA resulted in increased serum concentrations of EPA and DHA in a dose-dependent fashion. Greater dietary vitamin E intake resulted in increased serum vitamin E concentrations (P<0.01). Higher serum cholesterol was also associated with higher serum vitamin E concentrations (P<0.01). In turn, changes in serum cholesterol concentration were associated with diet-induced changes in serum FA concentrations (all P<0.01). At the beginning of the dietary treatment period the most significant predictor of urine 11-dehydro TXB(2) concentration was age, followed by lean-body mass. After dietary treatment with different amounts of fish oil, age (increases 11-dehydro TXB(2)) was followed by EPA concentration as a significant negative predictor of urine 11-dehydro TXB(2) concentration (increasing serum concentrations of EPA decrease 11-dehydro TXB(2)), and then lean-body mass (decreases 11-dehydro TXB(2)). Serum docosahexaenoyl-glycerophosphocholine concentration was increased by feeding fish oil in a dose-response manner. In summary, serum vitamin E concentration is enhanced primarily by feeding vitamin E and secondarily by serum cholesterol concentration. When feeding diets enriched with fish oil, the major negative predictor of urinary 11-dehydro TXB(2) concentration is serum EPA concentration. Plasma lysophospholipids can be dynamically regulated by dietary fish oil supplementation.

Postprandial hyperglycemia is a determinant of platelet activation in early type 2 diabetes mellitus.

BACKGROUND: Chronic hyperglycemia is a major contributor to in vivo platelet activation in diabetes mellitus. OBJECTIVES: To evaluate the effects of acarbose, an alpha-glucosidase inhibitor, on platelet activation and its determinants in newly diagnosed type 2 diabetic patients. METHODS: Forty-eight subjects (26 males, aged 61 +/- 8 years) with early type 2 diabetes (baseline hemoglobin A(1c) < or = 7% and no previous hypoglycemic treatment) were randomly assigned to acarbose up to 100 mg three times a day or placebo, and evaluated every 4 weeks for 20 weeks. The main outcome measures were urinary 11-dehydro-thromboxane (TX)B(2) (marker of in vivo platelet activation) and 8-iso-prostaglandin (PG)F(2alpha) (marker of in vivo lipid peroxidation) excretion rate, 2-h postprandial plasma glucose (PPG) after a test meal, and assessment of glucose fluctuations by mean amplitude of glycemic excursions (MAGE). RESULTS: Baseline measurements revealed biochemical evidence of enhanced lipid peroxidation and platelet activation. As compared with the placebo group, patients treated with acarbose had statistically significant reductions in urinary 11-dehydro-TXB(2) and 8-iso-PGF(2alpha) excretion rate as early as after 8 weeks and at each subsequent time point (between-group P < 0.0001 at 12, 16 and 20 weeks), following earlier decreases in PPG and MAGE. Multiple regression analyses in the acarbose group revealed that PPG was the only significant predictor of 11-dehydro-TXB(2) urinary excretion rate (beta = 0.39, P = 0.002) and MAGE the only predictor of 8-iso-PGF(2alpha) urinary excretion rate (beta = 0.42, P = 0.001). CONCLUSIONS: Postprandial hyperglycemia is associated with enhanced lipid peroxidation and platelet activation in early type 2 diabetes. A moderate decrease in PPG achieved with acarbose causes time-dependent downregulation of these phenomena, suggesting a causal link between early metabolic abnormalities and platelet activation in this setting.

Determinants of platelet activation in hypertensives with microalbuminuria.

Microalbuminuria is a predictor of adverse outcome in hypertension.We evaluated in vivo platelet activation, by urinary 11-dehydrothromboxane (TX)B2 and plasma P-selectin, in hypertensives with or without microalbuminuria, and its possible association with oxidative stress, by urinary 8-iso-prostaglandin (PG)F2alpha and endothelial dysfunction. Sixty essential hypertensive patients, with (n=30) or without (n=30) microalbuminuria, and 30 controls were studied. Endothelial function was assessed by nitric oxide products, intercellular adhesion molecule (ICAM)-1, and asymmetric dimethylarginine (ADMA) levels. Urinary 11-dehydro-TXB2 excretion was higher in microalbuminuric (median 805 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (414 and 291 pg/mg, respectively; P<0.0001). Plasma P-selectinwas significantly higher in patients with microalbuminuria (median 136 ng/ml) as compared to those without microalbuminuria or controls (85 and 65 ng/ml; P<0.0001). Urinary 8-iso-PGF2alpha excretion was also enhanced in microalbuminuric (median 279 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (157 and 146 pg/mg, respectively; P<0.0001). A significant impairment in endothelial function was found in microalbuminuric patients, with decreased nitric oxide and increased ICAM-1 and ADMA levels. Multivariate regression analysis showed that urinary 8-iso-PGF2alpha excretion (beta=0.49; P<0.0001) and microalbuminuria (beta=0.36; P<0.001) were independently related to 11-dehydro-TXB2 in hypertensives. Vitamin E supplementation (900 mg daily for 1 month) in 10 hypertensives with microalbuminuria was associated with normalization in median 11-dehydro-TXB2 and 8-iso-PGF2alpha. We conclude that lipid peroxidation is a major determinant of persistent platelet activation in hypertensive patients with microalbuminuria.

Effect of low dose antioxidant vitamin and trace element supplementation on the urinary concentrations of thromboxane and prostacyclin metabolites.

OBJECTIVE: This trial evaluated the effect of antioxidant supplementation on the urinary excretion of 11-dehydro TXB(2)/2,3 dinor 6 keto PGF(1alpha) ratio, a marker of the pathogenesis of thrombosis and arteriosclerosis. METHODS: This study was a randomised, double-blind, placebo-controlled trial involving 186 presumably healthy volunteers. One hundred received a multi-antioxidant supplementation and 86 a placebo for two years. Blood zinc, selenium, beta-carotene, vitamin C and E and urinary excretion of 11-dehydro TXB(2) and 2,3 dinor 6 keto PGF(1alpha) were measured. RESULTS: Baseline subject characteristics did not differ between the two groups. Blood zinc, selenium, and beta-carotene concentrations significantly increased between baseline and two years in the multi-antioxidant supplementation group supporting subject compliance (p < 0.05). At two years, the median urinary 11-dehydro TXB(2)/2,3 dinor 6 keto PGF(1alpha) ratio was significantly lower in the multi-antioxidant supplementation group (3.4 versus 2.78, p = 0.015). Serum selenium concentration was the only antioxidant studied that was significantly related to the urinary 11-dehydro TXB(2)/2,3 dinor 6 keto PGF(1alpha) ratio. CONCLUSIONS: These results support the hypothesis that a low-dose multi-antioxidant supplementation may contributes to a reduction in platelet activation which is beneficial for cardiovascular function.

Screening for aspirin resistance in stable coronary artery patients by three different tests.

BACKGROUND: Aspirin (ASA) failure to inhibit in vitro platelet function had been termed ASA resistance. The prevalence of this phenomenon as measured with different platelet function tests varies widely among studies. OBJECTIVES: In this study, we propose to determine the prevalence of ASA non-responsiveness in stable coronary artery patients using three different tests. PATIENTS AND METHODS: One hundred ninety-one patients with a stable coronary artery disease and receiving secondary ASA prophylaxis (250 mg/day) were tested. For each patient the ASA-induced platelet inhibition was determined using three different tests: Ivy Bleeding time (BT), collagen/epinephrine closure time (CEPI-CT; PFA-100, Dade-Behring) and urinary 11-dehydrothromboxane B2 (uTxB2) excretion level. The agreement between these tests was evaluated by kappa statistics test. RESULTS: The prevalence of biological ASA resistance was 15.7% (n=30), 20.4% (n=39) and 24.6% (n=47) by BT, PFA-100 and UTxB2, respectively. Only fourteen patients (7.3%) were non-responders for two tests: 6 (3.1%) BT/ PFA-100; 1 (0.5%) BT/UTxB2; 7 (3.7%) PFA-100/UTxB2). A poor agreement was found between these three methods and only 3 patients were resistant with all the tests (1.6%). CONCLUSION: The lack of agreement supposed that different types of aspirin resistance exist. Thus, combination of two tests or more could be a primary solution for a better identification of ASA resistant patients. This hypothesis must be confirmed by a large-scale randomized study with clinically well-defined endpoints.

Reduced in vivo oxidative stress following 5-methyltetrahydrofolate supplementation in patients with early-onset thrombosis and 677TT methylenetetrahydrofolate reductase genotype.

The protective role of folate in vascular disease has been related to antioxidant effects. In 45 patients with previous early-onset (at age <50 years) thrombotic episodes and the 677TT methylenetetrahydrofolate reductase genotype, we evaluated the effects of a 28 d-course (15 mg/d) of 5-methyltetrahydrofolate (MTHF) on homocysteine metabolism and on in vivo generation of 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a reliable marker of oxidative stress. At baseline, patients' fasting total homocysteine (tHcy) was 11.5 micromol/l (geometric mean) and urinary excretion of 8-iso-PGF2alpha was 304 pg/mg creatinine, with the highest metabolite levels in the lowest quartile of plasma folate distribution (P < 0.05). After 5-MTHF supplementation, plasma folate levels increased approximately 13-fold (P < 0.0001 versus baseline); tHcy levels (6.7 micromol/l, P < 0.0001) and urinary 8-iso-PGF2alpha (254 pg/mg creatinine, P < 0.001) were both significantly lowered, their reduction being proportional to baseline values (r = 0.98 and r = 0.77, respectively) and maximal in patients with the lowest pre-supplementation folate levels (P < 0.05). The effects on folate (P < 0.0001) and tHcy (P = 0.0004) persisted for at least up to 2 months after withdrawing 5-MTHF. In parallel with long-lasting tHcy-lowering effects, a short-course 5-MTHF supplementation reduces in vivo formation of 8-iso-PGF2alpha in this population, supporting the antioxidant protective effects of folate in vascular disease.

Daily prickly pear consumption improves platelet function.

Prickly pear is traditionally used by Pima Indians as a dietary nutrient against diabetes mellitus. We examined the effect of daily consumption of 250 g in 8 healthy volunteers and 8 patients with mild familial heterozygous hypercholesterolemia on various parameters of platelet function. Beside its action on lipids and lipoproteins, prickly pear consumption significantly reduced the platelet proteins (platelet factor 4 and beta-thromboglobulin), ADP-induced platelet aggregation and improved platelet sensitivity (against PGI2 and PGE1) in volunteers as well as in patients. Also plasma 11-DH-TXB2 and the WU-test showed a significant improvement in both patients and volunteers. In contrast, collagen-induced platelet aggregation and the number of circulating endothelial cells showed a significant response in patients only. No influence of prickly pear ingestion on peripheral platelet count was monitored. The dietary run-in period did not influence any of the parameters of haemostasis examined. No sex difference was seen. Prickly pear may induce at least part of its beneficial actions on the cardiovascular system via decreasing platelet activity and thereby improving haemostatic balance.

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology.

Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.

Effect of the ingestion of Ginkgo biloba extract on platelet aggregation and urinary prostanoid excretion in healthy and Type 2 diabetic subjects.

Enhanced platelet function, particularly in response to collagen, is a common occurrence in diabetes that increases the risk of cardiovascular disease. Ginkgo biloba extract is ingested primarily to improve mental focus but it possesses a blood-thinning potential, which has not been well characterized. This study was designed to compare the effect of ingesting G. biloba extract on platelet aggregation in platelet-rich plasma (PRP) and prostanoid urinary excretion in healthy volunteers and subjects with Type 2 diabetes mellitus (T2DM). Before and after ingesting 120 mg of standardized G. biloba extract for 3 months, platelet aggregation was studied in PRP and urinary metabolites of thromboxane B(2) (TXB(2)) and prostacyclin (PGI(2)) were measured. In healthy volunteers (age, 42+/-11 years; BMI, 28.4+/-4.8 kg/m(2); n=28), the ingestion of G. biloba extract significantly increased fasting insulin and C-peptide (10+/-4 vs. 12+/-6 microU/ml, p<0.007 and 1.3+/-0.8 vs. 2.1+/-1.1 ng/ml, p<0.001, respectively) and significantly reduced collagen but not PAF-mediated platelet aggregation, converting 21 of 28 subjects with [COL+/EPI+] platelets to the [COL-/EPI+] phenotype. This was accompanied by a reduction of 11-dehydro-TXB(2) from 12.4+/-6.1 to 10.3+/-6.1 ng/mg Cr (p<0.04) and PGI(2) metabolites (2,3-dinor-6-keto-PGF(1alpha) and 6-keto-PGF(1alpha)) from 2.2+/-0.8 to 1.8+/-0.8 ng/mg Cr (p<0.05). In the T2DM subjects (age, 54+/-8; BMI, 36.6+/-7.9 kg/m(2); n=19), G. biloba ingestion did not affect pancreatic beta-cell function but significantly reduced platelet aggregation, converting 16 of 19 [COL+/EPI+] platelets to the [COL-/EPI+] phenotype. Unlike the healthy volunteers, this was not accompanied by a reduced urinary prostanoid excretion. G. biloba-induced reduction of both classes of prostanoid metabolites in healthy volunteers, but not in T2DM subjects, may suggest a nonselective inhibition of COX-1-mediated TXA(2) in platelets and COX-2-mediated PGI(2) production by the endothelial cells and perhaps platelet-enriched levels of arachidonic acid or COX-1 activity, or both, in T2DM subjects.

Effects of intravenous oxygen on prostacyclin and thromboxane formation in patients with peripheral occlusive arterial disease.

Oxygen infusion is used in complementary medicine for treatment of peripheral occlusive arterial disease. The mechanism of action is unknown. Thus, we determined the effects of oxygen infusion on prostacyclin, thromboxane and nitric oxide synthesis. Twelve patients with peripheral occlusive arterial disease received oxygen 40 ml/d intravenously for 3 weeks. Study parameters, analyzed by gas chromatography-mass spectrometry on day 1, 3, 10, 16, 21: 2,3-dinor-6-oxo-PGF(1alpha), colour invisible 2,3-dinor-TXB2 and nitrate in one-hour-urine before and after oxygen infusion, reflecting prostacyclin, thromboxane and nitric oxide synthesis. Urinary 8-iso-PGF2alpha, indicating oxidative stress, was assessed in one patient. Urinary 2,3-dinor-6-oxo-PGF1alpha rose from baseline more than 4-fold after oxygen infusion. In contrast, urinary 2,3-dinor-TXB2 excretion remained unchanged. Oxygen infusion had no effect on urinary nitrate excretion. Urinary 8-iso-PGF(2alpha) was not influenced by oxygen infusion with and without diclofenac pretreatment. Our data demonstrate a shift of the prostacyclin/thromboxane ratio toward prostacyclin by oxygen infusion. Thus, a mechanism of action is provided and clinical trials with intravenous oxygen find a rational basis.

Oxidative stress and platelet activation in homozygous homocystinuria.

BACKGROUND: Severe hyperhomocysteinemia due to cystathionine beta-synthase deficiency (CbetaSD) is associated with early atherothrombotic vascular disease. Homocysteine may exert its effects by promoting oxidative damage. In the present study, we investigated whether in vivo formation of 8-iso-prostaglandin (PG) F(2alpha), a platelet-active product of arachidonic acid peroxidation, is enhanced in CbetaSD and whether it correlates with in vivo platelet activation, as reflected by thromboxane (TX) metabolite excretion. METHODS AND RESULTS: Urine and blood samples were obtained from patients with homozygous CbetaSD (n=13) and age-matched healthy subjects. Urinary 8-iso-PGF(2alpha) excretion was significantly higher in CbetaSD patients than in control subjects (640+/-384 versus 213+/-43 pg/mg creatinine; P=0.0015) and correlated with plasma homocysteine (rho=0.398, P=0.0076). Similarly, urinary 11-dehydro-TXB(2) excretion was enhanced in CbetaSD (1166+/-415 versus 324+/-72 pg/mg creatinine; P=0.0015) and correlated with urinary 8-iso-PGF(2alpha) (rho=0.362, P=0.0153). Vitamin E supplementation (600 mg/d for 2 weeks) was associated with a statistically significant increase in its plasma levels (from 16.6+/-4.6 to 40.4+/-8.7 micromol/L, P=0.0002) and with reductions in 8-iso-PGF(2alpha) (from 790+/-159 to 559+/-111 pg/mg creatinine, P=0.018) and 11-dehydro-TXB(2) (from 1273+/-383 to 913+/-336 pg/mg creatinine, P=0.028). A statistically significant inverse correlation was found between urinary 8-iso-PGF(2alpha) and plasma vitamin E levels (rho=-0.745, P=0.0135). CONCLUSIONS: The results of the present study suggest that enhanced peroxidation of arachidonic acid to form bioactive F(2)-isoprostanes may represent an important mechanism linking hyperhomocysteinemia and platelet activation in CbetaSD patients. Moreover, they provide a rationale for dose-finding studies of vitamin E supplementation in this setting.