Cyclooxygenase-1 haplotype C50T/A-842G does not affect platelet response to aspirin.

COX-1 polymorphism C50T, in complete linkage disequilibrium with the other polymorphism A-842G, has been depicted as a determinant of pharmacological response to aspirin treatment. Whether these polymorphisms exert an effect on response to aspirin both in vitro and ex vivo is still controversial. We genotyped a population of 148 healthy individuals for the C50T/A-842G haplotype. Thirty of them underwent low-dose aspirin (100 mg daily) treatment for four weeks and were followed up for seven days after withdrawal. In this subgroup, we evaluated the thromboxane-dependence of biochemical and functional indexes used to monitor the antiplatelet effect of low-dose aspirin. Among the 148 subjects studied, 10 were heterozygous for the C50T/A-842G haplotype (6.7%) and only one was homozygous for the 50T/-842G haplotype (0.67%). In the group on low-dose aspirin, serum thromboxane (TX) B(2) as well as urinary 11-dehydro-TXB(2) and arachidonic acid (AA)-induced aggregation were similarly suppressed in carriers and non-carriers of the 50T/-842G haplotype, with an increase until basal levels of all the parameters within seven days after withdrawal. We found no relationship between the 50T/-842G haplotype and the so-called phenomenon of aspirin resistance. Platelet cyclooxygenase activity, as reflected by serum TXB(2), was uniformly and persistently suppressed by low-dose aspirin in both carriers and non carriers of these polymorphisms.

Decreased plasma soluble RAGE in patients with hypercholesterolemia: effects of statins.

The receptor for advanced glycation endproducts (RAGE) is overexpressed at sites of vascular pathology. A soluble RAGE isoform (sRAGE) neutralizes the ligand-mediated damage by acting as a decoy. We hypothesized that in hypercholesterolemia up-regulation of the ligand-RAGE axis may bridge impairment of nitric oxide biosynthesis with oxidative stress. We measured in 60 hypercholesterolemic patients and 20 controls plasma total sRAGE levels, urinary 8-iso-prostaglandin (PG) F(2alpha) excretion, and plasma levels of asymmetric dimethylarginine (ADMA). The effects of two structurally different statins (pravastatin and atorvastatin) on these parameters were analyzed in 20 hypercholesterolemic subjects free of vascular disease. Plasma sRAGE was significantly lower, ADMA and urinary 8-iso-PGF(2alpha) were higher, in hypercholesterolemic versus normocholesterolemic patients. Patients on statin treatment with previous myocardial infarction had lower 8-iso-PGF(2alpha), higher sRAGE, and unchanged ADMA levels compared to subjects free of vascular disease. On multivariate regression analysis only 8-iso-PGF(2alpha) and ADMA predicted sRAGE levels. An 8-week treatment with either statin was associated with a significant reduction in urinary 8-iso-PGF(2alpha), whereas only atorvastatin raised sRAGE levels near to normal values, with no change in ADMA levels. sRAGE might serve as an endogenous protecting factor for accelerated atherosclerosis mediated by oxidative stress and endothelial dysfunction in hypercholesterolemia.

Targeted quantitative analysis of fatty acids in atherosclerotic plaques by high sensitivity liquid chromatography/tandem mass spectrometry.

The quantitative analysis of fatty acid composition in atherosclerotic plaques provides a way to monitor the underlying etiology of atherosclerosis. Previously, the method of choice for analyzing fatty acids in biological samples was gas chromatography/mass spectrometry (GC/MS); however, recent developments in electrospray ionization (ESI)/liquid chromatography (LC)/tandem mass spectrometry have made it a superior alternative. Previous research has largely focused on global analyses of intact lipids rather than more targeted analysis of the fatty acids themselves. We have now developed a targeted, stable isotope dilution LC-electrospray ionization/multiple reaction monitoring/MS method for the quantitative analysis of 10 fatty acids (myristic, palmitic, stearic, oleic, linoleic, alpha-linolenic, gamma-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids) using their trimethylaminoethyl ester (TMAE) derivatives to improve sensitivity. The method was validated, had a detection limit in the fmol range, and was used in the analysis of fatty acids in atherosclerotic plaques from carotid arteries.

LC/ESI/MS analysis of saturated and unsaturated fatty acids in rat intestinal epithelial cells.

Reactive oxygen species (ROS) can mediate damage to cellular macromolecules and lipids. Lipid peroxidation is considered to be a major pathway by which ROS can cause tissue damage and alterations in cell membranes. Other factors affecting oxidative damage include the target molecules such as fatty acids, which are readily oxidized by ROS. Thus, lipid peroxidation may depend upon the cellular fatty acid composition. Analysis of saturated fatty acids that are present by liquid chromatography/mass spectrometry (LC/MS) is difficult because they are poorly ionized under electrospray ionization (ESI) conditions. The separation of short to very long chain saturated and unsaturated fatty acids is also very challenging when LC is employed instead of gas chromatography. The use of trimethylaminoethyl (TMAE) ester iodide derivatization has been shown previously to improve the sensitivities of saturated fatty acids in the ESI mode. A reversed-phase LC method using a diphenyl column was employed to separate 14 fatty acids as their TMAE derivatives. Stable isotope dilution LC/ESI/multiple reaction monitoring/MS methodology was then developed for the quantitative analysis of seven saturated and seven unsaturated forms of short (C14) to very long (C26) chain fatty acids as their TMAE ester iodide derivatives. This methodology has allowed the analysis of fatty acid composition from parental rat intestinal epithelial cell and rat intestinal epithelial cells transfected with cyclooxygenase-2, a model system of oxidative stress.

Platelet cyclooxygenase inhibition by low-dose aspirin is not reflected consistently by platelet function assays: implications for aspirin "resistance".

OBJECTIVE: This study was conducted to assess the thromboxane (TX) dependence of biochemical and functional indexes used to monitor the effect of low-dose aspirin. BACKGROUND: Functional assays of the antiplatelet effects of low-dose aspirin variably reflect the TX-dependent component of platelet aggregation. Previous studies of aspirin resistance were typically based on a single determination of platelet aggregation. METHODS: We assessed the TXB(2) dependence of biochemical and functional indexes, as well as their intersubject and intrasubject variability during administration of the drug and after its withdrawal in 48 healthy volunteers randomized to receive aspirin 100 mg daily for 1 to 8 weeks. RESULTS: Serum TXB(2) was uniformly suppressed by 99% of baseline. Urinary 11-dehydro-TXB(2), arachidonic acid-induced aggregation, and VerifyNow Aspirin (Accumetrics Inc., San Diego, California) showed stable, incomplete inhibition (65%, 80%, and 35%, respectively). Adenosine diphosphate- and collagen-induced aggregation was highly variable and poorly affected by aspirin, with an apparent time-dependent reversal. Inhibition of platelet cyclooxygenase activity was nonlinearly related to inhibition of platelet aggregation. Platelet function largely recovered by day 3 post-aspirin, independently of treatment duration. With any functional assay, occasionally "resistant" subjects were found to be "responders" on previous or subsequent determinations. CONCLUSIONS: Platelet cyclooxygenase activity, as reflected by serum TXB(2) levels, is uniformly and persistently suppressed by low-dose aspirin in healthy subjects. However, the effect of aspirin is variably detected by functional assays, potentially leading to misclassification of "responder" as "resistant" phenotypes owing to poor reproducibility of functional measurements. The nonlinearity of the relationship between inhibition of TX production and inhibition of platelet function has important clinical implications.

Insulin resistance as a determinant of platelet activation in obese women.

OBJECTIVES: We tested the hypothesis that insulin resistance, per se, contributes to increased platelet activation in obesity, independently of underlying inflammation. BACKGROUND: Obesity, insulin resistance, and atherosclerosis are closely linked phenomena associated with low-grade inflammation. Obesity is associated with persistent platelet activation in otherwise healthy women. METHODS: We performed a cross-sectional study in 40 obese and 20 non-obese healthy women using urinary thromboxane metabolite excretion as a non-invasive index of platelet activation. An index of insulin sensitivity, S(I), and plasma adiponectin, C-reactive protein (CRP), and CD40 ligand (CD40L) levels were measured. RESULTS: Obese women had significantly (p < 0.0001) higher 11-dehydro-thromboxane B2 (11-dehydro-TXB2) excretion (median 718 vs. 211 pg/mg creatinine), CRP (1.13 vs. 0.48 mg/l), and CD40L levels (4.45 vs. 0.90 ng/ml) than controls. Obese women had lower S(I) (median 2.51 vs. 5.0 10(4) min(-1)/[microU/ml], p < 0.002) and adiponectin (6.3 vs. 10 microg/ml, p < 0.01) than control subjects. On multiple regression analysis, waist-to-hip ratio (beta = 0.27, p < 0.05) and S(I) (beta = -0.72, p < 0.04) predicted 11-dehydro-TXB2 excretion rate, independently of adiponectin, CRP, CD40L, and lipid patterns. In order to investigate the cause-effect relationship of these associations, we examined the effects of a 12-week weight loss program or a 3-week pioglitazone treatment on urinary 11-dehydro-TXB2 in 10 women with impaired S(I) and visceral obesity. Successful weight loss (0.6 kg loss/week) achieved in 5 subjects was associated with increased S(I) (+92%) and decreased CD40L (-27%), CRP (-37%), and 11-dehydro-TXB2 (-53%) (p < 0.05). Consistently, improvement of insulin sensitivity achieved with pioglitazone significantly decreased urinary 11-dehydro-TXB2 excretion (-43%, p < 0.05) without changes in body weight. CONCLUSIONS: Insulin resistance is a major determinant of platelet activation in female obesity.