A Chemometric Model Applied to Fatty Acid Determination in Blood

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Changes in fatty acid profiles have been associated with several pathophysiological processes. Gas chromatography-mass spectrometry (GC-MS) data monitoring of selected ions was used with principal components analysis (PCA), revealing a set of relevant ions for quantification and characterization of fatty acids. This protocol was successfully applied to the analyses of fatty acids in different human blood lipids, allowing the quantification of several fatty acids and revealing their unsaturation numbers. Moreover the presences of contaminants, artifacts and co-eluitions in the chromatogram were also revealed without any additional analyses. Thus, fatty acid constituents of triglycerides, phospholipids and esters of cholesterol present in plasma and erythrocyte membranes were accurately determined, with repeatability, low limits of determination and of quantification.24101599+Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Agencia Nacional do Petroleo (Brazilian Agency for Petroleum - ANP, SP, Brazil)University of FloridaNSFFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

eNOS T-786C polymorphism affects atorvastatin-induced changes in erythrocyte membrane fluidity

Statins have pleiotropic effects, including endothelial nitric oxide synthase (eNOS) upregulation and increased nitric oxide formation, which can be modulated by a genetic polymorphism in the promoter region of the eNOS gene (T-786C). Here, we report our investigation of whether this polymorphism modulates the effects of atorvastatin on the fluidity of erythrocyte membranes. We genotyped 200 healthy subjects (males, 18-60 years of age) and then randomly selected 15 of these with the TT genotype and 15 with the CC genotype to receive placebo or atorvastatin (10 mg/day oral administration) for 14 days. Cell membrane fluidity was evaluated by electron paramagnetic resonance (EPR) and spin-labeling method. The EPR spectra were registered on a VARIAN-E4 spectrometer. Thiobarbituric acid-reactive species (TBA-RS) and plasma membrane cholesterol were determined in the erythrocytes. Atorvastatin reduced membrane fluidity in CC subjects (P < 0.05) but not in those with the TT genotype (P > 0.05). While no significant differences were found in plasma membrane cholesterol concentrations, higher TBA-RS concentrations were found in the CC subjects than in the TT subjects (P < 0.05). These findings suggest that a short treatment with atorvastatin is disadvantageous to subjects with the CC genotype for the T-786C polymorphism compared to those with TT genotype, at least in terms of the hemorheological properties of erythrocytes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP-Brazil)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Brazil

Endothelial nitric oxide synthase gene haplotypes associated with circulating concentrations of nitric oxide products in healthy men

Objectives Controversy exists regarding the effects of polymorphisms in the endothelial nitric oxide synthase (eNOS) gene on nitrites/nitrates (NOx) plasma concentrations. In this study we compared the distribution of haplotypes involving three relevant eNOS polymorphisms (T-786C in the promoter region; b/a in intron 4, and Glu298Asp in exon 7) in healthy subjects with low and high circulating NOx levels. Methods We studied 154 healthy subjects (fasting, white males, who were non-smokers, 18-60 years of age, and not taking any medication). Genomic DNA was isolated from blood samples and genotypes were determined by PCR and restriction fragment length digestion. Circulating NOx was determined by chemiluminescence. Results Haplotype frequencies were compared in two groups of subjects: those with the 30 lowest NOx levels (group L) and those with the 30 highest NOx levels (group H). NOx levels in group L and H were 24.2 +/- 4.5 mu m and 80.9 +/- 8.9 mu m, respectively. Genotype frequencies for the three polymorphisms were not different when the two groups were compared (all P > 0.05, chi-squared test). However, the haplotype including the alleles C (promoter), 4b (intron 4), and Glu (exon 7) was significantly more common in group L (16%) than in group H (4%) (P=0.0047). The frequencies of the remaining haplotypes were not different among group L and H. Conclusions While eNOS genotypes are not significantly associated with changes in the circulating NOx concentrations, the specific eNOS haplotype that includes the 'C,'4b, and 'Glu' alleles is associated with lower circulating NOx concentrations.15856557

eNOS genotype is without effect on circulating nitrite/nitrate level in healthy male population

Introduction: Nitric oxide (NO) plays an important role in the regulation of the cardiovascular system. It is produced by endothelial nitric oxide synthase (eNOS), which exhibits genetic polymorphisms. Although the clinically relevant polymorphism T-786 C reduces eNOS-promoter activity, it is not clear whether circulating nitrite/nitrate (NOx) are affected by this polymorphism. Materials and Methods: We addressed this issue by studying a homogeneous group of 200 healthy subjects (mates, Caucasians, nonsmokers, 18-56 years of age, and not taking any medication). Genotypes were determined by restriction fragment length polymorphism and circulating NOx were determined by chemiluminescence. Results: We found nonsignificant effects of the T-786C potymorphism on circulating NOx (mean +/- S.D.=52.2 +/- 21.4, 49.0 +/- 17.8, and 45.9 +/- 16.8 mu mol/L for genotypes "TT", "TC", "CC", respectively) and on total plasma cholesterol concentrations (both P>.05). No correlation was found between circulating NOx, and total plasma cholesterol concentrations (P>.05). Conclusions: Our study provides strong evidence that the T-786C potymorphism does not affect plasma NOx concentrations, which are believed to reflect endogenous production of NO. Therefore, our results suggest that this polymorphism does not affect endogenous NO production. (C) 2004 Elsevier Ltd. All rights reserved.115537537

Consistent interethnic differences in the distribution of clinically relevant endothelial nitric oxide synthase genetic polymorphisms

A maldistribution of endothelial nitric oxide synthase (eNOS) genetic variants may explain differences in NO-mediated effects and response to drugs among black and white subjects. While interethnic differences in the distribution of eNOS genetic variants exist in the American population, it is not known whether such interethnic differences exist in other populations. To test this possibility, we examined the distribution of genetic variants of three clinically relevant eNOS polymorphisms (T-C-786 in the promoter, the VNTR in intron 4, and the Glu298Asp variant in exon 7) in 136 black and 154 white subjects from a Brazilian population, which is very heterogeneous. We also estimated the haplotype frequency and evaluated associations between these variants. The Asp298 variant was more common in whites (32.8%) than in blacks (15.1%) (P < 0.004). Similarly, the C-(786) variant was more common in whites (41.9%) than in blacks (19.5%) (P < 0.0004). However, the 4a variant was more common in blacks (32.0%) than in whites (17.9%) (P < 0.003). The most common predicted haplotype in both ethnic groups combined only wild-type variants. While the second most common haplotype in blacks includes the variant 4a and the wild-type variants for the remaining polymorphisms, the second most common haplotype in whites includes the variants Asp298 and C-786 and the wild-type variant for polymorphism in intron 4. The marked interethnic differences that we found in Brazilians are very similar to those previously reported in Americans. These findings strongly suggest a consistent difference in the distribution of eNOS genetic variants in blacks compared with whites and indicate that the interethnic differences do not vary with geographic origin. (c) 2005 Elsevier Inc. All rights reserved.12317718