Genotyping of the eNOS Polymorphism
DNA was extracted from the whole EDTA blood by means of a standard procedure (QIAmp Blood Kit, Qiagen). The variant of eNOS gene being analyzed is the G894T, in which a substitution of G to T at nucleotide position 894 results in a replacement of glutamic acid for aspartic acid at codon 298 (Glu298Asp) in the encoded protein. This 894G/T polymorphism in exon 7 of the eNOS gene was genotyped by a standard polymerase chain reaction (PCR) amplification and detected by restriction fragment length polymorphism. Approximately 10% of our samples were randomly selected for repeat PCR and genotyping for quality control of these assays.
Echocardiography and Determination of LVM
Two-dimensional guided M-mode echocardiograms were obtained with an ATL HDI 5000 echocardiography system (Advanced Technology Laboratories, Bothell, WA) equipped with a broadband, wide-angle phased-array transducer. All examinations were recorded on videotapes or directly stored in digital form on disks, from which the echo-parameters were calculated offline using the internal analysis software of the echocardiographic system. M-mode recordings were obtained from the left parasternal window, and LV internal dimension and interventricular septal and posterior wall thickness were measured at end-diastole and end-systole, in up to five consecutive cycles, according to the recommendations of the American Society of Echocardiography. In addition to the conventional LV structural and fimctional parameters, LVM (according to the Penn Convention) was calculated by Devereux formula and indexed (LVM index— LVMI) for body surface area (BSA). LVM was also indexed for body height raised to the 2.7th power, previously shown to provide a better normalization for LVM variability associated with body size and gender, independent of obesity. LVH was defined with the use of gender-specific partition points for LVMI by body surface area (g/m2) exceeding 134 in men and 110 in women or LVMI by height (g/height) exceeding 49.2 in men and 46.7 in women. generic levitra online
Data are expressed as mean values ± 1 SD, unless otherwise indicated. The M-mode and Doppler echocardiographic data represent the average of 3-5 consecutive beats. Their observer variability has been reported previously. Comparisons between BP (Generic Aceon used to treat high blood pressure and to prevent heart attack) groups were performed by use of the Student’s t-test for paired or unpaired data, the Fisher’s exact test or Chi-square contingency analysis (with Yates’ correction), where appropriate. All tests were two-sided, and a p value <0.05 was considered statistically significant. Comparison between the three G894T genotypes was performed by use of the one-way ANOVA and the Bonferroni correction for post hoc comparisons. A Chi-square test was also used to confirm that the observed genotype frequencies did not deviate from the Hardy-Weinberg equilibrium. Univariate relations between variables were assessed by linear regression analysis and nonlinear fitting routines. As an extension to this univariate analysis, a bivariate analysis was also applied to assess for the potential confounding influence of each covariate on the relation between G894T genotype and LVM. The variables of clinical importance, as identified by bivariate approach, were then proposed for inclusion into the final multiple regression models with LVM as dependent and G894T genotype as an independent variable, including age, gender, BMI, smoking and systolic BP (Drug Cardizem is used for treating supraventricular tachycardia, a rhythm disturbance of the heart).