| BackgroundElevated levels of serum homocysteine(Hcy) are an independent risk factor for coronary heart disease(CHD).However,the precise mechanism of homocysteine-associated cardiovascular disease is still unclear.Available data suggest that homocysteine thiolactone(HTL),as one of metabolites of Hcy,can react with lysine residues in proteins forming HTL adducts,damaging their structure and impairing their physiological activities.The most important pathogenesis of HTL adducts includes:1. harmful to human endothelial cell and adversely affecting vascular endothelium; 2.stimulating aggregation of low density lipoprotein(LDL),inducing LDL uptake by macrophages in culture and facilitating the formation of foam cells;3.HTL modified LDL has been found to be easier to form small dense LDL,one of the most important risk factors for CHD;4.HTLresult in the formation of thromboxane B2 and prostacyclin 6-keto-PGF1 alpha during thrombosis,which induces primary platelet aggregation and clot formation.HTL adducts are now considered as markers for cardiovascular diseases. Homocysteine thiolactone hydrolase(HTase),which is a calcium-dependent enzyme, may hydrolyze HTL to Hcy and thereby prevent the homocysteinylation of proteins. Experiments in vitro demonstrate that high activity forms of HTase afford better protection against protein homocysteinylation than low activity forms of HTase.Our research aimed to develop a stable and convenient assay system for measuring HTL adducts,and to evaluate the correlation between plasma HTL adducts and CHD. Homocysteine thiolactone adducts have been proposed as the culprit of homocysteine related cardiovascular diseases.We studied the association of these adducts in plasma, and the gene polymorphism ofparaoxonase-2 with CHD.Materials and Methods1.The antigen of HTL adducts was prepared by modifying the rabbit albumin in vitro.Then female New Zealand White rabbits were immunized using standard protocols. Special antibody against HTL adducts were developed and evaluated.An ELISA method was established for measuring HTL adducts;2.Two hundred and fifty-four patients and 308 controls were recruited for the study. HTL adducts were determined with ELISA.Detailed medical history and thorough physical examinations were performed on all participants.Weight,height and the body mass index(BMI) were recorded.The level of plasma HTL adducts was measured by ELISA method and plasma Hcy was detected by HPLC methods.Blood biochemical analyses were carried out by using automatic biochemistry analyzer.3.The codon 311 polymorphism of paraoxonase-2 gene was genotyped by using polymerase chain reaction and restrictive digestion.4.Statistical analysis:All calculations used SPSS 10.0 for Windows statistical analysis software.Measurement data was indicated by Mean±SD.Data among groups was comp rated by t-test or x~2 test according to the type of variables.Multiple stepwise logistic regression analysis,correlation analysis and multiple linear regression analysis were used.The distribution of the HTase gene polymorphism was in Hardy-Weinberg equilibrium.Results1.Human serum albumin and rabbit serum albumin were modified by HTL.The extent of modification was determined by DTNB and TNBS assays.TNBS assay showed that 20 percent of lysine residues of HSA and RSA were modified by HTL.2.Polyclonal antibody directed against HTL adducts was generated and its working concentration is about 1:500.In order to determine antibody specificity solid phase competition-based ELISA techniques were used.We conclude that the antibody we got is specifically directed against protein modified by HTL and not against native protein.3.The specificity and the sensitivity of the methods allow detection of HTL adducts quantities from 12.5 to 200 u/ml.High plasma HTL adducts was defined based the 90th percentile of the value of the control population(≥37.57 u/ml).Intra-assay coefficient of variation was 6.4%and inter-assay coefficient of variation was 8.9%.4.The plasma level of HTL adducts were significantly higher in patients than in controls(40.65±10.87 u/ml vs.30.58±10.20 u/ml,P＜0.01),with odds ratio 7.34,(95% confidence interval 4.020～13.406,P＜0.01),and increased according to the number of atherosclerotic coronary arteries:35.59±10.34 units/ml(n=76);41.88±8.83(n=70) and 43.13±11.47(n=108) in subjects with 1,2 and 3 affected arteries,respectively(r=0.174, P＜0.01).5.The frequency of CC genotype was significantly higher in patients with coronary heart disease(7.48%) than in controls(1.62%,P＜0.01),with adjusted odds ratio of 4.367 (95%confidence interval:1.178 to 16.191,P＜0.01),so was the C allele(23.2%vs. 14.9%,P=0.017).ConclusionHigh plasma HTL adducts and the CC 311 genotype of paraoxonase-2 gene may be the emerging risk factor for CHD. Background Growth-differentiation factor 15(GDF15) is a novel antihypertrophic factor in the heart,which is induced in response to cardiac injury and plays important regulatory role in the process of left ventricular hypertrophy.We hypothesized that genetic variants of GDF15 may associate with left ventricular mass and geometry in hypertension.Methods A community-based hypertensive population sample of 1527 persons was studied by Mono-mode echocardiography.Three single-nucleotide polymorphisms (SNPs),including one tagSNP -3148C＞G of a natural haplotype and two exonic SNP (+157A＞T and +2438C＞G) were genotyped.The SNP functions were studied by use of luciferase reporter assays and determination of GDF-15 serum levels.Results Only the tagSNP -3148G showed significantly association with lower risk of left ventricular hypertrophy(OR=0.78,95%CI 0.65-0.94,P=0.009).In multiple regression analyses,-3148G predicted statistically significant decrease in left ventricular end-diastolic diameter(β=-0.10,P=0.0001),end-systolic diameter(β=-0.09,P=0.0007), mass(β=-0.11,P＜0.0001) and indexed mass(β=-0.12,P＜0.0001).These effects were independent of conventional factors,including sex,age,body surface area,blood pressure,diabetes,and anti-hypertensive treatment,and cigarette and alcohol consumption.The SNP -3148G-associated GDF-15 promoter exhibited 192%(P＜0.001) of transcription activity than the SNP -3148C-associated promoter.The -3148G allele was also associated with a significant increase of GDF-15 serum level(P=0.04) in the hypertensive subjects.Conclusions—Genetic variation within the promoter region of GDF15 gene is strongly associated with left ventricular size,mass and hypertrophy status in human essential hypertension. |
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