敏感硫电极法在测定心血管组织细胞及血浆胱硫醚-γ-裂解酶/硫化氢的应用

目的:建立测定微量硫化氢(Hydrogen sulfide, H2S)的敏感硫电极法.方法:根据硫化氢的理化特性,应用化学反应将溶液中物理溶解和化学形式存在的硫化氢转变成硫离子(S2-),应用敏感硫电极检测微量S2-,换算出溶液中H2S,构建了敏感硫电极检测H2S的方法.并检测了大鼠及人血浆中H2S的浓度、大鼠心血管组织中内源性H2S的含量以及大鼠心血管组织和细胞胱硫醚-γ-裂解酶(cystathionine-γ-lyase, CSE)的活性.结果:敏感硫电极检测1～80 μmol/L的S2-有较好的指数相关关系,应用该方法检测到雄性和雌性大鼠血浆H2S的浓度分别为(40±4)和(41±5) μmol/L,差异无统计学意义,人类男性和女性静脉血血浆H2S浓度分别为(33±4) μmol/L 和(35±5) μmol/L,差异无统计学意义.雌、雄大鼠主动脉组织H2S的含量分别为每毫克蛋白(24±6)和(25±5) nmol,心肌组织含量分别为每毫克蛋白(19±4) 和(19±6) nmol,差异无统计学意义.采用敏感硫电极法测量主动脉组织CSE活性与传统方法测量结果差异无统计学意义,但可精确测量出血管平滑肌细胞CSE的活性.结论:敏感硫电极法可以应用于CSE/H2S信号通路的检测.

Application of sensitive sulphur electrode assay to measure and analyze cystathionine-γ-lyase/hydrogen sulfide pathway of cardiovascular tissues, cells and plasma in rats

OBJECTIVE: To construct a method of measurement microamount hydrogen sulfide (H(2)S) using sensitive sulphur electrode. METHODS: According to the physical and chemical characters of H(2)S, H(2)S, which in the fluids by mean of physical dissolve and chemical shape, is turned to sulphur ion (S(2-)) by chemical responses. After the microamount of S(2-) was measured by sensitive sulphur electrode, and the concentration of H(2)S was converted, a method was constructed to measure the H(2)S. It was used to analyze the concentrations of H(2)S of plasma in rats and humans, the endogenous concentration of H(2)S of cardiovascular tissue in rats, and CSE activity of cardiovascular tissues and cells. RESULTS: The exponential regression of S(2-) in the extent including 1 to 80 micromol/L was found using sensitive sulphur electrode. The H(2)S levels of plasma in male and female rats were 40+/-4 and 41+/-5 micromol/L, respectively, and significant difference was not found; those in venous blood plasma of men and women were 33+/-4 micromol/L and 35+/-5 micromol/L respectively, without significant difference. There were not significant differences in the aortic endogenous levels of H(2)S (24+/-6 and 25+/-5 nmol/mg pro) and myocardial levels (19+/-4 and 19+/-6 nmol/mg protein) between female and male rats. There were no different results of CSE activity in aortal tissue using sensitive sulphur electrode or traditional methods, however, the CSE activity of vascular smooth muscle cells could be accurately measured using sensitive sulphur electrode, which was difficult in using traditional method. CONCLUSION: The sensitive sulphur electrode assay was fit for the analysis of CSE/H(2)S pathway in cardiovascular research.