红细胞内源性硫化氢的测定

目的:建立测定红细胞内源性硫化氢(hydrogen sulfide, H2S)的方法.方法:根据红细胞硫化氢生成酶的理化特性,给予酶的底物β-巯基丙酮酸,并根据亚甲基蓝法测定释放硫化氢的量.结果:应用RT-PCR方法检测到红细胞内存在巯基丙酮酸转硫酶(3-mercaptopyruvate sulfurtransferase, MPST)的表达;红细胞内源性硫化氢的产率高达每10⁸个细胞(22.76±1.53) μmol/min,约是肝和肾组织内硫化氢生成率的5倍.而检测L-半胱氢酸(L-cysteine, L-cys)途径内源性硫化氢的产率,并以肝和肾作为阳性对照组织,发现红细胞内几乎检测不到硫化氢的产生.结论:红细胞内源性硫化氢的产生主要依赖MPST途径,本研究构建的方法可应用于红细胞生成硫化氢的检测.

Assay of endogenous hydrogen sulfide from erythrocytes

OBJECTIVE: To construct a detection method of endogenous hydrogen sulfide (H2S) from erythrocytes. METHODS: Prepared rat erythrocyte (10(6) cell) was added in 4 mL 2-amino-2-methyl-1,3-propanedid (225 mmol/L, pH=9.55) and transonically lysed for 3 times. The erythrocyte lysis was tranfered into a 25 mL Erlenmeyer flask and beta-mercaptopyruvate was added for final concentration of 2 mmol/L. Cryovial test tubes (2 mL) were used as the centre wells each contained 0.5 mL of 1% zinc acetate as trapping solution and a filter paper of 2.0x2.5 cm2 to increase the air/liquid contacting surface. The flasks containing reaction mixture and centre wells were flushed with N2 before being sealed with a double layer of Parafilm. Reaction was initiated by transferring the flasks from ice to a 37 degrees C shaking water bath. After incubation at 37 degrees C for 60 min, 0.5 mL of 50% trichloroacetic acid was added into the reaction mixture to stop the reaction. The flasks were sealed again and incubated at 37 degrees C for another 60 min to ensure a complete trapping of the H2S released from the mixture. Released H2S is absorbed by zinc acetate and generation zinc sulfide. The zinc sulfide formed is dissolved in a hydrochloric acid solution of paminodimethylaniline (N,N-dimethyl-p-phenylenediamine), and methylene blue is formed within 10 min at room temperature in the presence of ferric chloride. The blue color of methylene blue is measured at 670 or 650 nm on spectrophotometer. RESULTS: We first demonstrated the key enzymes of endogenous H2S generation-3-mercaptopyruvate sulfurtransferase (MPST) gene expression by RT-PCR. Endogenous H2S production from rat erythrocytes was 22.76+/-1.53 micromol/min/10(8) cells, about 4-folds as compared with to the liver and kidney tissues. However, the endogenous H2S production from erythrocyte by L-cysteine pathway was little detected. CONCLUSION: Endogenous H2S released from erythrocytes depended mainly on MPST pathway. Our method is effective to detect the erythrocytic endogenous H2S.