[Assay of endogenous hydrogen sulfide from erythrocytes]

To construct a detection method of endogenous hydrogen sulfide (H2S) from erythrocytes.

Prepared rat erythrocyte (10(6) cell) was added in 4 mL 2-amino-2-methyl-1,3-propanediol(225 mmol/L, pH=9.55) and ultrasonically lysed for 3 times. The erythrocyte lysis was transferred 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 amino-dimethylaniline (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.

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.

Endogenous H2S released from erythrocytes depended mainly on MPST pathway. Our method is effective to detect the erythrocytic endogenous H2S.