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[Modified methylene blue method for measurement of hydrogen sulfide level in plasma.] [Articl in Chinese]

ZHENG Yang, LIAO Feng, DU Junbao, Tang Chaoshu, XU Guoheng, GENG Bin

Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100191, China

Abstract

In past decade, hydrogen sulfide (H2S) as a novel gasotransmitter, covered many fields in biological and medical research. However, there is no effective, convenient and high-throughput method for determination of circulatory H2S until now. Here, we aim to develop an easy method for measurement of circulatory H2S by modified methylene blue method. In the present study, we added Zn(2+) to plasma sample to deposit H2S, HS(-) and S(2-), as well as plasma protein, then used NaOH to re-dissolve plasma protein. ZnS deposition was re-dissolved by the addition of N, N-dimethyl-p-phenylenediamine, and the remnant protein was deposited by trichloroacetic acid. After centrifugation, ferriammonium sulfate was added to the supernatant fluid to generate methylene blue, which was analyzed by spectrophotometer at 665 nm. Using the present method, we found that most ions including sulfate and thiosulfate did not affect detection of H2S concentration, but albumin (physiological concentration) reduced the detection value, which suggested the binding of serum albumin and a certain amount of H2S. The relative recovery ratio of present method is 81.9%, which implies that the method is relative accurate for the determination of H2S concentration in plasma or serum. H2S levels of frozen plasma samples from 65 healthy volunteers detected by the present method were (13.93 ± 4.98) µmol/L. There was no obvious difference between the detection values of fresh and frozen samples from the same SD rats. These results suggest the modified methylene blue assay is stable, sensitive, convenient and high-throughput. The method can be used to analyze the circulatory H2S in clinical and basic research.

Key words: hydrogen sulfide; plasma; methylene blue; GYY4137

Received: 2012-03-23　　Accepted: 2012-07-13

Corresponding author: 耿彬　　E-mail: bingeng@hsc.pku.edu.cn

Citing This Article：

ZHENG Yang, LIAO Feng, DU Junbao, Tang Chaoshu, XU Guoheng, GENG Bin. [Modified methylene blue method for measurement of hydrogen sulfide level in plasma.] [Articl in Chinese]. Acta Physiol Sin 2012; 64 (6): 681-686 (in Chinese with English abstract).