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

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 Zn²⁺ to plasma sample to deposit H₂S, HS⁻ and S²⁻, 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.