Measurement of Biologically Labile Hydrogen Sulfide Pools

摘要

A method to measure all relevant biologic hydrogen sulfide pools, namely free hydrogen sulfide, acid-labile sulfide, and bound sulfane sulfur, has been developed. This new method involves selective liberation, trapping and derivatization of labile hydrogen sulfide. The total labile sulfide, including the contribution of the bound sulfane sulfur pool, the acid-labile pool, and free H2S, was measured by incubating the sample with a reducing agent, TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), to reduce disulfide bonds in an acid solution. This method was used to measure the three sulfide pools in blood samples from mice and from humans. This method can be used for research, environmental, and clinical diagnostic purposes in determining hydrogen sulfide bioavailability in biological or other samples.

主权项

1. A method to measure in a sample the concentrations of free H2S, of acid-labile sulfide, and of sulfane-bound sulfur; said method comprising the following steps:
(a) placing separate portions X, Y, and Z of the sample into three individual evacuated containers, whereby most of the free H2S will partition into the headspaces of each of the respective containers; (b) combining portion X with an alkaline buffer, a chelating agent to chelate any trace metals that may be present in the sample, and monobromobimane, and incubating under low oxygen conditions; whereby free H2S will react with the monobromobimane to form a sulfide-dibimane product; chromatographically separating the sulfide-dibimane product from other components of the resulting mixture; and fluorimetrically measuring the amount of the separated sulfide-dibimane product as a measure of the concentration of free H2S in the sample (Result B); (c) combining portion Y with an acid buffer and incubating to release acid-labile sulfide as free H2S; combining portion Y with an alkaline buffer, a chelating agent to chelate any trace metals that may be present in the sample, and monobromobimane, and incubating under low oxygen conditions; whereby free H2S will react with the monobromobimane to form a sulfide-dibimane product; chromatographically separating the sulfide-dibimane product from other components of the resulting mixture; and fluorimetrically measuring the amount of the separated sulfide-dibimane product as a measure of the combined concentration of acid-labile sulfide and free H2S in the sample (Result A); (d) combining portion Z with an acid buffer and a reducing agent to release both acid-labile sulfide and sulfane sulfur as free H2S; combining portion Z with an alkaline buffer, a chelating agent to chelate any trace metals that may be present in the sample, and monobromobimane, and incubating under low oxygen conditions; whereby free H2S will react with the monobromobimane to form a sulfide-dibimane product; chromatographically separating the sulfide-dibimane product from other components of the resulting mixture; and fluorimetrically measuring the amount of the separated sulfide-dibimane product as a measure of the combined concentration of sulfane sulfur, acid-labile sulfide, and free H2S in the sample (Result C); and (e) determining the concentration of free H2S in the sample as equal to Result B; determining the concentration of acid-labile sulfide in the sample as equal to the difference between Result A and Result B; and determining the amount of sulfane sulfur as equal to the difference between Result C and Result A.