Despite several methods to measure H2S, physiological H2S serum concentrations remain highly variable and method-dependent. While modifying the classic methylene blue (MB) method to improve detection of serum H2S levels, we also probed potential sulfur-binding characteristics of serum albumin.
To avoid loss of serum H2S and to trap sulfide ions effectively in the MB method serum was strongly alkalized to completely ionize gaseous H2S, followed by incubation with zinc acetate at 37°C, to trap sulfide ions effectively. After incubation, N,N-Diethyl-p-phenylenediamine and FeCl3 were added to form MB product. Subsequently, trichloroacetic acid was added to remove matrix effects. Absorbance of MB product was at 670nm and compared to a dilution range of NaHS. Spectra (400-900nm) were taken to check quality of MB product and exclude matrix effects. Adaptations in experimental setup are mentioned below.
Applying the modified MB method produced the unexpected finding that yield of serum H2S increases with higher pH and longer incubation time. H2S synthesizing enzymes cystathionine-β-synthase and cystathionine -lyase were both ruled out as sources of serum H2S by separately treating serum with their respective inhibitors hydroxylamine and DL-propargylglycine. Human serum protein standard containing only albumin and -globulin, or pure bovine serum albumin (BSA), also yielded H2S. Albumin was confirmed as source of H2S by absence of increasing H2S yield from analbuminemic rat serum, as compared to normal rat serum. That yield of H2S increased at least twofold after treating both BSA and human serum with dithiolthreitol (DTT), a disulphide reducer, and close correlation between albumin level and yield of H2S during DTT incubation (r2=0.99), both suggest H2S storage in albumin as “labile sulphurs”.
The modified MB method allows measurement of release of H2S from albumin. Treating albumin with a disulphide reducer revealed H2S storage in albumin as sulfanes. Our work is consistent with suggestions that circulating 'pools' of biologically relevant sulfur/sulfide species exist. Thus, albumin may be a source of H2S, as has been observed for NO. How this is affected by disease states (e.g. chronic renal failure) remains to be seen.