Measurement of plasma hydrogen sulfide in vivo and in vitro

The gasotransmitter hydrogen sulfide is known to regulate multiple cellular functions during normal and pathophysiological states. However, a paucity of concise information exists regarding quantitative amounts of hydrogen sulfide involved in physiological and pathological responses. This is primarily due to disagreement among various methods employed to measure free hydrogen sulfide. In this article, we describe a very sensitive method of measuring the presence of H₂S in plasma down to nanomolar levels, using monobromobimane (MBB). The current standard assay using methylene blue provides erroneous results that do not actually measure H₂S. The method presented herein involves derivatization of sulfide with excess MBB in 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM DTPA) for 30 min in 1% oxygen at room temperature. The fluorescent product sulfide-dibimane (SDB) is analyzed by RP-HPLC using an eclipse XDB-C18 (4.6 × 250 mm) column with gradient elution by 0.1% (v/v) trifluoroacetic acid in acetonitrile. The limit of detection for sulfide-dibimane is 2 nM and the SDB product is very stable over time, allowing batch storage and analysis. In summary, our MBB method is suitable for sensitive quantitative measurement of free hydrogen sulfide in multiple biological samples such as plasma, tissue and cell culture lysates, or media.