Analysis of endogenous HS and HS in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (HS), have various physiological functions in mammalian cells. Although HS molecules have been considered as secondary metabolites derived from hydrogen sulfide (HS) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HS for simultaneous determination of HS, HS, HS and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of HS and HS standards under alkaline conditions (pH 9.5) induced significant decreases in HS and HS levels and a significant increase in the HS level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of HS and HS were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of HS, HS, and HS in mouse brain under physiological pH conditions. The concentrations of HS and HS were 0.030 ± 0.004μmol/g protein and 0.026 ± 0.002μmol/g protein, respectively. Although the level of HS was below the quantification limit of this method, HS was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous HS and HS in mammalian brain tissues. HS and HS exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.