Background and purpose: Hydrogen sulphide (H2S) is a labile, endogenous metabolite of cysteine, with multiple biological roles. The development of sulphide-based therapies for human diseases will benefit from a reliable method of quantifying H2S in blood and tissues. Experimental approach: Concentrations of reactive sulphide in saline and freshly drawn whole blood were quantified by reaction with the thio-specific derivatization agent monobromobimane, followed by reversed-phase fluorescence HPLC and/or mass spectrometry. In pharmacokinetic studies, male rats were exposed either to intravenous infusions of sodium sulphide or to H2S gas inhalation, and levels of available blood sulphide were measured. Levels of dissolved H2S/HS -were concomitantly measured using an amperometric sensor. Key results: Monobromobimane was found to rapidly and quantitatively derivatize sulphide in saline or whole blood to yield the stable small molecule sulphide dibimane. Extraction and quantification of this bis-bimane derivative were validated via reversed-phase HPLC separation coupled to fluorescence detection, and also by mass spectrometry. Baseline levels of sulphide in blood were in the range of 0.4-0.9 mM. Intravenous administration of sodium sulphide solution (2-20 mg·kg) or inhalation of H2S gas (50-400 ppm) elevated reactive sulphide in blood in a dose-dependent manner. Each 1 mg·kg -1 ·h -1 of sodium sulphide infusion into rats was found to be pharmacokinetically equivalent to approximately 30 ppm of H2S gas inhalation. Conclusions and implications:The monobromobimane derivatization method is a sensitive and reliable means to measure reactive sulphide species in whole blood. Using this method, we have established a bioequivalence between infused sodium sulphide and inhaled H2S gas.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Hydrogen sulphide (H2S) is a biological mediator and a potential therapeutic agent. In animal studies, the metabolism and pharmacokinetics of H2S have been characterized. WHAT THIS STUDY ADDS• This study is first to demonstrate the pharmacokinetics of an intravenously administered H2S formulation in humans, and to characterize the exhaled H2S response in humans. INTRODUCTIONHydrogen sulphide (H2S) is an endogenous gaseous signaling molecule and potential therapeutic agent. Emerging studies indicate its therapeutic potential in a variety of cardiovascular diseases and in critical illness. Augmentation of endogenous sulphide concentrations by intravenous administration of sodium sulphide can be used for the delivery of H2S to the tissues. In the current study, we have measured H2S concentrations in the exhaled breath of healthy human volunteers subjected to increasing doses sodium sulphide in a human phase I safety and tolerability study. METHODSWe have measured reactive sulphide in the blood via ex vivo derivatization of sulphide with monobromobimane to form sulphide-dibimane and blood concentrations of thiosulfate (major oxidative metabolite of sulphide) via ion chromatography. We have measured exhaled H2S concentrations using a custom-made device based on a sulphide gas detector (Interscan). RESULTSAdministration of IK-1001, a parenteral formulation of Na2S (0.005-0.20 mg kg -1 , i.v., infused over 1 min) induced an elevation of blood sulphide and thiosulfate concentrations over baseline, which was observed within the first 1-5 min following administration of IK-1001 at 0.10 mg kg -1 dose and higher. In all subjects, basal exhaled H2S was observed to be higher than the ambient concentration of H2S gas in room air, indicative of on-going endogenous H2S production in human subjects. Upon intravenous administration of Na2S, a rapid elevation of exhaled H2S concentrations was observed. The amount of exhaled H2S rapidly decreased after discontinuation of the infusion of Na2S. CONCLUSIONExhaled H2S represents a detectable route of elimination after parenteral administration of Na2S.

Our strategy for the identification of active species should be broadly applicable to other methods of generating complex libraries of small molecules. The selection from the library of a compound with desired biological properties augurs well for the potential value of generating and screening complex mixtures of small molecules in solution.

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Edward A. Wintner was born outside Philadelphia, PA, son of two Haverford College professors. He survived high school despite breakfast-table tutelage In English, German, and chemistry and went on to study at Yale, where he received a B.S. degree In chemistry. He graduated from Yale In 1991 and has since been a graduate student under Professor Rebek as an NSF predoctoral fellow. Still not tired of academic Hie, Ed hopes to pursue a career In organic chemistry as a university professor.M. Morgan Conn was born In 1968 In Ramapo, NY, and raised In Canada. He received his B.Sc. In chemistry and biochemistry from the University of Toronto In 1989. He joined the group of Prof. Julius Rebek, Jr., at MIT as an NSF predoctoral fellow and, subsequently, an NSERC predoctoral fellow and received his Ph.