A monobromobimane-based assay to measure the pharmacokinetic profile of reactive sulphide species in blood

Edward A. Wintner, Thomas L. Deckwerth, William Langston, Asa Bengtsson, Dina Leviten, Paul Hill, Michael A. Insko, Ronald Dumpit, Emily Vandenekart, Christopher F. Toombs, Csaba Szabo

Research output: Contribution to journalArticlepeer-review

172 Scopus citations

Abstract

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 H 2S/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 μM. Intravenous administration of sodium sulphide solution (2-20 mg·kg-1·h-1) 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.

Original languageEnglish (US)
Pages (from-to)941-957
Number of pages17
JournalBritish Journal of Pharmacology
Volume160
Issue number4
DOIs
StatePublished - Jun 2010
Externally publishedYes

Keywords

  • Amperometric detection
  • Bimane
  • Blood
  • Fluorescence detection
  • Hydrogen sulphide
  • Monobromobimane
  • Pharmacokinetics
  • Sodium sulphide

ASJC Scopus subject areas

  • Pharmacology

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