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

• Published in: British journal of pharmacology Vol. 160; no. 4; pp. 941 - 957
• Main Authors: Wintner, Edward A, Deckwerth, Thomas L, Langston, William, Bengtsson, Asa, Leviten, Dina, Hill, Paul, Insko, Michael A, Dumpit, Ronald, VandenEkart, Emily, Toombs, Christopher F, Szabo, Csaba
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.06.2010; Nature Publishing Group
• Subjects: amperometric detection; Analytic Sample Preparation Methods - methods; Animals; bimane; Biological and medical sciences; blood; Bridged Bicyclo Compounds - chemistry; Chromatography, High Pressure Liquid; fluorescence detection; Fluorescent Dyes - chemistry; Free Radicals - analysis; Free Radicals - blood; Free Radicals - chemistry; Humans; Hydrogen Sulfide - administration & dosage; Hydrogen Sulfide - blood; Hydrogen Sulfide - chemistry; Hydrogen Sulfide - pharmacokinetics; hydrogen sulphide; Kinetics; Limit of Detection; Male; Medical sciences; monobromobimane; pharmacokinetics; Pharmacology. Drug treatments; Rats; Rats, Sprague-Dawley; Research Papers; sodium sulphide; Spectrometry, Fluorescence; Spectrometry, Mass, Electrospray Ionization; Sulfhydryl Compounds - analysis; Sulfhydryl Compounds - blood; Sulfhydryl Compounds - chemistry; Sulfhydryl Reagents - chemistry; Sulfides - administration & dosage; Sulfides - blood; Sulfides - chemistry; Sulfides - pharmacokinetics; Sulfides - therapeutic use; Tandem Mass Spectrometry; Biological fluid; Fluorescence detector; Hydrogen Sulfides; bimane; fluorescence detection; sodium sulphide; Sodium Sulfides; amperometric detection; Blood; monobromobimane; Detection; Pharmacokinetics; hydrogen sulphide; Species
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/j.1476-5381.2010.00704.x

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 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.