A monobromobimane-based assay to measure the pharmacokinetic profile of reactive sulphide species in blood.
OBJECTIVE
Hydrogen sulphide (H(2)S) 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 H(2)S in blood and tissues.


METHODS
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 H(2)S gas inhalation, and levels of available blood sulphide were measured. Levels of dissolved H(2)S/HS(-) were concomitantly measured using an amperometric sensor.


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 microM. Intravenous administration of sodium sulphide solution (2-20 mg x kg(-1) x h(-1)) or inhalation of H(2)S gas (50-400 ppm) elevated reactive sulphide in blood in a dose-dependent manner. Each 1 mg x kg(-1) x h(-1) of sodium sulphide infusion into rats was found to be pharmacokinetically equivalent to approximately 30 ppm of H(2)S gas inhalation.


CONCLUSIONS
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 H(2)S gas.
Wintner
Edward A
EA
Ikaria, Seattle, WA, USA.
Deckwerth
Thomas L
TL
Langston
William
W
Bengtsson
Asa
A
Leviten
Dina
D
Hill
Paul
P
Insko
Michael A
MA
Dumpit
Ronald
R
VandenEkart
Emily
E
Toombs
Christopher F
CF
Szabo
Csaba
C
D053000:Analytic Sample Preparation Methods / Q000379:methods*; D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D005609:Free Radicals / Q000032:analysis / Q000097:blood / Q000737:chemistry; D006801:Humans; D006862:Hydrogen Sulfide / Q000008:administration & dosage / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D007700:Kinetics; D057230:Limit of Detection; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000097:blood* / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D013440:Sulfides / Q000008:administration & dosage / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics* / Q000627:therapeutic use; D053719:Tandem Mass Spectrometry
D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D005609:Free Radicals; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D013440:Sulfides; C028381:monobromobimane; C033479:sodium sulfide; D006862:Hydrogen Sulfide