Comprehensive analysis of how experimental parameters affect H2S measurements by the monobromobimane method

• Published in: Free radical biology & medicine 2019-05, Vol.136, p.146-158
• Main Authors: Ditrói, Tamás, Nagy, Attila, Martinelli, Diego, Rosta, András, Kožich, Viktor, Nagy, Péter
• Format: Article
• Language: English
• Subjects: Blood sulfide levels; Hydrogen sulfide; Monobromobimane method; Sulfide pool
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2019.04.006

A rapidly increasing number of studies report on widespread biological functions for endogenous hydrogen sulfide. However, the use of multiple, chemically distinct analytical methods to measure free hydrogen sulfide levels in biological samples accumulate data that are not in agreement with each other. In this work a widely appreciated technique, the monobromobimane method, was thoroughly investigated with the overall aims i) to demonstrate how results obtained by different versions of the method should be interpreted and ii) to provide an easy protocol for the community in order to obtain reliable and comparable results. We demonstrate that none of the previously published versions of the method measure free sulfide concentrations in blood serum or plasma samples due to significant interferences with the biomolecule-bound sulfide pool. On the other hand, we stress the biological relevance of these measurements in cases in which they are carefully conducted. To aid future studies, we extensively investigated the entire procedure from sample withdrawal through handling and storing of injection-ready samples until the detection protocol in order to pinpoint all parameters that can affect the final readouts. Based on our rigorous analytical investigations a set of recommendations were compiled that are necessary to ensure reliable, reproducible and comparable results in the field and a detailed standardized protocol is provided. [Display omitted] •Parameters affecting measured blood H2S levels by the MBB method were comprehensively analyzed.•EDTA tubes should not be used for blood collection; all chelators should be excluded from reagents.•Temperature, MBB concentration and alkylation time need to be tightly controlled.•UV–vis light should be excluded during sample preparation, but oxygen has negligible impact.•A protocol was suggested, which provided highly reproducible results in clinical samples.