Stable isotope dilution mass spectrometry quantification of hydrogen sulfide and thiols in biological matrices
Hind Malaeb,
Ibrahim Choucair,
Zeneng Wang,
Xinmin S. Li,
Lin Li,
W. Christopher Boyd,
Christopher Hine,
W.H. Wilson Tang,
Valentin Gogonea,
Stanley L. Hazen
Affiliations
Hind Malaeb
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Department of Chemistry, Cleveland State University, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA
Ibrahim Choucair
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA
Zeneng Wang
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA
Xinmin S. Li
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA
Lin Li
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA
W. Christopher Boyd
Department of Chemistry, Cleveland State University, Cleveland, OH, USA
Christopher Hine
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA
W.H. Wilson Tang
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA; Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic, Cleveland, OH, USA
Valentin Gogonea
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Department of Chemistry, Cleveland State University, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA; Corresponding author. Department of Chemistry, Cleveland State University, 2121 Euclid Ave., Cleveland, OH, 44115, USA.
Stanley L. Hazen
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; Department of Chemistry, Cleveland State University, Cleveland, OH, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH, USA; Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic, Cleveland, OH, USA; Corresponding author. Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., NC-10, Cleveland, OH, 44195, USA.
Background: Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices. Methods: After synthesizing [34S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota. Results: Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r2 > 0.997), recovery (90.9%–110%) and stability (90.0%–100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0–66.7% reduction with antibiotics). Conclusions: A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.
