BACKGROUND: Hydrogen sulfide (H2S) is established as the third gaseous signaling molecule and is known to be overproduced in down syndrome (DS) due to the extra copy of the CBS gene on chromosome 21, which has been suggested to contribute to the clinical manifestation of this condition. We recently discovered trimethylsulfonium (TMS) in human urine and highlighted its potential as a selective methylation metabolite of endogenously produced H2S, but the clinical utility of this novel metabolite has not been previously investigated. We hypothesize that the elevation of H2S production in DS would be reflected by an elevation in the methylation product TMS.
METHODS: To test this hypothesis, a case-control study was performed and the urinary levels of TMS were found to be higher in the DS group (geo. mean 4.5 nM, 95 % CI 2.4-3.9) than in the control (N) group (3.1 nM, 3.5-6.0), p-value 0.01, whereas the commonly used biomarker of hydrogen sulfide, thiosulfate, failed to reflect this alteration in H2S production (15 µM (N) vs. 13 µM (DS), p-value 0.24.
RESULTS: The observed association is in line with the proposed hypothesis and provides first clinical evidence of the utility of TMS as a novel and more sensitive biomarker for the endogenous production of the third gaseous signaling molecule than the conventionally used biomarker thiosulfate, which is heavily dependent on bacterial hydrogen sulfide production.
CONCLUSIONS: This work shows that TMS must be explored in clinical conditions where altered metabolism of hydrogen sulfide is implicated.

Although primarily considered as a toxic gas, hydrogen sulfide is naturally produced in humans from the amino acid cysteine in low quantities and is commonly referred to as the \"third gaseous signaling molecule\". There is a need to establish reliable biomarkers for exposure to hydrogen sulfide in human matrices not only at toxic levels but also at trace levels. Herein, we report optimized analytical methods for the direct determination of three compounds related to hydrogen sulfide metabolism in human urine without sample preparation; namely, trimethylsulfonium, thiosulfate, and cystine. The methods were based on ion-pair ultra-high performance liquid chromatography (UHPLC) with an anionic fluorinated acid or a cationic fluoroalkylamine, a member of a new generation of ion-pairing reagents, and detection by the electrospray ionization tandem mass spectrometry (ESI-MS/MS). Rapid chromatographic separation was achieved in 2-3 min with limits of detection of 0.1, 2.5, and 50 nmol L-1 (nM) for trimethylsulfonium, cystine, and thiosulfate, respectively. Isotopically labeled internal standards were used for each analyte to account for matrix effects and qualifier ions were employed to ensure selectivity. The method was validated for recovery (generally within 80-120%) and repeatability (RSD% 1.0-10%), and applied to investigate the normal concentrations of the three analytes in forty morning first-pass urine samples of healthy volunteers.