Abstract
The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.
Highlights
Thiol and thioether metabolites are a group of low-molecular weight molecules involved in one-carbon metabolism, amino acid biosynthesis, redox homeostasis, and transsulfuration reactions
We developed a method for the quantitative assessment of 10 biological thiol and thioether metabolites and creatinine, all of which are involved in B-vitamin metabolism relevant to folate, methionine and transsulfuration pathways (Figure 1a)
This study revealed that the distribution and redox state of thiol and thioether metabolites is specific to cell types
Summary
Thiol and thioether metabolites are a group of low-molecular weight molecules involved in one-carbon metabolism, amino acid biosynthesis, redox homeostasis, and transsulfuration reactions. Thiols and thioethers connect central pathways of metabolism by serving as substrates for enzymatic reactions (methionine, homocysteine, cysteine, cystathionine) [1], redox buffers (cysteine, glutathione) [2,3] and in the post-translational modification of cysteine residues in proteins (S-homocysteinylation, S-glutathionylation, S-cysteinylation) [4,5,6]. Mutations in genes encoding enzymes of the methionine, cobalamin, folate and transsulfuration pathways lead to elevated or reduced concentrations of enzymatically-produced thiol and thioether metabolites, which have diagnostic value. Inherited diseases of metabolism that are characterized by oxidative stress feature abnormal concentrations of thiol and thioether metabolites, such as glutathione and cysteine [12]. The concentration of certain thiol and thioether metabolites has predictive value to assess mortality and morbidity in chronic disorders, such as cardiovascular disease [17,18] and cancer [19,20]
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