Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine

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.

Vitamin B12 Status Upon Short-Term Intervention with a Vegan Diet—A Randomized Controlled Trial in Healthy Participants

“…Total levels of homocysteine, cysteine, cystathionine, glutathione, methionine, and methionine sulfoxide were measured by LC-MS/MS according to a published procedure [23]. Briefly, 20 µL of plasma was mixed with 20 µL internal standard ( S -methylglutathione, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and 20 µL 0.5 M dithiothreitol (DTT, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany).…”

Amino Assets: How Amino Acids Support Immunity

“…Different tissues and niches contain different amino acid repertoires (Behringer et al, 2019), a concept that is particu-larly relevant to migratory immune cells, which are likely to experience various microenvironments. Immune cells may elect not to indiscriminately use available amino acids for protein synthesis, but to selectively use them or store them.…”

Elevated Plasma Vitamin B12 in Patients with Hepatic Glycogen Storage Diseases

“…Total plasma homocysteine concentrations were measured by tandem mass spectrometry as described earlier [ 20 ]. Briefly, 20 µL of plasma were mixed with 20 µL of DTT 0.5 M to reduce all free and protein-bound disulfides, vortexed and allowed to react at room temperature for 15 min.…”

“…Twenty µL of internal standard D 4 -homocysteine (50 µM) were added and metabolites were extracted by addition of 100 µL of 0.1% formic acid in MeOH. The sample was centrifuged at 9447× g for 10 min at room temperature and the resulting supernatants transferred into HPLC vials for LC-MS/MS analysis as described [ 20 ]. Methylmalonic acid levels in plasma were determined using liquid chromatography- tandem mass spectrometry as described elsewhere [ 21 ].…”