Serum Methionine Cycle Metabolite Quantification

In this study, we detected three core serum methionine metabolites in the methionine cycle including SAM, SAH, and Hcy by HPLC–MS/MS methods (20 (link)–22 (link)) (Supplementary Table S1). Serum SAM/SAH was calculated as the methylation index.
Before testing, 10 μL of 50 mM DL-Dithiothreitol, and a 10 μL mixture of deuterium-labeled internal standards (²H₃-SAM, 500 nM; ²H₄-SAH, 500 nM; ²H₄-Hcy, 5 μM) were added to the serum samples (50 μL) in turn. The mixtures were vortexed for 5 s and incubated at 37°C for 15 min in the dark. Then 30 μL perchloric acid (1 M) was added to samples for protein precipitation. Subsequently, the samples were centrifuged at 15,000 × g for 10 min at 4°C. Finally, the supernatants were filtered by a 0.22 μm membrane. The methionine metabolites were separated through an Acquity BEH C18 column (2.1 × 50 mm; i.d. 1.7 μm) (Waters Corp., Milford, MA, USA), detected by Agilent 1290 Infinity II UHPLC system coupled with Agilent 6410 Triple Quadrupole LC/MS system, and quantified in multiple reaction monitoring modes. The peaks and concentrations of the targets were measured by UPLC -MS/MS (Agilent) in the positive-ion (ESI+) mode (Supplementary Figure S1). The linearity regression coefficients of SAM, SAH, and Hcy were more than 0.99, with all the inter- and intra-assay coefficients of variation <10% (Supplementary Tables S2, S3).