Abstract PO-006: Tracking S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) biosynthesis pathway using stable isotope-resolved metabolomics (SIRM)

Abstract

Abstract S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) are low abundance, key metabolites in one-carbon metabolism. The ratio of SAM/SAH controls epigenetic DNA and histone methylation, which is central to regulating gene expression in both normal and disease states including cancers (1,2). Stable isotope resolved metabolomics (SIRM) is a powerful approach for mapping metabolic networks in cells and tissues, which employs untargeted ultrahigh resolution MS1 to quantify a full set of isotopologues for a large number of metabolites (3). This demand makes it difficult to implement liquid chromatography (LC)-based analysis. We have developed a simple, rapid and robust LC-free method for analyzing SAM and SAH in mouse tissue, A549 cells, and human plasma. This method has sub nmol detection and is fully compatible with SIRM analysis of many other metabolites. Flash frozen mouse liver tissues or A549 cells were quenched and extracted with ice-cold acetonitrile:water:chloroform (2:0.75:1, v/v). Prior to extraction, A549 cells were grown for 24 h in the presence of either 13C6-glucose or 13CH3-methionine. No extraction was applied to human plasmas. SAM and SAH were captured by solid phase extraction (SPE) using a Bond-Elut phenylboronic acid (PBA) column (Agilent). SAM and SAH levels in liver tissues and plasma obtained by our method was comparable to those found by other methods (4). Tracing with 13C6 glucose showed extensive incorporation of label into the ribose ring and purine of the adenosyl subunit in both SAM and SAH. In contrast, tracing with 13CH3-methioinine showed predominant, single carbon label in SAM and none in SAH, suggesting the prevalence in the incorporation of exogenous methionine into SAM and turnover into SAH. Thus, it is practical to determine SAM and SAH turnover in the methionine cycle in various biological samples, which provides important insights into epigenetic metabolism. Funding: This work was supported in part by grants: 1R01ES022191-01 (to TWMF and RMH), 1P01CA163223-01A1 (to ANL and TWMF), and 1U24DK097215-01A1 (to RMH, TWMF, and ANL).