Abstract 2324: Small-scale microwave-assisted acid hydrolysis method for glycogen determination and turnover in tumors using Stable Isotope Resolved Metabolomics

Abstract

Abstract Introduction: Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose. Although glycogen primarily occurs in the liver and muscle, it can be found in most tissues throughout the body, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Methods: We have developed a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions were first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose in 1 N HCl for 10 minutes at 110 °C under a N2 atmosphere in a microwave-assisted synthesis reactor. The resulting glucose and its 13C isotopologues were then analyzed by UHR-FTMS and/or NMR. Results: We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 minutes at 110-115 °C to give > 90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We showed the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, both in terms of the 13C levels and fractional enrichment, thereby demonstrating the utility of the method in tracing glycogen turnover in cells and tissues. Conclusions: The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials. Keywords: glycogen turnover; 13C6-glucose, stable isotope resolved metabolomics (SIRM); microwave-assisted hydrolysis. Citation Format: Andrew N. Lane, Timothy L. Scott, Juan Zhu, Teresa A. Cassel, Sara Vicente-Munoz, Penghui Lin, Richard M. Higashi, Teresa W-M Fan. Small-scale microwave-assisted acid hydrolysis method for glycogen determination and turnover in tumors using Stable Isotope Resolved Metabolomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2324.