A Micro-Scale Analytical Method for Determining Glycogen Turnover by NMR and FTMS

Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose anchored to the protein glycogenin. Although glycogen primarily occurs in the liver and muscle, it is found in most tissues, 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 (13)C(6)-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Here we describe 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 (13)C positional isotopomer distributions, are first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose under N(2) in a focused beam microwave reactor, with subsequent analysis by UHR-FTMS and/or NMR. We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 min at 110–115 °C to give >90% recovery. The method was applied to track the fate of (13)C(6)-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of (2)H(7)-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We measured the incorporation of (13)C(6)-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, to demonstrate the utility of the method in tracing glycogen turnover in cells and tissues. The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials.