2490-P: Improved Positional Measurement of Deuterium Enrichment of Glucose Hydrogen Atoms by Gas Chromatography–Mass Spectrometry for Rigorous Determination of Gluconeogenesis

Introduction and Objective: Quantitative determination of gluconeogenesis (GNG) and glycogenolysis is critical for understanding glucose homeostasis in metabolic health and disease. 2H2O allows for estimation of relative sources of glucose production based on 2H enrichment of glucose in positions H2, H5 and H6. Position specificity can be obtained by nuclear magnetic resonance (NMR) spectroscopy, but it requires a large volume of analyte. Gas chromatography-mass spectrometry (GC-MS) based positional isotopic analysis provides a sensitive alternative based on either tandem fragmentation approaches or use of distinct derivatives. Yet, the bond between carbon 1 (C1) and C2 is challenging to fragment therefore limiting the positional insights of GC-MS based methodology. To improve measurement of 2H at C2 specifically, diethyl dithioacetal acetate (DEDTA) derivatization was incorporated into the workflow, enhancing position-specific accuracy. Methods: Plasma glucose was isolated from mice fed ad libitum or fasted mice and analyzed by GC-MS using common derivatives in combination with novel DEDTA protocol to monitor 2H enrichment at C1 and C2. DEDTA selectively stabilizes the anomeric carbon while preserving adjacent C2, allowing refined measurement of 2H incorporation. Mass isotopomers distributions from electron ionization GC-MS were used to quantify enrichment at C2. Results: Inclusion of DEDTA improved enrichment estimation of 2H at C2 compared with MOX, which captures combined C1-C2 enrichment but cannot isolate C2 independently. DEDTA allowed more precise and reproducible quantification of 2H incorporation at C2, enhancing the ability to detect GNG contributions to plasma glucose and improving estimate of glycogenolysis. Conclusion: Addition of DEDTA alongside established GC-MS derivatives provides superior position-specific estimation of 2H enrichment of glucose at C2, refining the accuracy of GNG flux analysis in metabolic disease. Disclosure J. Rajendran: None. H.H. Pak: None. N. Pudelko-Malik: None. S. Deja: None.