Quantitation of gluconeogenesis by ²H nuclear magnetic resonance analysis of plasma glucose following ingestion of ²H₂O

We present a simple ²H NMR assay of the fractional contribution of gluconeogenesis to hepatic glucose output following ingestion of ²H₂O. The assay is based on the measurement of relative deuterium enrichment in hydrogens 2 and 3 of plasma glucose. Plasma glucose was enzymatically converted to gluconate, which displays fully resolved deuterium 2 and 3 resonances in its ²H NMR spectrum at 14.1 T. The signal intensity of deuterium 3 relative to deuterium 2 in the gluconate derivative as quantitated by ²H NMR was shown to provide a precise and accurate measurement of glucose enrichment in hydrogen 3 relative to hydrogen 2. This measurement was used to estimate the fractional contribution of gluconeogenesis to hepatic glucose output for two groups of rats; one group was fasted for 7 h and the other was fasted for 29 h. Rats were administered ²H₂O to enrich total body water to 5% over the last 4-5 h of each fasting period. For the 7-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.32 ± 0.09 (n = 7). This indicates that gluconeogenesis contributed 32 ± 9% of total hepatic glucose output with glycogenolysis contributing the remainder. For the 29-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.81 ± 0.10 (n = 6), indicating that gluconeogenesis supplied the bulk of hepatic glucose output (81 ± 10%).