A simplified mass isotopomer approach to estimate gluconeogenesis rate in vivo using deuterium oxide

We compare a new simplified ²H enrichment mass isotopomer analysis (MIA) against the laborious hexamethylentetramine (HMT) method to quantify the contribution of gluconeogenesis (GNG) to total glucose production (GP) in calves. Both methods are based on the ²H labeling of glucose after in vivo administration of deuterium oxide. The ²H enrichments of plasma glucose at different C-H positions were measured as aldonitrile pentaacetate (AAc) and methyloxime-trimethylsilyl (MoxTMS) derivatives or HMT by gas chromatography/mass spectrometry (GC/MS). Two preruminating fasted Holstein calves (51 kg body mass, BM, age 7 days) received two oral bolus doses of ²H₂O (10g/kg BM, 70 atom% ²H) at 7:00 h and 11:00 h after overnight food withdrawal. Blood samples for fractional GNG determination were collected at -24 and between 6 and 9 h after the first ²H₂O dose. The ratio of ²H enrichments C5/C2 represents the contribution of GNG to GP. The ²H enrichment at C2 was calculated based on the ion fragments at m/z 328 (C1-C6) - m/z 187 (C3-C6) of glucose AAc. The ²H enrichment at C5 was approximated either by averaging the ²H enrichment at C5-C6 using the ion fragment of glucose MoxTMS at m/z 205 or by conversion of the C5 of glucose into HMT. The fractional GNG calculated by the C5-C6 average ²H enrichment method (41.4±6.9%) compared to the HMT method (34.3±11.4%) was not different (mean±SD, n = 6 replicates). In conclusion, GNG can be estimated with less laborious sample preparation by means of our new C5-C6 average ²H enrichment method using AAc and MoxTMS glucose derivatives.