[C-13] GC-C-IRMS analysis of methylboronic acid derivatives of glucose from liver glycogen after the ingestion of [C-13] labeled tracers in rats

Abstract : We developed a complete method to measure low [C-13] enrichments in glycogen. Fourteen rats were fed a control diet. Six of them also ingested either [U-C-13] glucose (n = 2) or a Mixture of 20 [U-C-13] amino acids (n = 4). Hepatic glycogen was extracted, digested to glucose and purified on anion-cation exchange resins. After the optimization of methylboronic acid derivatization using GC-MS, [C-13] enrichment of extracted glucose was measured by GC-C-IRMS. The accuracy was addressed by measuring the enrichment excess of a calibration curve, which observed values were in good agreement with the expected values (R = 0.9979). Corrected delta values were -15.6 +/- 1.6 delta C-13 (parts per thousand) for control rats (n = 8) and increased to -5 to delta C-13 (parts per thousand) parts per thousand and 12-14 delta C-13 (parts per thousand) parts per thousand after the ingestion of [U-C-13] amino acids or [U-C-13] glucose as oral tracers, respectively. The method enabled the determination of dietary substrate transfer into glycogen. The sequestration of dietary glucose in liver glycogen 4 h after the meal was 35% of the ingested dose whereas the transfer of carbon skeletons from amino acids was only 0.25 to 1%.