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

Abstract

We developed a complete method to measure low [ 13C] enrichments in glycogen. Fourteen rats were fed a control diet. Six of them also ingested either [U- 13C] glucose ( n = 2) or a mixture of 20 [U- 13C] 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, [ 13C] 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 δ 13C (‰) for control rats ( n = 8) and increased to −5 to 8 δ 13C (‰) ‰ and 12–14 δ 13C (‰) ‰ after the ingestion of [U- 13C] amino acids or [U- 13C] 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%.