Simple measurement of gluconeogenesis by direct ²H NMR analysis of menthol glucuronide enrichment from ²H₂O

Abstract

The contribution of gluconeogenesis to fasting glucose production was determined by a simple measurement of urinary menthol glucuronide (MG) 2 Key words: deuterium; gluconeogenesis; endogenous glucose production; menthol; glucuronide Deuterated water is widely considered a practical tracer of endogenous glucose production in humans. After ingestion, the deuterium distributes rapidly into bulk body water and is incorporated into numerous metabolites including hepatic glucose-6-phosphate (G6P). During fasting, the ratio of 2 H enrichment in positions 5 and 2 of G6P reflects the relative contribution of glycogenolysis and gluconeogenesis to hepatic G6P synthesis (1). The bulk of fasting plasma glucose is derived from hepatic G6P; therefore, both metabolites share a common 2 H enrichment pattern under isotopic steady-state conditions. On this basis, the contribution of gluconeogenesis to fasting glucose production can be obtained by quantifying 2 H enrichment in positions 5 and 2 of plasma glucose (1-3). This can be achieved by a very sensitive but labor-intensive dehomologation and mass-spectrometry procedure (1,4), or alternatively, by a less sensitive but more convenient 2 H NMR analysis of a monoacetone glucose (MAG) derivative (5-7). MAG is easily prepared from plasma glucose and has fully resolved 2 H NMR signals for all hydrogens attached to the hexose skeleton. Quantification of the 5:2 deuterium enrichment ratio from the relative areas of the 2 H NMR signals of hydrogen 5 and 2 is simple and provides estimates of gluconeogenesis that are consistent with the MS procedure (8). Due to rapid exchange between hepatic G6P and glucose-1-phosphate (G1P), the 5:2 deuterium enrichment ratio of G6P is also preserved in the hexose moieties of G1P, UDP-glucose, and glucuronide. Preliminary studies suggest that the 5:2 deuterium enrichment ratio of urinary paracetamol glucuronide is equal to that of plasma glucose under fasting conditions (9). The high abundance of urinary paracetamol glucuronide allows NMR collection times to be reduced by a factor of 10 or more in comparison to analysis of plasma glucose (9). However, paracetamol glucuronide has poorly dispersed hydrogen NMR signals; hence, it must be derivatized to MAG for 2 H NMR analysis. MAG preparation from urinary glucuronide is considerably more labor intensive than its preparation from plasma glucose (9) and is a significant obstacle for the routine analysis of a large number of urine samples. Therefore, a direct analysis of urinary glucuronide 2 H enrichment with minimal sample processing would be highly desirable for quantifying gluconeogenesis from 2 H 2 O. Menthol glucuronide (MG) is a suitable metabolite for such an analysis for the following reasons. First, the chemical shifts of its glucuronide hydrogens are highly dispersed, providing complete resolution of 2 H NMR signals at fields of 11.75 T or higher. Second, menthol glucuronide can be rapidly isolated from urine either as a crude ammonium salt or by simple ether extraction (10). Third, recent studi...

Postprandial and Fasting Hepatic Glucose Fluxes in Long-Standing Type 1 Diabetes

“…Based on previous studies (13,43–45), the low glucuronide yields in T1Di were unexpected and precluded a rigorous evaluation of fluxes for this group. Our previous experience is that T1Dp tend to generate the lowest yields of urinary glucuronides, in particular those with urinary glucose excretion.…”

Noninvasive Analysis of Hepatic Glycogen Kinetics Before and After Breakfast with Deuterated Water and Acetaminophen

“…Glucuronide G5/G2 values can be conveniently determined by 2 H nuclear magnetic resonance (NMR) following derivatization to monoacetone glucose (MAG) (15). Where applicable, the use of menthol in the form of peppermint oil as the glucuronidation probe can further simplify the procedure since urinary menthol glucuronide can be analyzed directly after a simple purification procedure (16).…”

Use of stable isotopes to evaluate the functional effects of nutrients

“…Initially, this was performed using paracetamol and nuclear magnetic resonance analysis of monoacetone glucose prepared from urinary paracetamol glucuronide [17]. A more convenient and simpler method using menthol glucuronide was recently published [18]. These methods should allow further studies of the effects of nutrients on gluconeogenesis.…”