Integration of [U‐¹³C]glucose and ²H₂O for quantification of hepatic glucose production and gluconeogenesis

Abstract

Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13 C]glucose and 2 H 2 O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13 C-isotopomers derived from the incorporation of [U-13 C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13 C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13 C]glucose and provided reasonable estimates (1.90 AE 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2 H-enrichment from 2 H 2 O by 2 H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 AE 0.14 and 1.03 AE 0.10 mg/kg/min, respectively). Hepatic glutamine 13 C-isotopomers representing multiply-enriched oxaloacetate and [U-13 C]acetyl-CoA were identified as multiplets in the 13 C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13 C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and $ 0.05% to carbon 4 of glutamine.

NMR Derivatives for Quantification of2H and13C‐Enrichment of Human Glucuronide from Metabolic Tracers

“…This requires the assumption that metabolism of the tracer will not generate significant levels of singly 13 C-enriched hexose isotopomers. [8] In cases where this assumption is not valid (e.g., metabolism of [2-13 C]glycerol), the 13 C excess enrichment can be obtained from the 1 H NMR spectrum, where signals from hydrogens attached to 13 C are split by 13 C-1 H coupling whereas those attached to 12 C are not. The background contributions from natural abundance 13 C can also be accounted for in this analysis.…”

“…[1 -6] While mass spectroscopy (MS) analysis is more established and widespread due to lower instrumentation cost and better sensitivity, NMR spectroscopy is becoming increasingly available due to the proliferation of medium field (400-600 MHz 1 H) spectrometers that have vastly improved sensitivity and performance over their predecessors. NMR is particularly effective for resolving complex enrichment patterns from mixtures of 13 C and 2 H tracers, [4,7,8] thereby providing the basis for the design of highly informative tracer studies for measurement of metabolic fluxes. Labeling information for each isotope is represented by NMR signals whose resolution and quantification rests on two main characteristics.…”

“…Below is the 13 C NMR spectrum of the 1,2-O-isopropylidene-a-Dglucofuranurono-6,3-lactone (MAGL) derivative (9,000 FIDs., 6.25 h). The carbon 2 and 5 resonances are shown in expanded view and their 13 C-isotopomer components are labeled as follows: S-NA ¼ natural abundance singlet; Q2 ¼ quartet signal from[1,2,[3][4][5][6][7][8][9][10][11][12][13] C 3 ]glucuronide; D12 ¼ doublet signal from [1,2-13 C 2 ]glucuronide; D23 ¼ doublet signal from [2,3-13 C 2 ]glucuronide; Q5 ¼ quartet signal from [4,5,6-13 C 3 ]glucuronide; D56 ¼ doublet signal from [5,6-13 C 2 ]glucuronide; D45 ¼ doublet signal from [4,5-13 C 2 ]glucuronide.…”

Sources of hepatic glycogen synthesis during an oral glucose tolerance test: Effect of transaldolase exchange on flux estimates

“…Plasma glucose and urinary menthol glucuronide 13 C resonances consist of singlet signals, mostly derived from natural abundance 13 C (1.11%), and the 13 C-13 C spin-spin coupled multiplets. In the C1 resonance of MAG, [U- 13 C]glucose is detected as a doublet of doublets arising from the splitting because of coupling between positions 1 and 2, and 1 and 5 of MAG (17). Plasma [U-13 C]glucose enrichments attained relatively constant levels; reaching the glucose load enrichment, at 2 and 3 h after the load (6.97 Ϯ 0.19% and 6.50 Ϯ 0.18%, respectively) ( Table 2).…”

A fast and sensitive 1H NMR method to measure the turnover of the H2 hydrogen of lactate

“…These methods are able to monitor, by 13 C NMR, the incorporation of deuterium into 13 C-labeled metabolites because covalent binding of 2 H results in characteristic 13 C-2 H heteronuclear couplings and geminal or vicinal isotopic shifts of the corresponding 13 C resonances (14,15). The methodology combines the inherent advantage of detecting deuterium incorporation with the positional selectivity and increased sensitivity of 13 C NMR, as compared to the direct 2 H NMR detection (16,17). However, it requires the use of relatively expensive 13 C-labeled substrates, as well as sufficiently long 13 C NMR acquisitions.…”