Search

p-Aminobenzoic acid (PABA) was evaluated for noninvasive sampling of UDP-glucose in the liver. Six healthy subjects ingested 550 mg PABA during a breakfast meal. Urine was collected 0-2 and 2-4 h after PABA ingestion. N-acetyl PABA glucuronide (NAPG) was identified with 522 ± 212 μmol recovered in the 2-4 h urines. One of the subjects ingested 2 g of 98% [U- 2 H 7 ]glucose alongside PABA and the NAPG was analyzed for positional 2 H-enrichment by 2 H NMR following derivatization to 5-O-acetyl monoacetone glucuronolactone. In conclusion, PABA is an effective agent for the chemical biopsy of hepatic UDP-glucose in humans., (Copyright © 2019. Published by Elsevier Inc.)

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.

Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.

13C NMR isotopomer analysis is a powerful method for measuring metabolic fluxes through pathways intersecting in the tricarboxylic acid cycle. However, the inherent insensitivity of 13C NMR spectroscopy makes application of isotopomer analysis to small tissue samples (mouse tissue, human biopsies, or cells grown in tissue culture) problematic. (1)H NMR is intrinsically more sensitive than 13C NMR and can potentially supply the same information via indirect detection of 13C providing that isotopomer information can be preserved. We report here the use of J-resolved HSQC (J-HSQC) for 13C isotopomer analysis of tissue samples. We show that J-HSQC reports isotopomer multiplet patterns identical to those reported by direct 13C detection but with improved sensitivity., (Copyright 2001 Academic Press.)

We present a simple (2)H NMR assay of the fractional contribution of gluconeogenesis to hepatic glucose output following ingestion of (2)H(2)O. The assay is based on the measurement of relative deuterium enrichment in hydrogens 2 and 3 of plasma glucose. Plasma glucose was enzymatically converted to gluconate, which displays fully resolved deuterium 2 and 3 resonances in its (2)H NMR spectrum at 14.1 T. The signal intensity of deuterium 3 relative to deuterium 2 in the gluconate derivative as quantitated by (2)H NMR was shown to provide a precise and accurate measurement of glucose enrichment in hydrogen 3 relative to hydrogen 2. This measurement was used to estimate the fractional contribution of gluconeogenesis to hepatic glucose output for two groups of rats; one group was fasted for 7 h and the other was fasted for 29 h. Rats were administered (2)H(2)O to enrich total body water to 5% over the last 4-5 h of each fasting period. For the 7-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.32 +/- 0.09 (n = 7). This indicates that gluconeogenesis contributed 32 +/- 9% of total hepatic glucose output with glycogenolysis contributing the remainder. For the 29-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.81 +/- 0.10 (n = 6), indicating that gluconeogenesis supplied the bulk of hepatic glucose output (81 +/- 10%)., (Copyright 2000 Academic Press.)

13C and 1H NMR spectroscopy of plasma glucose was used to resolve the isotopomer contributions from tracer levels of [1,6-13C2]glucose, a novel tracer of glucose carbon skeleton turnover, and [U-13C]propionate, a tracer of hepatic citric acid cycle metabolism. This allowed simultaneous measurements of hepatic glucose production and citric acid cycle fluxes from the NMR analysis of a single plasma glucose sample in fasted animals. Glucose carbon skeleton turnover, as reported by the dilution of [1,6-13C2]glucose, was 56 +/- 2 micromol/kg/min in the presence of labeling from [U-13C]propionate and 53 +/- 4 micromol/kg/min in its absence. Therefore, as expected, the labeling contributions from [U-13C]propionate metabolism did not have a significant effect on the measurement of glucose turnover. For the group infused with both tracers, citric acid cycle flux estimates from the analysis of glucose C2 isotopomer ratios were consistent with those from our recent experiments where only [U-13C]propionate was infused, verifying that the presence of [1,6-13C2]glucose did not interfere with these measurements. This integrated analysis of hepatic glucose output and citric acid cycle fluxes from plasma glucose isotopomers yielded a noninvasive estimate of hepatic citrate synthase flux of 74 +/- 12 micromol/kg/min for 24-h fasted rats., (Copyright 1998 Academic Press.)

The contribution of a 13C-enriched substrate to the acetyl-CoA pool in animal tissues is typically measured by analysis of glutamate enrichment from tissue extracts. 13C NMR analysis offers the advantages of minimal sample processing and high information content, but has a low analytical sensitivity compared to other methods of tracer analysis such as GC/MS. We present a sensitive, simple, and direct 1H NMR measurement of glutamate C4 enrichment from tissue extracts. The method is demonstrated with heart and hindlimb muscle tissue extracts of rats infused with [2,4,6,8-13C4]-octanoate, a source of [2-13C]acetyl-CoA. Glutamate C4 enrichment in extracts of individual hindlimb soleus muscles weighing approximately 150 mg and containing approximately 0.3 mumol of glutamate was quantified by 1H NMR within about 40 min. Glutamate C4 enrichment measurements by 1H NMR in heart and gastrocnemius muscle were also highly correlated with independent measurements obtained from 13C NMR isotopomer analysis.

13C NMR analysis of 13C-enriched glucose containing multiple isotopomers is hampered by chemical shift similarities of several carbon resonances and by the presence of two anomeric forms. A convenient and quantitative method of enzymatically oxidizing glucose to gluconate in tissue and perfusate extracts is presented. The six carbon resonances of the resulting 13C-enriched gluconate are fully resolved at high pH, thereby allowing a determination of the fractional population of each 13C isotopomer by 13C NMR. The utility of this method is demonstrated using the effluent from an isolated perfused liver containing 13C-enriched glucose produced by hepatic metabolism of sodium [1,2,3-13C3]propionate via the citric acid cycle and gluconeogenesis. An analysis of the gluconate C2 and C5 resonances in this sample showed that pentose phosphate activity was insignificant during this perfusion protocol. As demonstrated, this method provides a means of fully describing 13C isotopomer populations in enriched glucose samples where isotope may be derived from multiple metabolic pathways, thus expanding the scope of experimental design and enrichment strategies.