Hepatic triglyceride (HTG) accumulation from peripheral dietary sources and from endogenous de novo lipogenesis (DNL) was quantified in adult Sprague-Dawley rats by combining in vivo localized 1 H MRS measurement of total hepatic lipid with a novel ex vivo 2 H NMR analysis of HTG 2 H enrichment from 2 H-enriched body water. The methodology for DNL determination needs further validation against standard methodologies. To examine the effect of a high-fat diet on HTG concentrations and sources, animals (n ¼ 5) were given high-fat chow for 35 days. HTG accumulation, measured by in vivo 1 H MRS, increased significantly after 1 week (3.85 W 0.60% vs 2.13 W 0.34% for animals fed on a standard chow diet, P < 0.05) and was maintained until week 5 (3.30 W 0.60% vs 1.12 W 0.30%, P < 0.05). Animals fed on a high-fat diet were glucose intolerant (13.3 W 1.3 vs 9.4 W 0.8 mM in animals fed on a standard chow diet, for 60 min glycemia after glucose challenge, P < 0.05). In control animals, DNL accounted for 10.9 W 1.0% of HTG, whereas in animals given the high-fat diet, the DNL contribution was significantly reduced to 1.0 W 0.2% (P < 0.01 relative to controls). In a separate study to determine the response of HTG to weaning from a high-fat diet, animals with raised HTG (3.33 W 0.51%) after 7days of a high-fat diet reverted to basal HTG concentrations (0.76 W 0.06%) after an additional 7 days of weaning on a standard chow diet. These studies show that, in healthy rats, HTG concentrations are acutely influenced by dietary lipid concentrations. Although the DNL contribution to HTG content is suppressed by a high-fat diet in adult Sprague-Dawley rats, this effect is insufficient to prevent overall increases in HTG concentrations.
The contributions of hepatic glycogenolysis to fasting glucose production and direct pathway to hepatic glycogen synthesis were quantified in eight type 1 diabetic patients and nine healthy control subjects by ingestion of 2 H 2 O and acetaminophen before breakfast followed by analysis of urinary water and acetaminophen glucuronide. After overnight fasting, enrichment of glucuronide position 5 relative to body water (G5/body water) was significantly higher in type 1 diabetic patients compared with control subjects, indicating a reduced contribution of glycogenolysis to glucose production (38 ؎ 3 vs. 46 ؎ 2%). Following breakfast, G5/body water was significantly higher in type 1 diabetic patients, indicating a smaller direct pathway contribution to glycogen synthesis (47 ؎ 2 vs. 59 ؎ 2%). Glucuronide hydrogen 2 enrichment (G2) was equivalent to body water during fasting (G2/body water 0.94 ؎ 0.03 and 1.02 ؎ 0.06 for control and type 1 diabetic subjects, respectively) but was significantly lower after breakfast (G2/body water 0.78 ؎ 0.03 and 0.82 ؎ 0.05 for control and type 1 diabetic subjects, respectively). The reduced postprandial G2 levels reflect incomplete glucose-6-phosphate-fructose-6-phosphate exchange or glycogen synthesis from dietary galactose. Unlike current measurements of human hepatic glycogen metabolism, the 2 H 2 O/acetaminophen assay does not require specialized on-site clinical equipment or personnel. Diabetes
A multinuclear ( 1 H, 13 C, 17 O, 51 V) 1D and 2D NMR study of the complexation of L-lactic acid with vanadium(V) and hydrogen peroxide shows that four peroxo complexes are formed in aqueous solution in the pH range 1−7. Two isomeric 2:2:1 (metal:ligand:peroxo) complexes, together with a 2:2:2 species, are found over the entire pH range. At pH [a]
Quantification of 2 H and 13 C enrichment distributions in human urinary glucuronide following ingestion of 2 H 2 O and 13 C gluconeogenic tracers was achieved by NMR spectroscopy of the 1,2-O-isopropylidene-a-D-glucofuranurono-6,3-lactone and 5-O-acetyl-1,2-O-isopropylidene-a-D-glucofuranurono-6,3-lactone derivatives. The derivatization process is simple and can be applied to any glucuronide species. The derivatives are highly soluble in acetonitrile and generate well-resolved and narrow 2 H and 13 C NMR signals.The 1,2-O-isopropylidene-a-D-glucofuranurono-6,3-lactone derivative provided resolution of the six glucuronide 13 C signals and numerous 13 C isotopomer populations through one-and two-bond 13 C-13 C-coupling, while the 5-O-acetyl-1,2-Oisopropylidene-a-D-glucofuranurono-6,3-lactone derivative provided complete resolution of the 2 H NMR signals for the five glucuronide hydrogens. The isopropylidene methyl signals were also resolved and provided an internal 2 H enrichment standard following the acetonation of glucuronolactone with deuterated acetone.
Exchange of hepatic glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate via transaldolase modifies hepatic G6P enrichment from glucose or gluconeogenic tracers. Transaldolase exchange was quantified in five healthy, fed subjects following an oral bolus of [1,2,3-13 C 3 ]glycerol (25-30 mg/kg) and paracetamol (10 -12 mg/kg). 13 Hepatic glucose-6-phosphate (G6P) lies at the metabolic crossroads of hepatic glucose and glycogen metabolism. Under fasting conditions when the liver is a net producer of glucose, hepatic G6P is generated by the hydrolysis of glycogen and from gluconeogenesis and is then converted to glucose via G6P. Under fed conditions there is net hepatic glycogen synthesis from both glucose and from gluconeogenic precursors. These metabolic pathways converge at G6P and the hexose carbon skeletons are then incorporated into glycogen via glucose-1-phosphate and UDP-glucose. The gluconeogenic contribution to G6P synthesis is modified in a variety of human diseases, including insulin-and noninsulin-dependent diabetes, cirrhosis, and malaria (1-4). Therefore, quantifying the fraction of G6P derived from gluconeogenesis is a key parameter for defining hepatic carbohydrate metabolism under these and other pathophysiological conditions. In humans, several different tracer methods have been developed for quantifying the contribution of gluconeogenesis to hepatic G6P flux. Hepatic G6P enrichment from these tracers can be quantified noninvasively by analysis of urinary glucuronide enrichment (5-7).Implicit in all measurements of hepatic gluconeogenesis using labeled gluconeogenic substrates is the assumption that G6P molecules derived from non-gluconeogenic precursors (i.e., glycogenolysis) are not labeled with the tracer. 1 Likewise, when labeled glucose is used to determine the contribution of direct and indirect pathways of hepatic glycogen synthesis under fed conditions, dilution of the glucose tracer at the level of hepatic G6P is assumed to be entirely due to gluconeogenic G6P production.The possibility that transaldolase (TA) exchange could invalidate these assumptions was recognized by Landau and co-workers (8 -10). TA catalyzes the exchange between the glyceraldehyde-3-phosphate (GA3P) moiety (i.e., carbons 4, 5, and 6) of fructose-6-phosphate (F6P) and free GA3P in many tissues (11)(12)(13). This exchange is independent of oxidative pentose phosphate pathway (PPP) flux, hence tissues that have relatively low oxidative PPP utilization of G6P, such as liver, may nevertheless have significant TA exchange activity (13). Since G6P and F6P are in rapid exchange, G6P molecules derived from glucose or glycogen are exposed to TA activity. With gluconeogenic tracers that label GA3P, the effect of TA exchange is to transfer the label to G6P molecules derived from glucose or glycogen. Hence, the gluconeogenic contribution is overestimated relative to the contribution from
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...
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