Metabolomic Analysis of Liver Tissue from the VX2 Rabbit Model of Secondary Liver Tumors.

Purpose. The incidence of liver neoplasms is rising in USA. The purpose of this study was to determine metabolic profiles of liver tissue during early cancer development. Methods. We used the rabbit VX2 model of liver tumors (LT) and a control group consisting of sham animals implanted with Gelfoam into their livers (LG). After two weeks from implantation, liver tissue from lobes with and without tumor was obtained from experimental animals (LT+/LT-) as well as liver tissue from controls (LG+/LG-). Peaks obtained by Gas Chromatography-Mass Spectrometry were subjected to identification. 56 metabolites were identified and their profiles compared between groups using principal component analysis (PCA) and a mixed-effect two-way ANOVA model. Results. Animals recovered from surgery uneventfully. Analyses identified a metabolite profile that significantly differs in experimental conditions after controlling the False Discovery Rate (FDR). 16 metabolites concentrations differed significantly when comparing samples from (LT+/LT-) to samples from (LG+/LG-) livers. A significant difference was also shown in 20 metabolites when comparing samples from (LT+) liver lobes to samples from (LT-) liver lobes. Conclusion. Normal liver tissue harboring malignancy had a distinct metabolic signature. The role of metabolic profiles on liver biopsies for the detection of early liver cancer remains to be determined.

Disturbances in the glutathione/ophthalmate redox buffer system in the woodchuck model of hepatitis virus-induced hepatocellular carcinoma.

Purpose. The incidence of liver tumors is rising in USA. The purpose of this study was to evaluate liver oxido-reductive status in the presence of chronic liver disease and hepatocellular carcinoma (HCC). Methods. Glutathione species and ophthalmate (OA) concentrations were measured by LC-MS in processed plasma and red blood cells (RBC) from infected Woodchuck with hepatitis virus (WHV). Blood samples were obtained from: (i) infected animals with tumors (WHV+/HCC+), (ii) infected animals without tumors (WHV+/HCC-) and (iii) healthy animals (WHC-/HCC-). Results. The concentration of reduced glutathione (GSH) and the ratio GSH/GSG were lower in plasma from WHV+/HCC+ animals when compared to WHV+/HCC- and WHV-/HCC- (P < 0.01). In contrast, the concentration of oxidized glutathione (GSSG) was found to be higher in plasma from WHV+/HCC+ animals when compared to WHV+/HCC- and WHV-/HCC- (P < 0.01). The Glutathione species and its ratio from the RBC compartment were similar among all groups. OA concentration in both plasma and RBC was significantly higher from WHV+/HCC+ when compared to WHV+/HCC- and WHV-/HCC- (P < 0.01). Conclusions. Disturbances of the glutathione redox buffer system and higher concentrations of OA were found in the WCV+/HCC+ animal model. The role of these compounds as biomarkers of early tumor development in patients with end stage liver disease remains to be determined.

The dynamics of glutathione species and ophthalmate concentrations in plasma from the VX2 rabbit model of secondary liver tumors.

PURPOSE: Available tumor markers have low sensitivity/specificity for the diagnosis of liver tumors. The present study was designed to evaluate the oxidoreductive status of the liver as surrogates of tumor subsistence and growth. METHODS: Glutathione species (GSH:GSSG), ophthalmate (OA) concentrations, and their turnover were measured in plasma of rabbits (n = 6) in their healthy state and in the state of tumor growth after implantation of the VX2 carcinoma in their liver. Tumors were allowed to grow for a period of 14 days when rabbits were sacrificed. Livers were removed and cysteine concentration was measured in liver tissue. RESULTS: Tumor growth was found in 100% of the rabbits. Concentration and labeling of GSH/GSSG were similar in experimental animals before and after tumor implantation and to sham animals. In contrast, OA concentration increased significantly in experimental animals after tumor implantation when compared to same animals prior to tumor implantation and to sham animals (P < .05). The concentration of cysteine, a precursor of GSH, was found to be significantly lower in the liver tissue adjacent to the tumor (P < .05). CONCLUSION: Disturbances in the oxidoreductive state of livers appear to be a surrogate of early tumor growth.

Carnitine palmitoyltransferase II deficiency: successful anaplerotic diet therapy.

BACKGROUND: Carnitine palmitoyltransferase II (CPT II) deficiency is an important cause of recurrent rhabdomyolysis in children and adults. Current treatment includes dietary fat restriction, with increased carbohydrate intake and exercise restriction to avoid muscle pain and rhabdomyolysis. METHODS: CPT II enzyme assay, DNA mutation analysis, quantitative analysis of acylcarnitines in blood and cultured fibroblasts, urinary organic acids, the standardized 36-item Short-Form Health Status survey (SF-36) version 2, and bioelectric impedance for body fat composition. Diet treatment with triheptanoin at 30% to 35% of total daily caloric intake was used for all patients. RESULTS: Seven patients with CPT II deficiency were studied from 7 to 61 months on the triheptanoin (anaplerotic) diet. Five had previous episodes of rhabdomyolysis requiring hospitalizations and muscle pain on exertion prior to the diet (two younger patients had not had rhabdomyolysis). While on the diet, only two patients experienced mild muscle pain with exercise. During short periods of noncompliance, two patients experienced rhabdomyolysis with exercise. None experienced rhabdomyolysis or hospitalizations while on the diet. All patients returned to normal physical activities including strenuous sports. Exercise restriction was eliminated. Previously abnormal SF-36 physical composite scores returned to normal levels that persisted for the duration of the therapy in all five symptomatic patients. CONCLUSIONS: The triheptanoin diet seems to be an effective therapy for adult-onset carnitine palmitoyltransferase II deficiency.

An unusual cause for ketoacidosis.

A 22-year-old male developed a severe degree of metabolic acidosis (plasma pH 7.20, bicarbonate 8 mmol/l), with a large increase in the plasma anion gap (26 mEq/l). Ketoacidosis was suspected because of the odour of acetone on his breath and a positive qualitative test for acetone in plasma (to a 1:4 dilution). Later, his plasma beta-hydroxybutyrate concentration was found to be 4.5 mmol/l. After receiving an infusion of 1 l of half-isotonic saline and 1 l of 5% dextrose in water over 24 h, as well as curtailing his large oral intake of sweetened beverages, all blood tests became normal. Diabetic ketoacidosis, alcoholic ketoacidosis, starvation ketosis and hypoglycaemic ketoacidosis were all ruled out, and his toxin screen was negative for salicylates. Finding another possible cause for ketoacidosis became the focus of this case.

Assay of the concentration and 13C-isotopic enrichment of malonyl-coenzyme A by gas chromatography-mass spectrometry.

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of malonyl-CoA in tissues. The assay involves perchloric acid extraction of the tissue, spiking the extract with [U-13C3]malonyl-CoA or dimethylmalonyl-CoA internal standard, isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge, alkaline hydrolysis to malonate, trimethylsilyl derivatization, and analysis of the mass isotopomer distribution of malonate. The assay was applied to labeling of malonyl-CoA from various [13C]substrates in perfused rat livers and hearts. In livers perfused with [1,2-13C2]acetate, malonyl-CoA is doubly labeled from [1,2-13C2]acetate and singly labeled from 13CO2. In livers perfused with either NaH13CO3 or [3-13C]lactate + [3-13C]pyruvate, the half-lives of singly labeled malonyl-CoA were less than 20 s and 6.95 min, respectively. In rat heart, the half-life of malonyl-CoA, traced with NaH13CO3, was about 1.25 min. Thus, our assay allows us to measure the turnover of tissue malonyl-CoA, the contribution of various [13C]substrates to its production in lipogenic and nonlipogenic organs, and the cycling between acetyl-CoA and malonyl-CoA in nonlipogenic organs.

Acute hibernation decreases myocardial pyruvate carboxylation and citrate release.

In the well-perfused heart, pyruvate carboxylation accounts for 3-6% of the citric acid cycle (CAC) flux, and CAC carbon is lost via citrate release. We investigated the effects of an acute reduction in coronary flow on these processes and on the tissue content of CAC intermediates. Measurements were made in an open-chest anesthetized swine model. Left anterior descending coronary artery blood flow was controlled by a extracorporeal perfusion circuit, and flow was decreased by 40% for 80 min to induce myocardial hibernation (n = 8). An intracoronary infusion of [U-(13)C(3)]lactate and [U-(13)C(3)]pyruvate was given to measure the entry of pyruvate into the CAC through pyruvate carboxylation from the (13)C-labeled isotopomers of CAC intermediates. Compared with normal coronary flow, myocardial hibernation resulted in parallel decreases of 65% and 79% in pyruvate carboxylation and net citrate release by the myocardium, respectively, and maintenance of the CAC intermediate content. Elevation of the arterial pyruvate concentration by 1 mM had no effect. Thus a 40% decrease in coronary blood flow resulted in a concomitant decrease in pyruvate carboxylation and citrate release as well as maintenance of the CAC intermediates.

A probing dose of phenylacetate does not affect glucose production and gluconeogenesis in humans.

Phenylacetate ingestion has been used to probe Krebs cycle metabolism and to augment waste nitrogen excretion in urea cycle disorders. Phenylalkanoic acids, including phenylacetate, have been proposed as potential therapeutic agents in the treatment of diabetes. They inhibit gluconeogenesis in the liver in vitro and reduce the blood glucose concentration in diabetic rats. The effect of sodium phenylacetate ingestion on blood glucose and the contribution of gluconeogenesis to glucose production have now been studied in 7 type 2 diabetic patients. The study was not designed to test whether the changes in glucose metabolism observed in the rat could be reproduced in humans. After an overnight fast, over a period of 1 hour, 4.8 g phenylacetate was ingested, which is the highest dose used to probe Krebs cycle metabolism. Glucose production was measured by tracer kinetics using [6,6-(2)H2]glucose and gluconeogenesis by the labeling of the hydrogens of blood glucose on (2)H20 ingestion. The concentration of phenylacetate in plasma peaked by 2 hours after its ingestion, and about 40% of the dose was excreted in 5 hours. The plasma glucose concentration and production, and the contribution of gluconeogenesis to glucose production, were unaffected by phenylacetate ingestion at the highest dose used to probe Krebs cycle metabolism.

Partitioning of pyruvate between oxidation and anaplerosis in swine hearts.

The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts by infusing [U-(13)C(3)]lactate and/or [U-(13)C(3)] pyruvate into the left anterior descending (LAD) coronary artery. After 1 h, the tissue was freeze-clamped and analyzed by gas chromatography-mass spectrometry for the mass isotopomer distribution of citrate and its oxaloacetate moiety. LAD blood pyruvate and lactate enrichments and concentrations were constant after 15 min of infusion. Under near-normal physiological concentrations of lactate and pyruvate, pyruvate carboxylation and decarboxylation accounted for 4.7 +/- 0.3 and 41.5 +/- 2.0% of citrate formation, respectively. Similar relative fluxes were found when arterial pyruvate was raised from 0.2 to 1.1 mM. Addition of 1 mM octanoate to 1 mM pyruvate inhibited pyruvate decarboxylation by 93% without affecting carboxylation. The absence of M1 and M2 pyruvate demonstrated net irreversible pyruvate carboxylation. Under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the citric acid cycle flux despite considerable variation in the flux through pyruvate decarboxylation.

Dog model of therapeutic ketosis induced by oral administration of R,S-1,3-butanediol diacetoacetate.

A high-fat, almost carbohydrate-free diet is used in children with intractable epilepsy to help control seizures by inducing a permanent state of ketosis. Esters of ketone bodies have been previously studied for their potential as parenteral and enteral nutrients. We tested in conscious dogs whether ketosis could be induced by repeated ingestion of R,S-1,3-butanediol diacetoacetate with or without carbohydrates. This ester is a water-soluble precursor of ketone bodies. Two constraints were imposed on this preclinical study: The rate of ester administration was limited to one half of the daily caloric requirement and to one half of the capacity of the liver to oxidize butanediol derived from ester hydrolysis. Under these conditions, the level of ketosis achieved in this dog model (0.8 mM) was lower than the level measured in children whose seizures were controlled by the ketogenic diet (1-3 mM). However, because humans may have a lower capacity for ketone body utilization than dogs, the doses of R,S-butanediol diacetoacetate used in the present study might induce higher average ketone body concentrations in humans than in dogs.

Glutamate, a window on liver intermediary metabolism.

In isotopic experiments, the labeling pattern of glutamate opens a window on hepatic metabolism, particularly the citric acid cycle, gluconeogenesis and fatty acid oxidation. This is because glutamate is in isotopic equilibrium with alpha-ketoglutarate, whose labeling pattern is influenced by the following: 1) the contributions of glucose and fatty acids to acetyl-CoA, 2) the relative contributions of pyruvate carboxylase and pyruvate dehydrogenase to the entry of pyruvate carbon into the citric acid cycle, and 3) the rate of gluconeogenesis in relation to citric acid cycle activity. In humans and primates, hepatic glutamate can be sampled noninvasively via urinary phenylacetylglutamine, which is formed in liver from phenylacetate (a side product of phenylalanine catabolism) and glutamine (which equilibrates with liver glutamate and alpha-ketoglutarate). The (14)C- or (13)C-labeling pattern of the glutamate moiety of phenylacetylglutamine can be measured by sequential degradations to (14)CO(2), gas chromatography-mass spectrometry or nuclear magnetic resonance (NMR). When phenylacetylglutamine is labeled from singly labeled [(14)C]- or [(13)C]substrates, relative metabolic rates can be computed from the labeling pattern using Landau's model. In diabetic patients infused with [3-(13)C]pyruvate, the noninvasive sampling of hepatic glutamate via phenylacetylglutamine allows one to test the degree of liver insulinization via the (pyruvate carboxylase)/(pyruvate dehydrogenase) activity ratio. This ratio regulates gluconeogenesis in part. Its measurement may allow the identification of patients who might benefit from the intraperitoneal administration of insulin, or from recently developed antidiabetic drugs.

Integrative physiology of splanchnic glutamine and ammonium metabolism.

The substrates for hepatic ureagenesis are equimolar amounts of ammonium and aspartate. The study design mimics conditions in which the liver receives more NH(+)(4) than aspartate precursors (very low-protein diet). Fasted dogs, fitted acutely with transhepatic catheters, were infused with a tracer amount of (15)NH(4)Cl. From arteriovenous differences, the major NH(+)(4) precursor for hepatic ureagenesis was via deamidation of glutamine in the portal drainage system (rather than in the liver), because there was a 1:1 stoichiometry between glutamine disappearance and NH(+)(4) appearance, and the amide (but not the amine) nitrogen of glutamine supplied the (15)N added to the portal venous NH(+)(4) pool. The liver extracted all this NH(+)(4) from glutamine deamidation plus an additional amount in a single pass, suggesting that there was an activator of hepatic ureagenesis. The other major source of nitrogen extracted by the liver was [(14)N]alanine. Because alanine was not produced in the portal venous system, we speculate that it was derived ultimately from proteins in peripheral tissues.

Zonation of acetate labeling across the liver: implications for studies of lipogenesis by MIDA.

Measurement of fractional lipogenesis by mass isotopomer distribution analysis (MIDA) of fatty acids or cholesterol labeled from [(13)C]acetate assumes constant enrichment of lipogenic acetyl-CoA in all hepatocytes. This would not be the case if uptake and release of acetate by the liver resulted in transhepatic gradients of acetyl-CoA enrichment. Conscious dogs, prefitted with transhepatic catheters, were infused with glucose and [1, 2-(13)C(2)]acetate. Stable concentrations and enrichments of acetate were measured in artery (17 microM, 36%), portal vein (61 microM, 5. 4%), and hepatic vein (17 microM, 1.0%) and were computed for mixed blood entering the liver (53 microM, 7.4%). We also measured balances of propionate and butyrate across gut and liver. All gut release of propionate and butyrate is taken up by the liver. The threefold decrease in acetate concentration and the sevenfold decrease in acetate enrichment across the liver strongly suggest that the enrichment of lipogenic acetyl-CoA decreases across the liver. Thus fractional hepatic lipogenesis measured in vivo by MIDA may be underestimated.

15N enrichment of ammonium, glutamine-amide and urea, measured via mass isotopomer analysis of hexamethylenetetramine.

Ammonium is an important intermediate of protein metabolism and is a key component of acid-base balance. Investigations of the metabolism of NH(4)(+) in vivo using isotopic techniques are difficult because of the low concentration of NH(4)(+) in biological fluids and because of frequent artifactual isotopic dilution of the enrichment of NH(4)(+) during the assay. A new gas chromatographic mass spectrometric method was designed to monitor the (15)N enrichment and concentration of NH(4)(+) in vivo. These are both calculated from the mass isotopomer distribution of hexamethylenetetramine (HMT) formed by reacting NH(4)(+) with formaldehyde. The enrichment of NH(4)(+) is amplified four times since the HMT molecule contains four atoms of nitrogen derived from NH(4)(+). This allows the measurement of low (15)N enrichment of NH(4)(+), down to 0.1%. (15)N enrichment of urea and of the amide N of L-glutamine are measured by enzymatic release of NH(4)(+) and conversion of the latter to HMT. These new techniques facilitate in vivo investigations of the metabolism of NH(4)(+) and related compounds.

Contributions by kidney and liver to glucose production in the postabsorptive state and after 60 h of fasting.

Contributions of renal glucose production to whole-body glucose turnover were determined in healthy individuals by using the arteriovenous balance technique across the kidneys and the splanchnic area combined with intravenous infusion of [U-13C6]glucose, [3-(3)H]glucose, or [6-(3)H]glucose. In the postabsorptive state, the rate of glucose appearance was 11.5 +/- 0.6 micromol x kg(-1) x min(-1). Hepatic glucose production, calculated as the sum of net glucose output (9.8 +/- 0.8 micromol x kg(-1) x min(-1)) and splanchnic glucose uptake (2.2 +/- 0.3 micromol x kg(-1) x min(-1)) accounted for the entire rate of glucose appearance. There was no net exchange of glucose across the kidney and no significant renal extraction of labeled glucose. The renal contribution to total glucose production calculated from the arterial, hepatic, and renal venous 13C-enrichments (glucose M+6) was 5 +/- 2%. In the 60-h fasted state, the rate of glucose appearance was 8.2 +/- 0.3 micromol x kg(-1) x min(-1). Hepatic glucose production, estimated as net splanchnic output (5.8 +/- 0.7 micromol x kg(-1) x min(-1)) plus splanchnic uptake (0.6 +/- 0.3 micromol x kg(-1) x min(-1)) accounted for 79% of the rate of glucose appearance. There was a significant net renal output of glucose (0.9 +/- 0.3 micromol x kg(-1) x min(-1)), but no significant extraction of labeled glucose across the kidney. The renal contribution to whole-body glucose turnover calculated from the 13C-enrichments was 24 +/- 3%. We concluded that 1) glucose production by the human kidney in the postabsorptive state, in contrast to recent reports, makes at most only a minor contribution (approximately 5%) to blood glucose homeostasis, but that 2) after 60-h of fasting, renal glucose production may account for 20-25% of whole-body glucose turnover.

Limitations in estimating gluconeogenesis and Cori cycling from mass isotopomer distributions using [U-13C6]glucose.

Tayek and Katz proposed calculating gluconeogenesis's contributions to glucose production and Cori cycling from mass isotopomer distributions in blood glucose and lactate during [U-13C6]glucose infusion [Tayek, J. A., and J. Katz. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E476-E484, 1997]. However, isotopic exchange was not adequately differentiated from dilution, nor was condensation of labeled with unlabeled triose phosphates properly equated. We introduce and apply corrected equations to data from subjects fasted for 12 and 60 h. Impossibly low contributions of gluconeogenesis to glucose production at 60 h are obtained (23-41%). Distributions in overnight-fasted normal subjects calculate to only approximately 18%. Cori cycling estimates are approximately 10-15% after overnight fasting and 20% after 60 h of fasting. There are several possible reasons for the underestimates. The contribution of gluconeogenesis is underestimated because glucose production from glycerol and amino acids not metabolized via pyruvate is ascribed to glycogenolysis. Labeled oxaloacetate and alpha-ketoglutarate can exchange during equilibrium with circulating unlabeled aspartate, glutamate, and glutamine. Also, the assumption that isotopomer distributions in arterial lactate and hepatic pyruvate are the same may not be fulfilled.

Limitations of the mass isotopomer distribution analysis of glucose to study gluconeogenesis. Heterogeneity of glucose labeling in incubated hepatocytes.

We previously reported (Previs, S. F., Fernandez, C. A., Yang, D., Soloviev, M. V., David, F., and Brunengraber, H. (1995) J. Biol. Chem. 270, 19806-19815) that glucose made in isolated livers from starved rats perfused with physiological concentrations of lactate, pyruvate, and either [2-13C]- or [U-13C3]glycerol had a mass isotopomer distribution incompatible with glucose being made from a homogeneously labeled pool of triose phosphates. Similar data were obtained in live rats infused with [U-13C3]glycerol. We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver. We concluded that [13C]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments of gluconeogenic substrates. Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate + pyruvate) infusion ratio ranged from 0.23 to 3. 60. Metabolic and isotopic steady states were achieved in all cases. The apparent contribution of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were 13C-labeled, f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([13C]glycerol)/(lactate + pyruvate) increased, f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([13C]lactate + [13C]pyruvate) increased, f decreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in f show that the 13C labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and enrichments. We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol in rat liver.

Assay of low deuterium enrichment of water by isotopic exchange with [U-13C3]acetone and gas chromatography-mass spectrometry.

A sensitive assay of the 2H-enrichment of water based on the isotopic exchange between the hydrogens of water and of acetone in alkaline medium is described and validated. For low 2H-enrichments (0.008 to 0.5%), the sample is spiked with [U-13C3]acetone and NaOH. After exchange, 2H-enriched [U-13C3]acetone is extracted with chloroform and assayed by gas chromatography-mass spectrometry. With some instruments, ion-molecule reactions, resulting in increased baseline enrichment, are minimized by lowering the electron ionization energy from the usual 70 to 10 eV. The 2H-enrichment of water is amplified nearly sixfold in the M4/M3 ratio of [U-13C3]acetone. For high 2H-enrichments (0.25 to 100%), the use of unlabeled acetone suffices. After exchange, the mass isotopomer distribution of acetone is analyzed, yielding the 2H-enrichment of water. The assay with [U-13C3]acetone allows measuring the 2H-enrichment of water even in biological samples containing acetone. This technique is more rapid and economical than the classical isotope ratio mass spectrometric assay of the enrichment of hydrogen gas derived from the reduction of water.

Non-invasive tracing of liver intermediary metabolism in normal subjects and in moderately hyperglycaemic NIDDM subjects. Evidence against increased gluconeogenesis and hepatic fatty acid oxidation in NIDDM.

To test whether gluconeogenesis is increased in non-insulin-dependent diabetic (NIDDM) patients we infused (post-absorptive state) healthy subjects and NIDDM patients with [6,6-2H2]glucose (150 min) and [3-13C]lactate (6 h). Liver glutamine was sampled with phenylacetate and its labelling pattern determined (mass spectrometry) after purification of the glutamine moiety of urinary phenylacetylglutamine. After correction for 13CO2 re-incorporation (control test with NaH13CO3 infusion) this pattern was used to calculate the dilution factor (F) in the hepatic oxaloacetate pool and fluxes through liver Krebs cycle. NIDDM patients had increased lactate turnover rates (16.18+/-0.92 vs 12.14+/-0.60 micromol x kg(-1) x min(-1), p < 0.01) and a moderate rise in glucose production (EGP) (15.39+/-0.87 vs 12.52+/-0.28 micromol x kg(-1) x min(-1) , p = 0.047). Uncorrected contributions of gluconeogenesis to EGP were 31+/-3 % (control subjects) and 17+/-2 % (NIDDM patients). F was comparable (1.34+/-0.02 and 1.39 0.09, respectively) and the corrected percent and absolute contributions of gluconeogenesis were not increased in NIDDM (25+/-3 % and 3.8+/-O.5 micromol x kg(1) x min[-1]) compared to control subjects (41+/-3 % and 5.1+/-0.4 micromol x kg(-1) x min(-1]). The calculated pyruvate carboxylase over pyruvate dehydrogenase activity ratio was comparable (12.1+/-2.6 vs 11.2+/-1.4). Lastly hepatic fatty oxidation, as estimated by the model, was not increased in NIDDM (1.8+/-0.4 vs 1.6+/-0.1 micromol x kg(-1) x min[-1]). In conclusion, in the patients studied we found no evidence of increased hepatic fatty oxidation, or, despite the increased lactate turnover rate, an increased gluconeogenesis.

Methods for measuring gluconeogenesis in vivo.

Recent developments in stable isotope technology have led to the conception of new protocols for measuring the contribution of gluconeogenesis to glucose production in humans. Earlier techniques were subject to variable underestimations resulting from isotopic exchanges occurring during the transfer of carbon label from the tracer to glucose. This review concentrates on four novel techniques: (1) mass isotopomer distribution analysis of glucose labelled from [13C]glycerol or [13C]lactate; (2) mass isotopomer distribution analysis of glucose and lactate during infusion of [U-13C6]glucose; (3) 2H-enrichment of body water by ingestion of 2H2O, and measuring the 2H-labelling on C5 and C2 of glucose, and (4) difference between glucose turnover and rate of hepatic glycogenolysis measured by nuclear magnetic resonance spectroscopy. The advantages and limitations of the four protocols are discussed. The 2H2O technique is the most practical; it is not subject to artifacts resulting from isotopic exchanges, and is not affected by zonation of hepatic metabolism.