Rajan S. Kombu

Catabolism of 4-hydroxyacids and 4-hydroxynonenal via 4-hydroxy-4-phosphoacyl-CoAs.

4-Hydroxyacids are products of ubiquitously occurring lipid peroxidation (C9, C6) or drugs of abuse (C4, C5). We investigated the catabolism of these compounds using a combination of metabolomics and mass isotopomer analysis. Livers were perfused with various concentrations of unlabeled and labeled saturated 4-hydroxyacids (C4 to C11) or 4-hydroxynonenal. All the compounds tested form a new class of acyl-CoA esters, 4-hydroxy-4-phosphoacyl-CoAs, characterized by liquid chromatography-tandem mass spectrometry, accurate mass spectrometry, and 31P-NMR. All 4-hydroxyacids with five or more carbons are metabolized by two new pathways. The first and major pathway, which involves 4-hydroxy-4-phosphoacyl-CoAs, leads in six steps to the isomerization of 4-hydroxyacyl-CoA to 3-hydroxyacyl-CoAs. The latter are intermediates of physiological β-oxidation. The second and minor pathway involves a sequence of β-oxidation, α-oxidation, and β-oxidation steps. In mice deficient in succinic semialdehyde dehydrogenase, high plasma concentrations of 4-hydroxybutyrate result in high concentrations of 4-hydroxy-4-phospho-butyryl-CoA in brain and liver. The high concentration of 4-hydroxy-4-phospho-butyryl-CoA may be related to the cerebral dysfunction of subjects ingesting 4-hydroxybutyrate and to the mental retardation of patients with 4-hydroxybutyric aciduria. Our data illustrate the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.

Parenteral and enteral metabolism of anaplerotic triheptanoin in normal rats. II. Effects on lipolysis, glucose production, and liver acyl-CoA profile

The anaplerotic odd-medium-chain triglyceride triheptanoin is used in clinical trials for the chronic dietary treatment of patients with long-chain fatty acid oxidation disorders. We previously showed (Kinman RP, Kasumov T, Jobbins KA, Thomas KR, Adams JE, Brunengraber LN, Kutz G, Brewer WU, Roe CR, Brunengraber H. Am J Physiol Endocrinol Metab 291: E860-E866, 2006) that the intravenous infusion of triheptanoin increases lipolysis traced by the turnover of glycerol. In this study, we tested whether lipolysis induced by triheptanoin infusion is accompanied by the potentially harmful release of long-chain fatty acids. Rats were infused with heptanoate +/- glycerol or triheptanoin. Intravenous infusion of triheptanoin at 40% of caloric requirement markedly increased glycerol endogenous R(a) but not oleate endogenous R(a). Thus, the activation of lipolysis was balanced by fatty acid reesterification in the same cells. The liver acyl-CoA profile showed the accumulation of intermediates of heptanoate beta-oxidation and C(5)-ketogenesis and a decrease in free CoA but no evidence of metabolic perturbation of liver metabolism such as propionyl overload. Our data suggest that triheptanoin, administered either intravenously or intraduodenally, could be used for intensive care and nutritional support of metabolically decompensated long-chain fatty acid oxidation disorders.

Metabolomic and Mass Isotopomer Analysis of Liver Gluconeogenesis and Citric Acid Cycle I. INTERRELATION BETWEEN GLUCONEOGENESIS AND CATAPLEROSIS; FORMATION OF METHOXAMATES FROM AMINOOXYACETATE AND KETOACIDS

We conducted a study coupling metabolomics and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. Rat livers were perfused with lactate or pyruvate ± aminooxyacetate or mercaptopicolinate in the presence of 40% enriched NaH13CO3. Other livers were perfused with dimethyl [1,4-13C2]succinate ± mercaptopicolinate. In this first of two companion articles, we show that a substantial fraction of gluconeogenic carbon leaves the liver as citric acid cycle intermediates, mostly α-ketoglutarate. The efflux of gluconeogenic carbon ranges from 10 to 200% of the rate of liver gluconeogenesis. This cataplerotic efflux of gluconeogenic carbon may contribute to renal gluconeogenesis in vivo. Multiple crossover analyses of concentrations of gluconeogenic intermediates and redox measurements expand previous reports on the regulation of gluconeogenesis and the effects of inhibitors. We also demonstrate the formation of adducts from the condensation, in the liver, of (i) aminooxyacetate with pyruvate, α-ketoglutarate, and oxaloacetate and (ii) mercaptopicolinate and pyruvate. These adducts may exert metabolic effects unrelated to their effect on gluconeogenesis.

Metabolomic and Mass Isotopomer Analysis of Liver Gluconeogenesis and Citric Acid Cycle II. HETEROGENEITY OF METABOLITE LABELING PATTERN

In this second of two companion articles, we compare the mass isotopomer distribution of metabolites of liver gluconeogenesis and citric acid cycle labeled from NaH13CO3 or dimethyl [1,4-13C2]succinate. The mass isotopomer distribution of intermediates reveals the reversibility of the isocitrate dehydrogenase + aconitase reactions, even in the absence of a source of α-ketoglutarate. In addition, in many cases, a number of labeling incompatibilities were found as follows: (i) glucose versus triose phosphates and phosphoenolpyruvate; (ii) differences in the labeling ratios C-4/C-3 of glucose versus (glyceraldehyde 3-phosphate)/(dihydroxyacetone phosphate); and (iii) labeling of citric acid cycle intermediates in tissue versus effluent perfusate. Overall, our data show that gluconeogenic and citric acid cycle intermediates cannot be considered as sets of homogeneously labeled pools. This probably results from the zonation of hepatic metabolism and, in some cases, from differences in the labeling pattern of mitochondrial versus extramitochondrial metabolites. Our data have implications for the use of labeling patterns for the calculation of metabolic rates or fractional syntheses in liver, as well as for modeling liver intermediary metabolism.

Dynamics of glutathione and ophthalmate traced with 2H-enriched body water in rats and humans

We developed a LC-MS-MS assay of the (2)H labeling of free glutathione (GSH) and bound glutathione [GSSR; which includes all DTT-reducible forms, primarily glutathione disulfide (GSSG) and mixed disulfides with proteins] and ophthalmate (an index of GSH depletion) labeled from (2)H-enriched body water. In rats whose body water was 2.5% (2)H enriched for up to 31 days, GSH labeling follows a complex pattern because of different rates of labeling of its constitutive amino acids. In rats infused with [(13)C(2),(15)N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 micromol.min(-1).kg(-1), and the half-life of plasma GSH/GSSR was 6-8 min. In healthy humans whose body fluids were 0.5% (2)H enriched, the (2)H labeling of GSH/GSSR and ophthalmate can be precisely measured after 4 h, with GSH being more rapidly labeled than GSSR. Since plasma GSH/GSSR derives mostly from liver, this technique opens the way to 2) probe noninvasively the labeling pattern and redox status of the liver GSH system in humans and 2) assess the usefulness of ophthalmate as an index of GSH depletion.

Analysis of the Citric Acid Cycle Intermediates Using Gas Chromatography-Mass Spectrometry

Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer useful information to understanding interrelationships between the CAC and metabolic pathways under various physiological and pathological conditions. This chapter presents a relatively simple method that is sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using gas chromatography-mass spectrometry.

Polyethylene glycol modified-albumin enhances the cold preservation properties of university of wisconsin solution in rat liver and a hepatocyte cell line

Liver grafts preserved in cold storage undergo changes mainly manifested by morphological modifications of the sinusoidal endothelium that result in poor graft function upon reperfusion. The present studies aimed to determine if the addition of polyethylene glycol-albumin to University of Wisconsin (Peg-AlbUW) solution ameliorates the cold preservation injuries of liver grafts. Rat livers were preserved cold with various preservation solutions and evaluated for weight changes and endothelial morphology. Solutions that preserved graft weight and endothelial morphology were tested in the isolated perfused rat liver model to assess graft function. A rat hepatocyte cell line was evaluated for both viability and glutathione concentrations emulating cold preservation and reperfusion conditions. Liver grafts preserved with Peg-AlbUW maintained their initial weight and showed a conserved endothelial morphology compared with liver grafts preserved in UW for 30 h (P<0.05). Liver grafts preserved with Peg-AlbUW had improved portal blood flow and bile secretion compared with liver grafts preserved in UW for 30 h (P<0.05). In vitro we noted comparable hepatocyte viability when cells were preserved in Peg-AlbUW versus UW under similar preservation conditions (P>0.05); glutathione concentrations (reduced and total) were significantly increased in hepatocytes preserved in 3% Peg-AlbUW compared with other preservation solutions (P<0.05). The addition of Peg-Alb to UW preservation solution ameliorated the cold preservation injuries of rat liver grafts as shown by stable liver graft weight, a better preservation of the endothelial morphology, improved portal vein blood flow, and increased bile secretion. Peg-Alb-UW solution improved the integrity of the glutathione redox buffer system of a hepatocyte cell line after cold storage and reperfusion.

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.

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.