4-Hydroxyacids are products of ubiquitously occurring lipid peroxidation (C 9 , C 6 ) or drugs of abuse (C 4 , C 5 ). 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 (C 4 to C 11 ) 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 31 P-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-phosphoacylCoAs, leads in six steps to the isomerization of 4-hydroxyacylCoA to 3-hydroxyacyl-CoAs. The latter are intermediates of physiological ␤-oxidation. The second and minor pathway involves a sequence of ␤-oxidation, ␣-oxidation, and ␤-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.4-Hydroxy-n-acids are involved in different areas of mammalian metabolism. Some unsaturated 4-hydroxyacids are derived from 4-hydroxynonenal and 4-hydroxyhexenal, which are products of lipid peroxidation (1). The metabolism of 4-hydroxynonenal has been extensively studied, especially its conjugation with glutathione (2), covalent modification of proteins (3, 4), and conversion to 4-hydroxynonenoate, 4-hydroxynonanoate and 1,4-dihydroxynonene, as well as its role in inflammatory processes (1, 5-11). However, the catabolism of its carbon skeleton has not been unraveled. The four-carbon 4-hydroxybutyrate is a physiological neurotransmitter derived from ␥-aminobutyrate. Humans with inborn disorder of succinic semialdehyde dehydrogenase have high 4-hydroxybutyrate concentrations in body fluids, mental retardation, and seizures (12). 4-Hydroxybutyrate is also a drug of abuse that impairs the capacity to exercise judgment for unknown reasons. 4-Hydroxybutyrate is used for the treatment of narcolepsy (13). Its known metabolism (14, 15) proceeds via oxidation to succinic semialdehyde and then to succinate, an intermediate of the citric acid cycle. The five-carbon 4-hydroxypentanoate is also a drug of abuse (16). The calcium salt of a compound closely related to 4-hydroxypentanoate, levulinate (4-ketopentanoate, 4-ketovalerate), is used as an oral or intravenous source of calcium in humans.We conducted a study on the catabolism of C 4 to C 11 4-hydroxyacids in perfused rat livers using a combination of metabolomics (17,18) and mass isotopomer analysis 2 (19)....

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 Ϯ glycerol or triheptanoin. Intravenous infusion of triheptanoin at 40% of caloric requirement markedly increased glycerol endogenous Ra but not oleate endogenous Ra. 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 ␤-oxidation and C5-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.INHERITED FATTY ACID OXIDATION DISORDERS (FOD) can affect the carnitine transporter, the "carnitine cycle" [carnitine palmitoyltransferase (CPT) I, translocase, CPT II], or the mitochondrial ␤-oxidation spiral (for a review, see Ref. 17). Although the phenotype of long-chain FOD is variable, patients often suffer from muscle weakness, hypotonia, cardiac arrhythmia, and cardiomyopathy. Acute episodes, triggered by an infection or trauma, often involve hypoketotic hypoglycemia, massive rhabdomyolysis with release of creatine kinase in plasma, shock, and death (22).Since the early 1980s, the chronic dietary treatment of long-chain FOD involved 1) a moderately high-carbohydrate diet with cornstarch at bed time, 2) decreasing long-chain fats to about 20% of the calories, including essential fatty acids, and 3) providing medium-chain triglycerides (1) since the corresponding C 8 and C 10 fatty acids enter mitochondria as carboxylates, which, after activation, require only those ␤-oxidation enzymes with medium-and short-chain specificity (10). The treatment with medium-chain triglycerides is restricted to long-chain FOD and is contraindicated for medium-and shortchain FOD (17).In 2002, we proposed to replace the even-medium-chain triglycerides with odd-chain triheptanoin (19). Heptanoate, like octanoate, enters mitochondria without passing through the CPT system (10). Unlike octanoate, which is ␤-oxidized to acetyl-CoA, heptanoate is oxidized to acetyl-CoA and propionyl-CoA (Fig. 1). The latter is anaplerotic for the citric acid cycle. We reasoned that, during episodes of metabolic decompensation, such as long-chain FOD, the release of large molecules from cells, e.g., creatine kinase, is probably accompanied by the release of small molecules, including citric acid cycle intermediates. The latter carry acetyl groups as they are oxidized to CO 2 . This would explain the muscle weakness often encountered in long-chain FOD patients. Chronic anaplerotic therapy with triheptanoin improved the clinical status and quality of life of a number of patients (18,20).In another study (8), we explored the metabolism of triheptanoin administered to r...

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

In this second of two companion articles, we compare the mass isotopomer distribution of metabolites of liver gluconeogenesis and citric acid cycle labeled from NaH 13 CO 3 or dimethyl [1,4-13 C 2 ]succinate. The mass isotopomer distribution of intermediates reveals the reversibility of the isocitrate dehydrogenase ؉ aconitase reactions, even in the absence of a source of ␣-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.This second of two companion articles concentrates on a comparison of the mass isotopomer distributions of metabolites of gluconeogenesis and the citric acid cycle in livers perfused with precursors of [1-13 C]PEP. 2 One substrate was NaH 13 CO 3 that labels liver GNG from lactate or pyruvate via carboxylation and isotopic exchange reactions (1). The second substrate was dimethyl [1,4-13 C 2 ]succinate that labels PEP via reactions of the citric acid cycle and PEPCK. We modulated the rates of GNG from lactate, pyruvate, or [1,[4][5][6][7][8][9][10][11][12][13] C 2 ]succinate using mercaptopicolinate (MPA), an inhibitor of PEPCK (2, 3), or aminooxyacetate (AOA), an inhibitor of the glutamate-aspartate shuttle (4 -6). Our data reveal major incompatibilities in the labeling of gluconeogenic intermediates extracted from the whole rat liver. EXPERIMENTAL PROCEDURESMaterials-The materials and rat liver perfusion experiments are described in detail in the accompanying article (28). Briefly, livers from 18-h fasted rats (180 -220 g) were perfused (7) with nonrecirculating bicarbonate buffer (40 ml/min) containing the following: (i) 40% enriched NaH 13 CO 3 and 5 mM lactate, or 2 mM pyruvate Ϯ 0.3 mM MPA, or 0.5 mM AOA (protocol I), or (ii) dimethyl [1,4-13 C 2 ]succinate Ϯ 0.3 mM MPA (protocol II). In orientation experiments, we found that the labeling of gluconeogenic and CAC intermediates as well as glucose production were two to four times greater with dimethyl [1,4-13 C 2 ]succinate than with [1,4-13 C 2 ]succinate (not shown). Similar ratios in glucose production from dimethyl succinate and succinate were reported by Rognstad (8). Therefore, we conducted all the experiments of this group with 0.5 mM dimethyl [1,4-13 C 2 ]succinate Ϯ 0.3 mM MPA. Sample Preparation-Powdered fr...