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Mitochondrial enoyl-CoA isomerase (ECU) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Ecil-deficient knockout (KO) mice to explore a potential presentation of human ECU deficiency. Upon food withdrawal, Ecil-deficient mice displayed normal blood β-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:l acylcarnitine (WT 0.03 µM vs. KO 0.09 µM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:l acylcarnitine levels (WT 0.01 oM vs. KO 0.04 µM, P<0.01). Overall, the phenotypic presentation of Ecil-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Ecil-deficient fibroblasts caused a more pronounced accumulation of C12:l acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECU deficiency in humans. [ABSTRACT FROM AUTHOR]

Abstract: Rat hearts perfused for up to 60min in the working mode with palmitate, but not with glucose, resulted in substantial formation of palmitoylcarnitine and stearoylcarnitine. To test whether lipolysis of endogenous lipids was responsible for the increased stearoylcarnitine content or whether some of the perfused palmitate underwent chain elongation, hearts were perfused with hexadecanoic-16,16,16-d 3 acid (M+3). The pentafluorophenacyl ester of deuterium labeled stearoylcarnitine had an M+3 (639.4 m/z) compared to the unlabeled M+0 (636.3 m/z) consistent with a direct chain elongation of the perfused palmitate. Furthermore, the near equal isotope enrichment of palmitoyl- (90.2±5.8%) and stearoylcarnitine (78.0±7.1%) suggest that both palmitoyl- and stearoyl-CoA have ready access to mitochondrial carnitine palmitoyltransferase and that most of the stearoylcarnitine is derived from the perfused palmitate. [Copyright &y& Elsevier]

According to current concepts, GNG from lactate involves the transfer of mitochondrial oxaloacetate to the cytosol via the glutamate-aspartate shuttle. This concept was established by showing that AOA, an inhibitor of aminotransferases, markedly decreases GNG from lactate but not from pyruvate (in GNG from pyruvate, oxaloacetate leaves mitochondria as malate). In rat livers perfused with non-recirculating buffer containing 5 mM lactate ± 0.5 mM AOA, we found that AOA decreased the tissue pyruvate concentration 20-fold. We suspected that AOA (carboxymethoxylamine) forms a carboxymethoxamate with pyruvate. We synthesized this methoxamate and set up its assay by LC-MS-MS. In the effluent of livers perfused with 5 mM lactate ± 0.3 mM AOA, we found pyruvate carboxymethoxamate in the presence of AOA (18 ± 3 µM), but not in its absence. When unlabeled lactate was replaced by [13C3]lactate, M3 [13C3]pyruvate carboxymethoxamate (21 µM) was formed in the presence of AOA. In perfusions with 2 mM pyruvate, GNG was not inhibited by 0.5 mM AOA, but we found pyruvate carboxymethoxamate in the effluent perfusate (216 ± 29 µM). The excess pyruvate prevented the inhibition of GNG by AOA. In conclusion, the inhibition of GNG from lactate by AOA involves the trapping of pyruvate as carboxymethoxamate. This trapping occurs upstream of the inhibition of the glutamate-aspartate shuttle by AOA. [ABSTRACT FROM AUTHOR]

Heinen et al reported the use of [U-13C6]gluconolactone to trace the PPP in microorganisms (Appl. Environ. Microbiology 07/2006, p 4743). We adapted this technique for use in perfused hearts and livers from rats. The tissue was extracted in acetonitrile:water (50:50) and the supernatant applied to an ion exchange solid phase extraction cartridge. The sugar phosphates were eluted with 50 mM ammonium formate in 50% methanol-water (recovery was 90%). We used a hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems) coupled with HPLC to monitor the sugar phosphates using the enhanced resolution mode. We used hydrophilic interaction liquid chromatography to avoid ion pairing reagents and to increase the detection sensitivity. The flux through the PPP is calculated as (uptake of [U-13C6]gluconolactone)/(M6 enrichment of 6-P-gluconate). The latter can be used in tracer amounts because of low background of analytes at M6 and M5. The production of NADPH in the PPP is equal to twice its flux. The contribution of the oxidative branch of the PPP is the enrichment ratio (M5 ribose-5-P)/(M6 6-P-gluconate). [ABSTRACT FROM AUTHOR]

We present a new LC-MS-MS technique for measuring the concentration and stable isotope enrichment of reduced and oxidized glutathione (GSH and GSSG) in biological fluids. Because of differences in signal sensitivity and stability of GSH and GSSG, we assay both compounds as similar GSH thioethers. The assay involves (i) spiking the sample with homoglutathione internal standard (glutamate-cysteine-alanine), (ii) reaction with iodoacetate to form a stable thioether with extant GSH, (iii) reaction with mercaptoethanol to reduce GSSG to GSH, and to trap excess iodoacetate (iv) reaction with excess iodoacetonitrile to form a stable thioether nitrile with GSH derived from GSSG reduction, and (v) electrospray-ionization mass spectrometry of thioethers on a 4000 QTrap (Applied Biosystems) coupled to Agilent HPLC. In addition to enhanced mass spectra, ions monitored are 366.1/237.2 for GSH, 347.2/272.1 for GSH derived from GSSG reduction, and 380.1/251.2 for homoglutathione internal standard (carboxymethyl derivative). The method was applied to the measuring of the turnover of plasma GSH/GSSG in rats whose body fluids were 2% 2H-enriched for 2.5 days. GSH and GSSG showed similar patterns of enrichment with half lives of 21 and 23 hr, respectively. [ABSTRACT FROM AUTHOR]

GHB is both a brain metabolite and a drug of abuse (date rape drug). We conducted a metabolomic study of GHB metabolism liver, using GCMS, GC-combustion-IRMS, and LC-MS-MS. Rat livers were perfused with recirculating albuminated buffer containing 4 mM glucose and 2 mM of either unlabeled GHB, [U-13C4]GHB, or [²H6]GHB. In the perfusate, we identified by GC-MS 2,4-dihydroxybutyrate, 3,4-dihydroxybutyrate, and 3-hydroxypropionate. The mass isotopomer distribution of these compounds in livers perfused with GHB, [U-13C4]GHB, or [²H6]GHB confirms that they are derived from the partial β-oxidation of GHB. 3-Hydroxypropionate may derive from the omega-oxidation of 3,4-dihydroxybutyrate (or its CoA ester). GC-combustion-IRMS detected 5 unknown metabolites of [U-13C4]GHB (to be identified). In livers perfused with [U13C4]GHB, acetyl-CoA was unlabeled (even in the absence of glucose), confirming the incomplete β-oxidation of GHB. Citric acid cycle intermediates were labeled from [U-13C4]GHB consistent with entry into the cycle via succinate. LC-MS-MS of liver acyl-CoAs detected an unknown compound (m/z 934 with CoA signature daughter ion at m/z 428). This compound, which becomes M4 with [U-13C4]GHB, and M6 with [²H6]GHB, contains the 4 C and the 6 H of GHB. In livers perfused with unlabeled GHB and either [U-13C6]glucose or [1,2-13C3]acetate, the CoA ester was not labeled. Based on the above and on its rapid migration on the reverse phase LC column, the unknown CoA ester was tentatively identified as 4-phospho-GHB-CoA. [ABSTRACT FROM AUTHOR]

Dietary therapy with triheptanoin, a precursor of anaplerotic propionyl-CoA, is used for the chronic treatment of long-chain fatty acid oxidation disorders (Roe, JCI 110: 252, 2002) and pyruvate carboxylase deficiency (Mochel, Mol Gen Metab 84: 305, 2005). Heptanoate is partially converted in liver to C5-ketone bodies (β-ketopentanoate and β-hydroxypentanoate. These are precursors of propionyl-CoA in peripheral tissues. We tested whether heptanoate can be taken up as such by the brain. Anesthetized mice were infused with [5,6,7-13C3]heptanoate (20 to 100 nmol/g x min) for 30 min before freezing the brains. We assayed the concentration and mass isotopomer distribution of brain acyl-CoAs by LC-MS-MS. We found that the brain concentrations of [5,6,7-13C3]heptanoyl-CoA, [3,4,5-13C3]pentanoyl-CoA, and [1,2,3-13C3]propionyl-CoA increased with the rate of infusion of [5,6,7-13C3]heptanoate. The spectrum of triply-labeled acyl-CoAs demonstrates that heptanoate is taken up as such by the brain, and that it is anaplerotic in this organ. Our data support the hypothesis, presented in (Mol Gen Metab 84: 305, 2005) that the neurological status of a patient with pyruvate carboxylase deficiency was improved by anaplerosis from heptanoate in the brain. These investigations are extended to mice deficient in very long-chain acyl-CoA dehydrogenase deficiency. [ABSTRACT FROM AUTHOR]