Assay of the concentration and stable isotope enrichment of short‐chain fatty acids by gas chromatography/mass spectrometry

Powers, Lisa; Osborn, Melissa; Yang, Dongyan; Kien, C. Lawrence; Murray, Robert; Beylot, M.; Brunengraber, Henri

doi:10.1002/jms.1190300514

Cited by 43 publications

(31 citation statements)

References 14 publications

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“…an indicator of mitochondrial acetyl-CoA, ␤-hydroxybutyrate (BHB) (22), free acetate (29), and formate (29), were assayed as described previously. Exact mass analysis of 4-P-butyryl-CoA and 4-P-pentanoyl-CoA was run on a Thermo Finnigan Fourier transform LTQ mass spectrometer.…”

Section: Methodsmentioning

confidence: 99%

Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl-CoAs

Zhang

Kombu

Kasumov

et al. 2009

Journal of Biological Chemistry

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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)....

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Section: Methodsmentioning

confidence: 99%

Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl-CoAs

Zhang

Kombu

Kasumov

et al. 2009

Journal of Biological Chemistry

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“…The 13 C-labelling of the C1 + 2 fragment of BHB (another probe of mitochondrial acetyl-CoA [7,8]) was calculated as the difference between the labelling of the whole molecule and that of the C3 + 4 fragment [15]. The 13 C-labelling of liver malonyl-CoA [12] and of acetate in the effluent perfusate [16] were assayed as described previously. The labelling of acetylcarnitine was measured by LC-MS (liquid chromatography-MS) [17].…”

Section: Analytical Proceduresmentioning

confidence: 99%

Probing peroxisomal β-oxidation and the labelling of acetyl-CoA proxies with [1-13C]octanoate and [3-13C]octanoate in the perfused rat liver

Kasumov

Adams

Bian

et al. 2005

Biochemical Journal

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We reported previously that a substantial fraction of the acetyl groups used to synthesize malonyl-CoA in rat heart is derived from peroxisomal β-oxidation of long-chain and very-long-chain fatty acids. This conclusion was based on the interpretation of the 13 Clabelling ratio (malonyl-CoA)/(acetyl moiety of citrate) measured in the presence of substrates that label acetyl-CoA in mitochondria only (ratio < 1.0) or in both mitochondria and peroxisomes (ratio > 1.0). The goals of the present study were to test, in rat livers perfused with [1-13 C]octanoate or [3-13 C]octanoate, (i) whether peroxisomal β-oxidation contributes acetyl groups for malonylCoA synthesis, and (ii) the degree of labelling homogeneity of acetyl-CoA proxies (acetyl moiety of citrate, acetate, β-hydroxybutyrate, malonyl-CoA and acetylcarnitine). Our data show that (i) octanoate undergoes two cycles of peroxisomal β-oxidation in liver, (ii) acetyl groups formed in peroxisomes contribute to malonyl-CoA synthesis, (iii) the labelling of acetyl-CoA proxies is markedly heterogeneous, and (iv) the labelling of C1 + 2 of β-hydroxybutyrate does not reflect the labelling of acetyl-CoA used in the citric acid cycle.

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“…A conservative estimate of flux rates, using the mean P i concentration (0.64 Ϯ 0.11 mol/g of tissue), gave a 2-fold increase in the ATP synthesis rates in the UCP3KO mice (0.120 Ϯ 0.10 mol/g of muscle/s) compared with normal mice (0.059 Ϯ 0.10 mol/g of muscle/s). Steady-state plasma [2][3][4][5][6][7][8][9][10][11][12][13] C]acetate enrichments (normal mice, 21.1 Ϯ 4.6; UCP3KO, 18.7 Ϯ 2.4, p ϭ NS) and concentrations were reached within 5 min. The mean basal plasma acetate concentrations were slightly but not significantly higher in the UCP3KO mice (controls, 0.087 Ϯ 0.006 mM; UCP3KO, 0.108 Ϯ 0.006 mM).…”

Section: Table I Phosphorous Metabolites In Skeletal Muscle Of Wild-tmentioning

confidence: 99%

“…Analysis of [2][3][4][5][6][7][8][9][10][11][12][13] C]Acetate and [ 13 C]Glutamate-Plasma acetate concentration and 13 C isotopic enrichment were determined by GC-MS as described in detail elsewhere (12). Briefly, 50 l of plasma was spiked with an equal volume of an internal concentration standard containing 50 M sodium acetic-d 3 acid.…”

Section: Tca Cycle Flux Rates In Skeletal Musclementioning

confidence: 99%

In Vivo Effects of Uncoupling Protein-3 Gene Disruption on Mitochondrial Energy Metabolism

Cline¹,

Vidal-Puig

Dufour

et al. 2001

Journal of Biological Chemistry

133

123

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To clarify the role of uncoupling protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using 31 P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of 13 C enrichment in C-4 and C-2 of glutamate during an infusion of [2-13 C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from P i more than 4-fold under fasting conditions (wild type, 2.2 ؎ 0.6 versus knockout, 9.1 ؎ 1.4 mol/g of muscle/min, p < 0.001) with no change in TCA cycle flux rate (wild type, 0.74 ؎ 0.04 versus knockout, 0.71 ؎ 0.03 mol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05) increase in the ratio of ATP to ADP in the muscle of UCP3KO mice (5.9 ؎ 0.3) compared with controls (4.5 ؎ 0.4). The data presented here provide the first evidence of uncoupling activity by UCP3 in skeletal muscle in vivo.Uncoupling protein-1 (UCP1) 1 has been shown to regulate non-shivering thermogenesis in brown adipose tissue by allowing proton flux across the inner mitochondrial membrane to bypass ATP synthase (1, 2). The high sequence homology (57%) of UCP1 to UCP3, a protein expressed primarily in muscle, is suggestive of a common physiological function of the two proteins (3, 4). However, the results of in vitro and in vivo studies of UCP3 casts doubt on the hypothesis that the primary role of UCP3 is the regulation of energy efficiency in muscle (3-6). Hence, the functional role of UCP3 in the regulation of mitochondrial oxidative phosphorylation in skeletal muscle remains uncertain. For example, the up-regulation of UCP3 protein levels during fasting, when energy efficiency should be increased, is inconsistent with UCP3 acting as an uncoupling protein in vivo. Also, studies of energy metabolism in UCP3 knockout mice (UCP3KO) were unable to demonstrate any phenotypic changes in whole body energy metabolism (3, 4). These mice showed no differences in body composition, growth characteristics, exercise tolerance, fatty acid oxidation, or coldinduced thermogenesis. The clearest evidence to date that UCP3 might have the capacity to act as an uncoupling protein comes from measurements in isolated mitochondria. These studies established for the first time that oxidative ATP production in mitochondria isolated from the muscle of UCP3KO mice were more coupled compared with normal littermates (3, 4) and that proton leak over a range of mitochondrial membrane potentials was decreased (4). Conversely, overexpression of UCP3 in muscle uncoupled oxidative phosphorylation in isolated mitochond...

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Assay of the concentration and stable isotope enrichment of short‐chain fatty acids by gas chromatography/mass spectrometry

Cited by 43 publications

References 14 publications

Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl-CoAs

Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl-CoAs

Probing peroxisomal β-oxidation and the labelling of acetyl-CoA proxies with [1-13C]octanoate and [3-13C]octanoate in the perfused rat liver

In Vivo Effects of Uncoupling Protein-3 Gene Disruption on Mitochondrial Energy Metabolism

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