Assay of the Concentration and 13C Enrichment of Acetate and Acetyl-CoA by Gas Chromatography-Mass Spectrometry

David, F.; Beylot, M.; Reider, Masha; Anderson, Vernon E.; Brunengraber, Henri

doi:10.1006/abio.1994.1153

Cited by 13 publications

(14 citation statements)

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“…We had described previously an assay of the 13 C-enrichment of acetyl-CoA after conversion into acetylglycine or acetylsarcosine [19]. Acetylsarcosine was more practical than acetylglycine, because it can be extracted in organic solvent.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…Notations-Let us call [C y Met] CzAc the amount of the metabolite (Met) formed and labeled on its carbon y (where 1 Յ y Յ 5) arising from acetate labeled on its carbon z, where z is equal to 1 or 2 because we used [1][2][3][4][5][6][7][8][9][10][11][12][13] Table I.…”

Section: Discussionmentioning

confidence: 99%

“…The flasks contained 1 ml of the suspension plus 3 ml of Krebs-Henseleit medium (12), either supplemented or not with substrates, i.e. 5 mM (final concentration) [1-14 C]acetate or [2-14 C]acetate (10 3 Bq/flask), [1][2][3][4][5][6][7][8][9][10][11][12][13] C]acetate or [2- 13 C]acetate in the absence and the presence of 5 mM L-alanine. These differently labeled acetates were used in an attempt to completely define the fate of the two acetate carbons.…”

Section: Preparation Of Kidney Tubules and Incubationmentioning

confidence: 99%

“…Its blood concentration is low (less than 0.2 mM) in fed and starved humans and starved herbivores but may reach the millimolar range in humans after alcohol consumption and in fed herbivorous species (1-7). The sources of blood acetate are on the one hand absorption of the acetate formed as a result of gastrointestinal bacterial fermentation, and on the other hand, the acetate formed and released by various tissues containing acetyl-CoA hydrolase activity (2,3,6,8).On the basis of experiments performed in vivo with labeled acetate, it has been shown that the turnover of circulating acetate is rapid and that, depending on the species and nutritional state, the oxidation of this compound provides from 6 to 70% of the whole body energy expenditure (3,4,7,9). This means that acetate is removed and metabolized by peripheral tissues.…”

mentioning

confidence: 99%

“…On the basis of experiments performed in vivo with labeled acetate, it has been shown that the turnover of circulating acetate is rapid and that, depending on the species and nutritional state, the oxidation of this compound provides from 6 to 70% of the whole body energy expenditure (3,4,7,9). This means that acetate is removed and metabolized by peripheral tissues.…”

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confidence: 99%

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The Anaplerotic Substrate Alanine Stimulates Acetate Incorporation into Glutamate and Glutamine in Rabbit Kidney Tubules

Conjard

Dugelay

Chauvin

et al. 2002

Journal of Biological Chemistry

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Although acetate, the main circulating volatile fatty acid in humans and animals, is metabolized at high rates by the renal tissue, little is known about the precise fate of its carbons and about the regulation of its renal metabolism. Therefore, we studied the metabolism of variously labeled [13 C]acetate and [ 14 C]acetate molecules and its regulation by alanine, which is also readily metabolized by the kidney, in isolated rabbit renal proximal tubules. With acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of acetate were released as CO 2 ; with acetate plus alanine, the corresponding values were decreased to 49 and 25%. The only other important products formed from the acetate carbons were glutamine, and to a smaller extent, glutamate. By combining 13 C NMR and radioactive and enzymatic measurements with a novel model of acetate metabolism, fluxes through the enzymes involved were calculated. Thanks to its anaplerotic effect, alanine caused a stimulation of acetate removal and a large increase in fluxes through pyruvate carboxylase, citrate synthase, and the enzymes involved in glutamate and glutamine synthesis but not in flux through ␣-ketoglutarate dehydrogenase. We conclude that the anaplerotic substrate alanine not only accelerates the disposal of acetate but also prevents the wasting of the latter compound as CO 2 .Acetate is the main circulating volatile fatty acid in humans and other mammalian species. Its blood concentration is low (less than 0.2 mM) in fed and starved humans and starved herbivores but may reach the millimolar range in humans after alcohol consumption and in fed herbivorous species (1-7). The sources of blood acetate are on the one hand absorption of the acetate formed as a result of gastrointestinal bacterial fermentation, and on the other hand, the acetate formed and released by various tissues containing acetyl-CoA hydrolase activity (2,3,6,8).On the basis of experiments performed in vivo with labeled acetate, it has been shown that the turnover of circulating acetate is rapid and that, depending on the species and nutritional state, the oxidation of this compound provides from 6 to 70% of the whole body energy expenditure (3,4,7,9). This means that acetate is removed and metabolized by peripheral tissues. Indeed, acetyl-CoA synthetase, the enzyme that initiates acetate degradation, has been demonstrated to be active in many tissues including the liver, kidney, heart, brain, adipose tissue, and skeletal muscle (3). It has been found that, besides the heart, the kidney contains a high activity of this enzyme (3).In agreement with this observation, we have shown in a recent study that acetate is readily metabolized by suspensions of rabbit renal proximal tubules (10). In the same study (10), we have demonstrated that acetate significantly altered the metabolism of alanine, a major precursor of glutamine in these tubules. For this, we used 13 C-labeled alanine and unlabeled acetate in combination with enzymatic and 13 C NMR spectroscopy measurements to calculate metabo...

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

et al. 1995

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Isotope dilution gas chromatographic mass spectrometric assays were developed for the concentration of shortchain fatty acids and of lactate in plasma. The assays involve spiking the samples with 'H-and/or "C-labeled internal standards and direct derivatization with 2,4difluoroaniline, using 1,3-dicyclohexylcarbiimide as a coup ling agent. The assay can be used to determine the isotopic enrichment or mass isotopomer distribution of the fatty acids and of lactate. Blanks by ubiquitous formate, acetate and lactate are minimized by the one-step derivatization procedure without deproteinization, using reagents dissolved in hydrocarbons. The assays were applied to experiments conducted in live dogs, pigs and monkeys, and in perfused rat livers. These techniques, in conjunction with appropriate stable isotope models, can be used to study carbohydrate fermentation in humans, ruminants and non-ruminant mammals.

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Assay of the Concentration and 13C Enrichment of Acetate and Acetyl-CoA by Gas Chromatography-Mass Spectrometry

Cited by 13 publications

References 0 publications

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

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

The Anaplerotic Substrate Alanine Stimulates Acetate Incorporation into Glutamate and Glutamine in Rabbit Kidney Tubules

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

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