Assay of Blood and Tissue Oxaloacetate and α-Ketoglutarate by Isotope Dilution Gas Chromatography-Mass Spectrometry

Laplante, A.; Comte, Blandine; DesRosiers, Christine

doi:10.1006/abio.1995.1090

Cited by 20 publications

(9 citation statements)

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“…Subcellular fractionation of the heart was performed as described under "Experimental Procedures." The MIDs of citrate from (i) effluent perfusate collected immediately prior to freeze-clamping of the heart, (ii) whole tissue homogenates, (iii) tissue fractions enriched in mitochondria, and (iv) tissue fractions enriched in cytosol were corrected for natural abundance of heavy isotopes (6,43) and are expressed as molar percent enrichment (MPE). Data are means Ϯ S.E.…”

Section: Resultsmentioning

confidence: 99%

“…Calculation of Relative Fluxes-The MIDs of the acetyl and Rat hearts were perfused for 40 min under normoxia with nonrecirculating buffer containing 11 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 0.2 mM octanoate, and 0.5 mM [U- 13 C 5 ]glutamate. The MID of metabolites, isolated from freeze-clamped hearts, was corrected for natural abundance of heavy isotopes (6,43). Data are expressed as molar percent enrichment (MPE) and are means Ϯ S.E.…”

Section: Labeling Of the Acetyl And Oaa Moieties Of Citrate-thementioning

confidence: 99%

“…Alternatively, the relative contributions of these 13 C-substrates can be estimated from the M1 and M2 enrichments of the acetyl moiety of citrate, 88.0 Ϯ 0.3 and 12.0 Ϯ 0.7%, respectively (Table IIIC). temperature programs, elution times, and ions monitored for the analysis of the MID of CAC metabolites were previously reported (6,18,(37)(38)43). Areas under each fragmentogram were determined by computer integration and corrected for naturally occurring heavy isotopes (6 -7, 18, 36).…”

Section: Ma) the Derivatization Agent N-methyl-n-(t-butyldimethylsilmentioning

confidence: 99%

“…GCMS Methodology-The MID of tissue and effluent perfusate citrate and other CAC metabolites (␣KG, OAA, succinate, fumarate, malate) was determined by published GCMS methods (6,18,(37)(38)43). For subcellular fractionation, tissue was homogenized in Tris buffer 50 mM with sucrose 250 mM (pH 7.4) (15% w/v), followed by sequential 10 min centrifugation at 4°C, at 1,000 ϫ g, and at 10,000 ϫ g (44).…”

Section: Ma) the Derivatization Agent N-methyl-n-(t-butyldimethylsilmentioning

confidence: 99%

“…After 30 min at room temperature, the solution was acidified with concentrated HCl to destroy residual NaBH 4 , and the pH was readjusted to 7.6 before the addition of 750 l of 500 mM triethanolamine buffer (pH 7.4) containing 100 mM MgSO 4 , 50 mM EDTA, 50 mol hydroxylamine-hydrochloride, and 2.5 units of citrate lyase. After 5 min at 37°C, samples were sonicated (for complete conversion of OAA to its oxime derivative (43)). After addition of 350 l of saturated sulfosalicylic acid and centrifugation at 3,000 ϫ g for 15 min, the oxime was extracted from the supernatant with 3 ϫ 18 ml of diethyl ether.…”

Section: Ma) the Derivatization Agent N-methyl-n-(t-butyldimethylsilmentioning

confidence: 99%

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Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the 13C Labeling of Citrate Released by Perfused Rat Hearts

Comte

Vincent

Bouchard

et al. 1997

Journal of Biological Chemistry

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We present a strategy for simultaneous assessment of the relative contributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylation, and fatty acid oxidation to citrate formation in the perfused rat heart. This requires perfusing with a mix of 13 C-substrates and determining the 13 C labeling pattern of a single metabolite, citrate, by gas chromatography-mass spectrometry. The mass isotopomer distributions of the oxaloacetate and acetyl moieties of citrate allow calculation of the flux ratios: (pyruvate carboxylation)/(pyruvate decarboxylation), (pyruvate carboxylation)/(citrate synthesis), (pyruvate decarboxylation)/(citrate synthesis) (pyruvate carboxylation)/(fatty acid oxidation), and (pyruvate decarboxylation)/(fatty acid oxidation). Calculations, based on precursor-product relationship, are independent of pool size. The utility of our method was demonstrated for hearts perfused under normoxia with [U-13 C 3 ](lactate ؉ pyruvate) and [1-13 C]octanoate under steady-state conditions. Under these conditions, effluent and tissue citrate were similarly enriched in all 13 C mass isotopomers. The use of effluent citrate instead of tissue citrate allows probing substrate fluxes through the various reactions non-invasively in the intact heart. The methodology should also be applicable to hearts perfused with other 13 C-substrates, such as 1-13 C-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.Tracing of pyruvate metabolism and of other reactions feeding into (anaplerosis) or out (cataplerosis) of the citric acid cycle (CAC) 1 with radioactive or stable isotopes is complicated by label recycling and exchange reactions between CAC intermediates and other metabolites such as aspartate and glutamate. Mathematical models of increasing complexity were developed for the study of the CAC in various organs or tissues (1-12), including the heart (13-17). Solving for flux parameters in equations derived from these models requires measuring the incorporation of 14 C-or 13 C-labeled substrate(s) into various CAC metabolites (8, 9) or the distribution of label between carbons of given molecules such as glutamate (1,(13)(14)(15)(17)(18)(19) or citrate (4, 6). The use of 13 C-enriched labeled substrate(s) and measurements of 13 C labeling of CAC intermediates or related metabolites by nuclear magnetic resonance (NMR) or gas chromatography-mass spectrometry (GCMS) offers several advantages over classical 14 C methods. Also, these two techniques are complementary. One advantage of GCMS over NMR is its sensitivity. Thus, the 13 C labeling of the actual CAC intermediates can be determined.Investigations on the cardioprotective effects of pyruvate have emphasized the concerted regulation of pyruvate decarboxylation and fatty acid oxidation (14 -15, 17, 20 -23). For this purpose, elegant 13 C NMR techniques were developed to quantitate from the 13 C labeling of carbons of glutamate, the relative contributions of pyruvate, fatty acid, and ketone ...

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Section: Resultsmentioning

confidence: 99%

Section: Labeling Of the Acetyl And Oaa Moieties Of Citrate-thementioning

confidence: 99%

Section: Ma) the Derivatization Agent N-methyl-n-(t-butyldimethylsilmentioning

confidence: 99%

Section: Ma) the Derivatization Agent N-methyl-n-(t-butyldimethylsilmentioning

confidence: 99%

Section: Ma) the Derivatization Agent N-methyl-n-(t-butyldimethylsilmentioning

confidence: 99%

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Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the 13C Labeling of Citrate Released by Perfused Rat Hearts

Comte

Vincent

Bouchard

et al. 1997

Journal of Biological Chemistry

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Mass spectrometry for metabolic flux analysis

Wittmann

Heinzle

1999

Biotechnol. Bioeng.

164

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Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by ¹ H-detected ¹⁵ N nuclear magnetic resonance

et al. 2015

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NMR spectra of mixtures of metabolites extracted from cells or tissues are extremely complex, reflecting the large number of compounds that are present over a wide range of concentrations. Although multidimensional NMR can greatly improve resolution as well as improve reliability of compound assignments, lower abundance metabolites often remain hidden. We have developed a carbonyl selective aminooxy probe that specifically reacts with free keto and aldehyde functions, but not carboxylates. By incorporating 15N in the aminooxy functional group, 15N-edited NMR was used to select exclusively those metabolites that contain a free carbonyl function while all other metabolites are rejected. Here we demonstrate that the chemical shifts of the aminooxy adducts of ketones and aldehydes are very different, which can be used to discriminate between aldoses and ketoses for example. Utilizing the 2 or 3 bond 15N-1H couplings, the 15N-edited NMR analysis was optimized first with authentic standards and then applied to an extract of the lung adenocarcinoma cell line A549. More than 30 carbonyl containing compounds at NMR detectable levels, 6 of which we have assigned by reference to our database. As the aminooxy probe contains a permanently charged quaternary ammonium group, the adducts are also optimized for detection by mass spectrometry. Thus, this sample preparation technique provides a better link between the two structural determination tools, thereby paving the way to faster and more reliable identification of both known and unknown metabolites directly in crude biological extracts.

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Assay of Blood and Tissue Oxaloacetate and α-Ketoglutarate by Isotope Dilution Gas Chromatography-Mass Spectrometry

Cited by 20 publications

References 0 publications

Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the 13C Labeling of Citrate Released by Perfused Rat Hearts

Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the 13C Labeling of Citrate Released by Perfused Rat Hearts

Mass spectrometry for metabolic flux analysis

Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by ¹ H-detected ¹⁵ N nuclear magnetic resonance

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Assay of Blood and Tissue Oxaloacetate and α-Ketoglutarate by Isotope Dilution Gas Chromatography-Mass Spectrometry

Cited by 20 publications

References 0 publications

Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the 13C Labeling of Citrate Released by Perfused Rat Hearts

Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the 13C Labeling of Citrate Released by Perfused Rat Hearts

Mass spectrometry for metabolic flux analysis

Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by 1 H-detected 15 N nuclear magnetic resonance

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Product

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Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by ¹ H-detected ¹⁵ N nuclear magnetic resonance