Analysis of the Citric Acid Cycle Intermediates Using Gas Chromatography-Mass Spectrometry

Department of Nutrition, Mouse Metabolic Phenotyping Center, Case Western Reserve University, Cleveland, OH, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2011; 708:147-57. DOI: 10.1007/978-1-61737-985-7_8
Source: PubMed


Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer useful information to understanding interrelationships between the CAC and metabolic pathways under various physiological and pathological conditions. This chapter presents a relatively simple method that is sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using gas chromatography-mass spectrometry.

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Available from: Rajan Kombu
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    • "In recent years, several techniques have emerged that could measure CAC intermediates with greater precision, albeit still with limited coverage. In particular these include LC–MS/MS techniques developed by Luo et al. 2007 (Luo et al. 2007) and Koubaa et al. 2013 (Koubaa et al. 2013), GC/MS methodologies (Dunn and Winder 2011; Kombu et al. 2011), HPLC-fluorescence methodology (Kubota et al. 2005), capillary electrophoresis coupled to a mass spectrometer (CE/MS) (Soga et al. 2003; Wakayama et al. 2010), as well as 1 H NMR (Xu et al. 2011). 1 H NMR permits a greater coverage of the CAC intermediates as well as other metabolites (Xu et al. 2011) and does not require isotopically labeled internal standards. However, it is limited by the low micromolar sensitivity and potential spectral overlap. "
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    ABSTRACT: The quantitative profiling of the organic acid intermediates of the citric acid cycle (CAC) presents a challenge due to the lack of commercially available internal standards for all of the organic acid intermediates. We developed an analytical method that enables the quantitation of all the organic acids in the CAC in a single stable isotope dilution GC/MS analysis with deuterium-labeled analogs used as internal standards. The unstable α-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable α-deutero-α-hydroxy acids and these, along with their unlabeled analogs and other CAC organic acid intermediates, are converted to their tert-butyldimethylsilyl derivatives. Selected ion monitoring is employed with electron ionization. We validated this method by treating an untransformed mouse mammary epithelial cell line with well-known mitochondrial toxins affecting the electron transport chain and ATP synthase, which resulted in profound perturbations of the concentration of CAC intermediates. Electronic supplementary material The online version of this article (doi:10.1007/s11306-013-0521-1) contains supplementary material, which is available to authorized users.
    Full-text · Article · Oct 2013 · Metabolomics
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    • "ions were decanted and reserved for the measurements of concentrations and [ 13 C ] label of acetyl - CoA enrichment ( Zhang et al . 2013a ) . The tissue pellets were further extracted using a mixture of acetonitrile and 2 - propanol ( 3 : 1 ) , centrifuged , and then analyzed for CAC , amino acids , and related intermediates ( Yang et al . 2008 ; Kombu et al . 2011 ) . Extracts were then dried by nitrogen gas for 1 – 2 h and chemically derivatized using MTBSTFA + 1% TBDMCS reagent ( N - methyl - N - ( tert - butyldimethylsilyl )"
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    ABSTRACT: The coupling of upstream oxidative processes (glycolysis, beta-oxidation, CAC turnover) to mitochondrial oxidative phosphorylation (OXPHOS) under the driving conditions of energy demand by the cell results in the liberation of free energy as ATP. Perturbations in glycolytic CAC or OXPHOS can result in pathology or cell death. To better understand whole body energy expenditure during chronic ketosis, we used a diet-induced rat model of ketosis to determine if high-fat-carbohydrate-restricted "ketogenic" diet results in changes in total energy expenditure (TEE). Consistent with previous reports of increased energy expenditure in mice, we hypothesized that rats fed ketogenic diet for 3 weeks would result in increased resting energy expenditure due to alterations in metabolism associated with a "switch" in energy substrate from glucose to ketone bodies. The rationale is ketone bodies are a more efficient fuel than glucose. Indirect calorimetric analysis revealed a moderate increase in VO2 and decreased VCO2 and heat with ketosis. These results suggest ketosis induces a moderate uncoupling state and less oxidative efficiency compared to glucose oxidation.
    Full-text · Article · Jul 2013 · Advances in Experimental Medicine and Biology
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    • "The mass spectra of the TMS-derivatized compounds formed in the EI source led to fragment ions predicted from their chemical structure. The monitored ions were in accordance with research in which the analytes were determined using the same derivatizing reagent [16] [18]. The quantitation ions for all target compounds, including the internal standard, are shown in Table 2, which also includes the chromatographic retention times. "
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    ABSTRACT: Metabolites involved in the tricarboxylic acid (TCA) cycle have previously been proposed as cardiovascular biomarkers. This cycle plays a key role in cell metabolism and the levels of the involved metabolites can also be affected by other physiological factors. The influence of three cardiovascular risk factors such as obesity, hypercholesterolemia, and smoking habit on serum levels of TCA-cycle metabolites has been studied in patients diagnosed with significant coronary lesion. For this purpose, a method based on GC-MS for determination of the target metabolites (viz. citric/isocitric, pyruvic, aconitic, oxaloacetic, malic, fumaric and succinic acids) in serum has been developed. The high accuracy and throughput analysis featuring the method have allowed application to a cohort of 223 patients, 172 of them with significant coronary lesion. Multifactor analysis of variance has revealed interactions between the occurrence or not of a coronary lesion and the risk factors considered in this study. These interactions were crucial to explain the levels of target TCA metabolites. Statistical evaluation by ROC curves allowed discrimination of the capability of significant metabolites with the occurrence of coronary lesions.
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