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Metabolomics. 2013;9(5):1019-1030. Epub 2013 Apr 17.

The complete targeted profile of the organic acid intermediates of the citric acid cycle using a single stable isotope dilution analysis, sodium borodeuteride reduction and selected ion monitoring GC/MS.

Author information

1
Metabolomics Core Facility, Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC Canada.
2
Department of Biochemistry and Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC Canada.
3
Department of Biochemistry and Rosalind and Morris Goodman Cancer Research Centre, McGill University, 3655 Promenade Sir William Osler, Office: Room 713A, Montreal, QC H3A 1A3 Canada.
4
Metabolomics Core Facility, Rosalind and Morris Goodman Cancer Research Centre, McGill University, 1160 Pine Avenue West, Room 418, Montreal, QC H3A 1A3 Canada.

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.

KEYWORDS:

Citric acid cycle; GC/MS; Metabolite profiling; Mitochondrial toxins

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