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.

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 ?-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable ?-deutero-?-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.

SUBMITTER: Mamer O 

PROVIDER: S-EPMC3855487 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature