A method for estimation of Krebs cycle and related intermediates in animal tissues by gas chromatography

Abstract

1. 1. A method is described for the separation and estimation by gasliquid chromatography of pyruvate, lactate, β-hydroxybutyrate, and some Krebs cycle intermediates in rat tissues. The use of a 14C internal standard for each acid permits corrections for losses. After a simple extraction and purification procedure the intermediates are chromatographed as their methyl esters, the effluent peaks are collected, and their 14C content is measured to enable correction for losses of the individual acids during extraction and chromatography. The mean coefficient of variation of replicate estimations of the individual acids in pooled tissue sample ranged from 3 to 7.1%. Combined gas chromatography-mass spectrometry has been used to confirm the identity of the peaks in tissue chromatograms. 2. 2. Observations on the effect of sampling technique on the concentrations of the intermediates suggest that the combination of ether anesthesia with in situ freezing of tissue is the most satisfactory method when studying these intermediates in rat liver. 3. 3. The method described is also suitable for the simultaneous determination of the concentration and specific activity of tissue intermediates after injection of labeled precursors.