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A Method for Estimation of Krebs Cycle and Related Intermediates in Animal Tissues by Gas Chromatography

Journal/Book: Analytical Biochemistry 26 68-84 (1968) Received February 26 1968. 1968;

Abstract: D. BARNETT 1 R. D. COHEN 1 C. N. TASSOPOULOS J. R. TURTLE 2 A. DIMITRIADOU AND T. RUSSELL FRASER Department of Medicine Royal Post- graduate Medical School London W. 12 England Received February 26 1968 1 Present address: Medical Unit The London Hospital London E.1 England 2 Present address: Department of Medicine The University of Sydney N.S.W. Australia Gas chromatography has been successfully applied to the separation of Krebs cycle and related carboxylic acids in standard solution (1-6) plant extracts (7-9) and tissues (10 11). Accurate measurement by this method of the low concentrations of intermediates present in tissues is hindered by the losses that accompany the purification and concentration of extracts necessary to achieve satisfactory chromatograms. Esters of the intermediates may be affected to different degrees as noted by Kuksis and Prioreschi (11) who found that when extracts contained less than 5 gm liver the concentration procedures necessary resulted in significant losses of the more volatile esters. Although the use of derivatives less volatile than methyl esters might be advantageous it is nevertheless necessary to account for the losses which can occur during purification of the tissue extract. We describe in this paper a method for extraction purification and measurement by gas chromatography of the intermediates present in liver heart muscle kidney adipose tissue and blood. The problem of variable recovery has beep overcome by the use of a radioactive standard for each acid measured and-sufficient sensitivity has thus been achieved to measure the concentration of intermediates in as little as 0.25 gm tissue. ... schö

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