Micro-determination of isocitric and cis-aconitic acids in biological material.

oxaloacetate +pyruvate ci 8-acontate = ocitrat. In order to be able to examine the new hypothesis we have worked out methods for the micro-determination of ci8-aoonitic and i8ocitric acids, a preliminary account ofwhich has been published in the Proceedings of the Biochemical Society (Krebs & Eggleston, 1943 b). So far no specific micro-methods for the estimation of the two. acids have been described. The polarimetric method (Martius, 1938) for the determination of i8ocitric acid, based on the high rotation of the molybdate complex of the acid, is unspecific and is thereforenot generally applicable. Moreover, this method requires comparatively large quantities-about 25-50 mg.-if the determination is to be reasonably accurate. The determination of isocltric acid by isolation of its dimethylester lactone requires even larger quantities. The microdetermination of cia-aconitic acid (Johnson, 1939), depending on the manometric measurement of the hydrogen required for quantitative hydrogenation, is likewise unspecific and applicable only if other unsaturated compounds are absent. The following principle permits the determination of a few milligrams of (-)isocitric and ci8-aconitic acids in biological material provided that citric acid is not present in very much larger quantities. Three portions ofneutral tissue extract or tissue suspension are measured. The first is incubated with 'aconitase' whereby i8ocitric and cs8-aconitic acids are converted into citric acid, the latter being determined according to Pucher, Vickery& Leavenworth (1934). In the second portion citric acid is determined directly. From the third portion cis-aconitic acid is removed by conversion into the trans-form; it is then treated like the first sample. The difference in the citric acid content between the first and second samples indicates the sum of (-)i8ocitric and c8aconitic acids in the sample; the difference between the second and third the amount of (-)i8ocitric acid; the difference between the first and third the amount of cis-aconitic acid. Thus the determination of the two acids requires three determinations of citric acid. If only one of the acids is present (apart from citric acid) two citric acid determinations are required.