Microdetermination of alpha-keto acids with special reference to malic, lactic, and glutamic dehydrogenases in brain.

Previous studies have indicated marked differences in the quantitative distribution of a number of enzymes, as well as other substances, in various histologically distinct areas of the nervous system (l-4). An earlier method (2) for MDH, LDH, and GDH’ in brain required 0.3, 1.5, and 10 y of brain, dry weight, respectively, for the determination of these enzymatic activities. Some of the histological heterogeneity of the central nervous system may be circumvented through the use of bits of tissue of this small size, permitting histological-biochemical correlations to be made. It would be advantageous to have methods requiring even less tissue (0.1 -r) to allow sampling from the thin layers of the retina, for example, or from parts of the brain where the many histological components are intimately mixed together. Since MDH, LDH, and GDH convert the respective substrates to cr-keto acids, a sensitive method for the determination of cr-keto acids was desired. lt was found that cr-keto acids when allowed to react with QH give products having very high absorption maxima in the ultraviolet (305 mp). The reagent has been shown to be applicable to the measurement of as little as 4 X 10-l” mole of thirteen or-keto acids. It has also been shown that cr-keto acids may be determined in the presence of @-keto acids, ketones, and aldehydes, because the latter compounds give unstable products with absorptions at 305 rnp which virtually disappear with time. The reagent is even more sensitive when used for the measurement of the activities of the three enzymes because the DPNH produced during the enzymatic reaction has in acid an appreciable absorption at 305 rnp which is greater in the presence of QH. MDH and LDH have been measured in 0.1 y or