Fluorometric determination of alpha-ketosuccinamic acid in rat tissues

A method for the fluorometric determination of alpha-ketosuccinamic acid, the alpha-keto acid analog of asparagine, is described. The procedure involves the hydrolysis of alpha-ketosuccinamate to oxaloacetate by omega-amidase followed by NADH-dependent reduction of oxaloacetate to malate by malate dehydrogenase. A correction for endogenous oxaloacetate is made by using control samples lacking omega-amidase. Of the rat tissues investigated, liver contained the highest concentration, followed by kidney (53 +/- 6 (n = 11) and 18 +/- 3 (n = 3) mumol/kg wet wt, respectively). alpha-Ketosuccinamate was not detected in brain (less than 8 mumol/kg wet wt). Some chemical properties of alpha-ketosuccinamate were investigated. Concentrated solutions of sodium alpha-ketosuccinamate frozen for extended periods and the solid sodium salt of alpha-ketosuccinamate dimer heated to 130 degrees C are converted to at least 10 products by processes involving dimerization, dehydration, and decarboxylation. Isobutane chemical ionization mass spectral analysis (170-230 degrees C) of the free acid monomer yielded similar products. Many of the breakdown products were identified as di- and monoheterocyclic compounds, some of which are known to be of biological importance.