Stephen P. Raps’s scientific contributions

The levels of the antioxidants, glutathione and ascorbate were measured in primary cultures of murine astrocytes and neurons. The concentration of glutathione (reduced, GSH + oxidized, GSSG) was high in cultured, differentiated (i.e. treated with dBcAMP) and undifferentiated (i.e. untreated) astrocytes:≈25 (n = 2)and16.0 ± 5.0 (n = 7)nmol/mg protein, respectively. In contrast, glutathione levels in neurons were low:≦1.0 (n = 7)nmol/mg protein. Ascorbate could not be detected (<2nmol/mg protein) in either cell type. The apparent lack of defense mechanisms against oxidative stress may in part account for the ‘fragility’ of neurons in culture. The physiological implications of glutathione compartmentation in brain are discussed.

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.

A method for the fluorometric determination of α-ketosuccinamic acid, the α-keto acid analog of asparagine, is described. The procedure involves the hydrolysis of α-ketosuccinamate to oxaloacetate by ω-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 ω-amidase. Of the rat tissues investigated, liver contained the highest concentration, followed by kidney (53 ± 6 (n = 11) and 18 ± 3 (n = 3) μmol/kg wet wt, respectively). α-Ketosuccinamate was not detected in brain (<8 μmol/kg wet wt). Some chemical properties of α-ketosuccinamate were investigated. Concentrated solutions of sodium α-ketosuccinamate frozen for extended periods and the solid sodium salt of α-ketosuccinamate dimer heated to 130°C are converted to at least 10 products by processes involving dimerization, dehydration, and decarboxylation. Isobutane chemical ionization mass spectral analysis (170–230°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.