EVALUATION OF THE IMPORTANCE OF TRANSAMINATION VERSUS DEAMINATION IN ASTROCYTIC METABOLISM OF [U-C-13]GLUTAMATE

Glutamate metabolism was studied in primary cultures of cerebral corti cal astrocytes to determine the significance of transamination for the oxidative metabolism of glutamate. Cultures were incubated with [U-C- 13]glutamate (0.5 mM) in the presence and absence of the transaminase inhibitor aminooxyacetic acid (AOAA) and in some cases with methionine sulfoximine, an inhibitor of glutamine synthetase. Perchloric acid ex tracts of the cells as well as redissolved lyophilized incubation medi a were subjected to nuclear magnetic resonance spectroscopy to identif y C-13-labeled metabolites. Additionally, biochemical analyses were pe rformed to quantify amino acids, lactate, citrate, and ammonia. Glutam ine released into the medium and intracellular glutamate were labeled uniformly to a large extent, but the C-3 position showed not only the expected apparent triplet but also a doublet due to C-12 incorporation into the C-4 and C-5 positions. Incorporation of C-12 into the C-4 an d C-5 positions of glutamate and glutamine as well as labeling of lact ate, citrate, malate, and aspartate could only arise via metabolism of [U-C-13]glutamate through the tricarboxylic acid (TCA) cycle. Entry o f the carbon skeleton of glutamate into the TCA cycle must proceed via a-oxoglutarate. This conversion can occur as a transamination or an o xidative deamination. After blocking transamination with AOAA, metabol ism of glutamate through the TCA cycle was still taking place since la ctate labeling was only slightly reduced. Glutamate and glutamine synt hesis from 2-oxoglutarate could, however, not be detected under this c ondition. It therefore appears that while glutamate dehydrogenase is i mportant for glutamate degradation, glutamate biosynthesis occurs main ly as a transamination. (C) 1996 Wiley-Liss, Inc.