Analysis and physiological implications of renal 2-oxoglutaramate metabolism

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Abstract
The relative significance of the flux through the glutamine aminotransferase (glutaminase II) pathway to renal ammoniagenesis is poorly understood. A basic and unresolved question is whether 2-oxoglutaramate (2-OGM), a product of the glutaminase II reaction, is deamidated to yield 2-oxoglutarate and NH3, or whether 2-OGM accumulates as an unreactive lactam, depending on the environmental pH. In the current studies we utilized C-13 n.m.r. as well as N-15 n.m.r. to demonstrate that 2-OGM occurs as a lactam, i.e. 5-hydroxypyroglutamate, regardless of the environmental pH. Our additional aims were to determine whether human kidney cells (HK cells) in culture can produce 2-OGM and to ascertain a pH-dependent relationship between NH3 and 2-OGM production from glutamine. We therefore developed an isotope dilution assay for 2-OGM utilizing 5-hydroxy[4-C-13,1-N-15]pyroglutamate as the labelled species. Incubations of HK cells in minimal essential medium supplemented with 1 mM-[2-N-15]glutamine demonstrated significantly higher production of 2-OGM at pH 6.8 and lower production at pH 7.6 compared with pH 7.4. Similarly both (NH3)-N-15 and [N-15]alanine formation were significantly higher in acute acidosis (pH 6.8) and lower in acute alkalosis (pH 7.6) compared with that at physiological pH. Addition of 1 mM-amino-oxyacetate to the incubation medium at pH 7.4 significantly diminished [N-15]alanine and 2-OGM production, but the production of (NH3)-N-15 via the glutamate dehydrogenase pathway was significantly stimulated. The current observations indicate that the glutaminase II pathway plays a minor role and that flux through glutamate dehydrogenase is the predominant site for regulation of ammoniagenesis in human kidney.