The metabolism of (5-15N)glutamine and (2-15N)
glutamine has been studied by isolated hepatocytes obtained from either control, chronically acidotic, or alkalotic rats. The main goal was to elucidate the mechanism(s) by which altered
acid-base state affects hepatic ureagenesis from
glutamine. Isolated hepatocytes were incubated in Krebs
buffer (pH 7.4) supplemented with 0.1 mM
ornithine plus either 1 mM (5-15N)glutamine or (2-15N)glutamine. To elucidate the role of
glutamine cycling in net
ammonia metabolism, a separate series of experiments were performed with 1 mM unlabeled
glutamine plus 1 mM (15N)H4Cl. Net
glutamine utilization was significantly lower in hepatocytes obtained from chronically acidotic rats compared with control or alkalotic rats. The sum of the rates of 15NH3 and (15N)
urea production from (5-15N)glutamine was decreased in
acidosis compared with
alkalosis. After incubations of 50 min, approximately 75, 65, or 90% of the N in
carbamoyl-phosphate was derived from the 5-N of
glutamine in control,
acidosis, or
alkalosis respectively. In experiments with (2-15N)glutamine, the production of singly and doubly labeled (15N)
urea as well as (15N)
aspartate and (15N)H3 was significantly smaller in
acidosis compared with
alkalosis. Furthermore, a correlation was observed between production rates of (15N)
aspartate and (15N)
urea, suggesting that alterations in
urea production may depend on
aspartate formed from
glutamine. However, the production of (15N)
alanine was higher in
acidosis compared with
alkalosis with apparent correlation between the production of (15N)
alanine and
2-oxoglutaramate, a product of the
glutamine aminotransferase pathway. In addition, the rate of
glutamine recycling was significantly higher in
acidosis compared with control or
alkalosis, indicating that both flux through
glutamine aminotransferase and flux through
glutamine synthetase were elevated in
acidosis compared with
alkalosis. These data suggest that decreased formation of
aspartate from
glutamine may limit ureagenesis in chronic
metabolic acidosis. The formation of
aspartate may depend on the availability of
oxaloacetate rather than diminished flux through
transaminase reaction. The enhancement of
alanine production and
glutamine synthesis may provide an alternate route of N disposal in cases of diminished
urea formation.