Cooper A J, Nieves E, Coleman A E, Filc-DeRicco S, Gelbard A S
J Biol Chem. 1987 Jan 25;262(3):1073-80.
The short-term metabolic fate of [13N]ammonia in the livers of adult male, anesthetized rats was determined. Following a bolus injection of tracer quantities of [13N]ammonia into the portal vein, the single pass extraction was approximately 93%, in good agreement with the portal-hepatic vein difference of approximately 90%. High performance liquid chromatographic analysis of deproteinized liver samples indicated that labeled nitrogen is exchanged rapidly among components of: mitochondrial aspartate aminotransferase and glutamate dehydrogenase reactions and cytoplasmic aspartate aminotransferase and alanine aminotransferase reactions (t1/2 for the exchange of label toward equilibrium is on the order of seconds). Comparison of specific activities of glutamate and ammonia suggests that at 5 s most labeled glutamate was mitochondrial, whereas at 60 s approximately 93% was cytosolic; this change is presumably brought about by the combined action of the mitochondrial and cytosolic aspartate aminotransferases and the aspartate carrier of the malate-aspartate shuttle. Specific activity measurements of glutamate, alanine, and aspartate are in accord with the proposal by Williamson et al. (Williamson, D.H., Lopes-Vieira, O., and Walker, B. (1967) Biochem. J. 104, 497-502) that the components of the aspartate aminotransferase reaction are in thermodynamic equilibrium, whereas the components of the alanine aminotransferase reaction are in equilibrium but compartmented in the rat liver. Despite considerable label in citrulline at early time points, no radioactivity (less than or equal to 0.25% of the total) was detected in carbamyl phosphate, suggesting very efficient conversion to citrulline with little free carbamyl phosphate accumulating in the mitochondria. Our data also show that some portal vein-derived ammonia is metabolized to glutamine in the rat liver, but the amount is small (approximately 7% of that metabolized to urea) in part because liver glutamine synthetase is located in a small population of perivenous cells "downstream" from the urea cycle-containing periportal cells. Finally, no tracer evidence could be found for the participation of the purine nucleotide cycle in ammonia production from aspartate. The present work continues to emphasize the usefulness of [13N]ammonia for short-term metabolic studies under truly tracer conditions, particularly when turnover times are on the order of seconds.
测定了成年雄性麻醉大鼠肝脏中[¹³N]氨的短期代谢命运。向门静脉一次性注射示踪量的[¹³N]氨后，单次通过提取率约为93%，与门静脉 - 肝静脉差值约90%高度一致。对脱蛋白肝脏样品的高效液相色谱分析表明，标记的氮在以下成分之间快速交换：线粒体天冬氨酸转氨酶和谷氨酸脱氢酶反应以及细胞质天冬氨酸转氨酶和丙氨酸转氨酶反应（标记向平衡交换的半衰期约为秒级）。谷氨酸和氨比活性的比较表明，在5秒时，大多数标记的谷氨酸在线粒体中，而在60秒时，约93%在细胞质中；这种变化可能是由线粒体和细胞质天冬氨酸转氨酶以及苹果酸 - 天冬氨酸穿梭的天冬氨酸载体的共同作用引起的。谷氨酸、丙氨酸和天冬氨酸的比活性测量结果与Williamson等人（Williamson, D.H., Lopes-Vieira, O., and Walker, B. (1967) Biochem. J. 104, 497 - 502）的提议一致，即天冬氨酸转氨酶反应的成分处于热力学平衡，而丙氨酸转氨酶反应的成分处于平衡但在大鼠肝脏中是分隔的。尽管在早期时间点瓜氨酸中有相当多的标记，但在氨甲酰磷酸中未检测到放射性（小于或等于总量的0.25%），这表明转化为瓜氨酸的效率非常高，线粒体中几乎没有游离氨甲酰磷酸积累。我们的数据还表明，一些来自门静脉的氨在大鼠肝脏中代谢为谷氨酰胺，但量很小（约为代谢为尿素量的7%），部分原因是肝脏谷氨酰胺合成酶位于含尿素循环的门静脉周围细胞“下游”的一小群肝静脉周围细胞中。最后，没有示踪证据表明嘌呤核苷酸循环参与天冬氨酸产生氨的过程。目前的工作继续强调[¹³N]氨在真正示踪条件下进行短期代谢研究的有用性，特别是当周转时间约为秒级时。
