19 N as a Tracer for Studying Ammonia
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Uptake and Metabolism in the Brain ARTHUR J. L. COOPER and THOMAS E. DUFFY Departments of Neurology and Biochemistry, Cornell University Medical College, New York,NY10021 JOSEPH M. McDONALD and A L A N S. GELBARD Biophysics Laboratory, Memorial Sloan-Kettering Cancer Center, New York,NY10021 The uptake and metabolism of [ N]ammonia in the rat brain have been investigated. The results show that blood– borne ammonia (and cerebrospinal fluid ammonia) enters the brain largely by diffusion of the free base. On entering the brain much of the [ N]ammonia is rapidly metabolized (t = 1-3 s); 99% is incorporated into glutamine and only a small fraction appears in glutamate (1%). Specific activity measurements of metabolites, following infusion of [ N[am monia, confirm the hypothesis that the brain contains at least two separate compartments of glutamate metabolism. A computer model that simulates the flow of N into various compartments and metabolites following either a bolus injec tion or continuous infusion of [ N]ammonia has been devised, and the results of this simulation agree with experi mental observations. 13
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In recent years there has been a great deal of interest i n understanding the mechanisms involved i n ammonia metabolism in brain. The reasons for this interest are twofold: ammonia is thought to be a major toxin contributing to the symptoms of encephalopathy associated with liver disease ( 1 , 2 ) and of Reye's disease ( 3 ) ; and earlier experiments with N-labeled ammonia suggested the existence of at least two distinct metabolic pools in brain (4). Ammonia as a Neurotoxin. Several compelling reasons underlie the belief that ammonia is a major toxin contributing to the symptoms of hepatic encephalopathy (1,2,5,6,7). Ammonia is toxic to the brain in 15
0065-2393/81/0197-0369$05.00/0 © 1981 American Chemical Society
Root and Krohn; Short-Lived Radionuclides in Chemistry and Biology Advances in Chemistry; American Chemical Society: Washington, DC, 1982.
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hereditary hyperammonemia (congenital defects of the urea cycle) (8). Electroencephalographic changes and neuropathological abnormalities that resemble those of human hepatic encephalopathy can be induced i n alert monkeys by chronic infusions of ammonium salts (9). Ammonia concentrations in blood and cerebrospinal fluid (CSF) show a high correlation with hepatic encephalopathy in patients (10). Rats made chronically hyperammonemic, by the surgical construction of a portacaval anastamosis, are extremely sensitive to a small ammonia challenge ( I I ) . The currently most effective treatments for patients with hepatic coma are those directed toward reducing the plasma ammonia concentration (12). The Two-Pool Hypothesis Based on Studies of Brain Ammonia Metabolism. Schoenheimer and colleagues, in a series of classical experi ments, administered [ N]ammonia or N-labeled amino acids to rats and were the first to demonstrate that nitrogen derived from ammonia was incorporated into urea, the amide group of glutamine, other amino acids, and creatine (cf. 13). It had originally been assumed that deamination of L-amino acids in vivo was accomplished by the consecutive action of a specific a-ketoglutarate-dependent transaminase and glutamate dehydrogenase (14) and that incorporation of N , derived from [ N]ammonia, into amino acids occurred by the reversal of this reaction. However, Duda and Handler showed that, in the brain and other organs, the major fate of N , whether administered intravenously as [ N]ammonia or derived from endogenously produced [ N]ammonia (as in the breakdown of D-[ N]leucine), was incorporation into the amide group of glutamine (15). Later Waelsch and coworkers carried out a number of investigations in which they determined the metabolic fate of C-labeled glutamate in the brain (16,17). Comparison of the specific activities of glutamate vs. glutamine led to the conclusion that glutamate metabolism was compartmentalized in the brain. In later experiments, N-labeled ammonia was infused into the right common carotid artery of cats (4) and the incorporation of the label into the a-amino group of glutamine, the amide group of glutamine, and the a-amino group of glutamate was determined for the brain and liver. Analysis of the results indicated that blood-borne ammonia was converted to glutamine in the brain in a rapidly turning over compartment of glutamate; this com partment was metabolically distinct from a more slowly turning over, larger glutamate compartment (4). In contrast to the brain, the liver was shown to contain only one pool of glutamate (4). These early experi ments were the starting point for a large number of metabolic studies designed to characterize further the nature of the two cerebral compart ments. Thus, C-labeling patterns obtained after administration of C-acetate indicated the existence of separate T C A cycles in the small and large pools (18,19,20,21). 15
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Root and Krohn; Short-Lived Radionuclides in Chemistry and Biology Advances in Chemistry; American Chemical Society: Washington, DC, 1982.
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Ammonia Uptake and Metabolism in Brain
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A criticism of the early studies with N-labeled ammonia and ammonia precursors is that they were often carried out under nonphysiological conditions. We reinvestigated the metabolic fate of blood-borne ammonia at physiological concentrations of ammonia in awake rats, using N as a tracer (22). The findings confirm the existence of the two pools for brain glutamate metabolism and emphasize the importance of gluta mine synthetase in the brain for ammonia detoxification. These tracer studies also showed that the metabolic compartmentation was lost after inhibition of brain glutamine synthetase by methionine sulfoximine (22). Use of N as a Tracer for Ammonia in the Brain. With the intro duction of newer, more sensitive, and more accurate detection techniques, minute quantities of N can be analyzed. However, limitations to the use of N as a tracer would seem to be its natural abundance (0.37%) and the small variation of N-content in nature. A small amount of metabolite of high N-content, entering a large unlabeled pool might raise the N-content of endogenous metabolite only slightly above back ground. Such a small increase of N would be difficult to quantitate accurately. In contrast, because there is no natural N , relatively small amounts of high-specific-activity [ N] ammonia can be administered to experimental animals without disruption of the physiological steady state. Furthermore, because N gives rise to gamma radiation via positron annihilation, it is easier to quantitate than N and has the added advan tage that it can be detected in vivo by external imaging devices. N has two major drawbacks as a biological tracer: its use is limited to those institutions that possess a cyclotron; and since it has a short half-life (10 min), it can only be used in experiments of relatively short duration. Although few studies have been carried out using N as a tracer for uptake and metabolism of ammonia by the brain (e.g., 22,23, 24,25), initial findings indicate that N , despite its short half-life, is eminently suitable for this purpose. 15
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Production of
[^N'lAmmonia
N-Labeled ammonia has been generated by the bombardment of methane gas (26) or metal carbides (27) with high-energy deuterons. The method that is now used at the Memorial Sloan-Kettering Cancer Center is to bombard water with protons. The chemical form of the generated N is mostly nitrate, with smaller amounts of nitrite and very small amounts of ammonia (28). However, the [ N]nitrate and [ N]nitrite are readily converted to [ N]ammonia with a Devarda's alloy-NaOH mixture (29). The [ N]ammonia produced by the reduction reaction is flushed into 3 m L of physiological saline or buffer. Theoretically, the label should be carrier-free. However, it is impossible to exclude ammonia completely. Some unlabeled ammonia apparently arises from the De13
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Root and Krohn; Short-Lived Radionuclides in Chemistry and Biology Advances in Chemistry; American Chemical Society: Washington, DC, 1982.
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varda's alloy-sodium hydroxide mixture, so that dilution of the specific activity always occurs (22). Typically, after flushing into buffer the concentration of ammonia is in the range 80-200/AM and the total N radioactivity is in the range 20-100 mCi (22,25). 1 3
Dynamics of [^N] Ammonia Uptake and Metabolism by the Brain as Assessed by External Imaging Studies on the Whole Brain. The dynamics of uptake of label by the brain in vivo following a bolus injection of [ N]ammonia have been investigated in animals and in human beings (23,24,25). Phelps et al. found that the single-pass extraction of [ N]ammonia by cerebral capil laries (internal carotid artery injection) was inversely related to blood flow and was limited by the permeability of the blood-brain barrier to ammonia (23,24). In a comparable experiment in which [ N]ammonia was administered intravenously, Lockwood et al. determined that the single-pass extraction of ammonia in the human brain was 47% ( 25). Moreover, Lockwood et al. showed that: In 5 normal subjects and in 17 patients with liver disease, the rate of [ N]ammonia clearance from the vascular compartment and the brain ammonia utilization rate were linear functions of the arterial ammonia concentration, at least over the range of 50-250/xM; the ammonia utilization reaction(s) appeared to take place in a compartment that included
