Metabolism of glutamine and glutamate by rat renal tubules. Study with 15N and gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.     

Early events in the initiation of ammonia formation in kidney

Experiments were designed to examine the early events in the initiation of glutamate deamination in kidney. Perfused kidneys from methionine sulfoximine-treated rats formed ammonia from [15N]glutamate via the purine nucleotide cycle. The turnover of the 6-amino group of adenine nucleotides to yield ammonia occurred at the rate of 0.30 mumol/g of kidney/min. This rate is 3-4 times larger than in liver and is in agreement with published rates of the purine nucleotide cycle in kidney. The addition of 0.1 mM fluorocitrate to glutamate perfusions stimulated ammonia formation 3 1/2-fold. The turnover of the 6-amino group of adenine nucleotides increased during the first 5 min after adding fluorocitrate to form ammonia predominately from tissue glutamate and aspartate. This turnover correlates with a 3 1/2-fold increase in kidney tissue IMP levels. As the ATP/ADP ratio fell the purine nucleotide cycle was inhibited and glutamate dehydrogenase was stimulated to form ammonia stoichiometric with glutamate taken up from the perfusate. Ammonia formation via glutamate dehydrogenase occurred at a rate of 1.0 mumol/g of kidney/min. Fluorocitrate completely blocked ammonia formation from aspartate in perfusions. The perfused kidney formed ammonia from aspartate via the purine nucleotide cycle at a rate of 1.0 mumol/g of kidney/min. The results indicate a discrete role for aspartate in renal metabolism. Ammonia formation via the purine nucleotide cycle can occur at significant rates and equal to the rate of ammonia formation from glutamate via glutamate dehydrogenase.     

The purine nucleotide cycle and ammoniagenesis in rat kidney tubules

The contribution of the purine nucleotide cycle to renal ammoniagenesis was examined in cortical tubule suspensions prepared from acidotic rats and incubated with [alpha-15N]glutamine, [15N]glutamate, or [15N]aspartate. Labeling of ammonia and adenine nucleotides was determined after enzymatic transformations designed to circumvent the technical problem that 15NH3 and H2O have the same nominal mass. Labeling of the adenine nucleotide was undetectable (less than 10%) even after 1 h of incubation. From the measured concentrations of adenine nucleotides and ammonia and the labeling of the ammonia, the flux through the purine nucleotide cycle was calculated to account for less than 1% of the deamination of alpha-amino groups from all three substrates. The glutamate dehydrogenase reaction is therefore the likely pathway for deamination. The rate of 15NH3 production from [alpha-15N]glutamine was two or three times greater than from added [15N]glutamate, indicating a preference for intracellularly generated glutamate. 15NH3 production from added [15N]aspartate was similar to and perhaps slightly greater than that from added [15N]glutamate.     

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.     

Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N.

We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.     

Avian mitochondrial glutamine metabolism.

Intact avian liver mitochondria were shown to synthesize glutamine from glutamate in the absence of exogenous ATP and ammonia. With L-[U-14C]glutamate as the substrate, there was an approximate 1:1 stoichiometry between glutamate deaminated (as measured by the release of 14CO2 due to alpha-keto-[14C]glutarate oxidation) and glutamate amidated. With L-[15N]glutamate as the substrate, the isolated glutamine was shown by low and high resolution mass spectrometry of its phenylisothiocyanate derivative to contain 15N in both the alpha-amino and amide groups. Thus, for each mole of glutamate taken up, approximately 0.5 mol is deaminated and the other 0.5 mol serves as a substrate for glutamine synthetase previously localized in these mitochondria (Vorhaben, J. E., and Campbell, J. W. (1972) J. Biol. Chem. 247,2763). The permeability of L-glutamine to intact avian liver mitochondria was studied by a rapid centrifugation technique. Efflux as well as influx of L-glutamine were both rapid and appeared to occur by a passive, energy-independent process. These results indicate that the mitochondrial glutamine synthetase present in uricotelic species represents the primary ammonia detoxication reaction in that ammonia released intramitochondrially during amino acid catabolism is converted to glutamine for efflux to the cytosol where it may serve as a substrate for purine (uric acid) biosynthesis.     

Nitrogen-13 flux from L-[13N]glutamate in the isolated rabbit heart: effect of substrates and transaminase inhibition

Kinetic and biochemical parameters of nitrogen-13 flux from L-[13N]glutamate in myocardium were examined. Tissue radioactivity kinetics and chemical analyses were determined after bolus injection of L-[13N]glutamate into isolated arterially perfused interventricular septa under various metabolic states, which included addition of lactate, pyruvate, aminooxyacetate (a transaminase inhibitor), or a combination of aminooxyacetate and pyruvate to the standard perfusate containing insulin and glucose. Chemical analysis of tissue and effluent at 6 min allowed determination of the composition of the slow third kinetic component of the time-activity curves. 13N-labeled aspartate, alanine and glutamate accounted for more than 80% of the tissue nitrogen-13 under the experimental conditions used. Specific activities for these amino acids were constant, but not identical to each other, from 6 through 15 min after administration of L-[13N]glutamate. Little labeled ammonia (1.9%) and glutamine (4.7%) were produced, indicating limited accessibility of exogenous glutamate to catabolic mitochondrial glutamate dehydrogenase and glutamine synthetase, under control conditions. Lactate and pyruvate additions did not affect tissue amino acid specific activities. Aminooxyacetate suppressed formation of 13N-labeled alanine and aspartate and increased production of L-[13N]glutamine and [13N]ammonia. Formation of [13N]ammonia was, however, substantially decreased when aminooxyacetate was used in the presence of exogenous pyruvate. The data support a model for glutamate compartmentation in myocardium not affected by increasing the velocity of enzymatic reactions through increased substrate (i.e., lactate or pyruvate) concentrations but which can be altered by competitive inhibition of transaminases (via aminooxyacetate) making exogenous glutamate more available to other compartments.     

A 15N-n.m.r. study of cerebral, hepatic and renal nitrogen metabolism in hyperammonaemic rats.

1. Rats were infused with 15NH4+ or L-[15N]alanine to induce hyperammonaemia, a potential cause of hepatic encephalopathy. HClO4 extracts of freeze-clamped brain, liver and kidney were analysed by 15N-n.m.r. spectroscopy in combination with biochemical assays to investigate the effects of hyperammonaemia on tissue concentrations of ammonia, glutamine, glutamate and urea. 2. 15NH4+ infusion resulted in a 36-fold increase in the concentration of blood ammonia. Cerebral glutamine concentration increased, with 15NH4+ incorporated predominantly into the gamma-nitrogen atom of glutamine. Incorporation into glutamate was very low. Cerebral ammonia concentration increased 5-10-fold. The results suggest that the capacity of glutamine synthetase for ammonia detoxification was saturated. 3. Pretreatment with the glutamine synthetase inhibitor L-methionine DL-sulphoximine resulted in 84% inhibition of [gamma-15N]glutamine synthesis, but incorporation of 15N into other metabolites was not observed. The result suggests that no major alternative pathway for ammonia detoxification, other than glutamine synthetase, exists in rat brain. 4. In the liver 15NH4+ was incorporated into urea, glutamine, glutamate and alanine. The specific activity of 15N was higher in the gamma-nitrogen atom of glutamine than in urea. A similar pattern was observed when [15N]alanine was infused. The results are discussed in terms of the near-equilibrium states of the reactions involved in glutamate and alanine formation, heterogeneous distribution in the liver lobules of the enzymes involved in ammonia removal and their different affinities for ammonia. 5. Synthesis of glutamine, glutamate and hippurate de novo was observed in kidney. Hippurate, as well as 15NH4+, was contributed by co-extracted urine. 6. The potential utility and limitations of 15N n.m.r. for studies of mammalian metabolism in vivo are discussed.     

Ammonia assimilation by Aspergillus nidulans: [15N]ammonia study

15N kinetic labelling studies were done on liquid cultures of wild-type Aspergillus nidulans. The labelling pattern of major amino acids under 'steady state' conditions suggests that glutamate and glutamine-amide are the early products of ammonia assimilation in A. nidulans. In the presence of phosphinothricin, an inhibitor or glutamine synthetase, 15N labelling of glutamate, alanine and aspartate was maintained whereas the labelling of glutamine was low. This pattern of labelling is consistent with ammonia assimilation into glutamate via the glutamate dehydrogenase pathway. In the presence of azaserine, an inhibitor of glutamate synthase, glutamate was initially more highly labelled than any other amino acid, whereas its concentration declined. Isotope also accumulated in glutamine. Observations with these two inhibitors suggest that ammonia assimilation can occur concurrently via the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways in low-ammonia-grown A. nidulans. From a simple model it was estimated that about half of the glutamate was synthesized via the glutamate dehydrogenase pathway; the other half was formed from glutamine via the glutamate synthase pathway. The transfer coefficients of nine other amino acids were also determined.     

Refixation of photorespiratory ammonia and the role of alanine in photorespiration: Studies with15N

Summary Labelling experiments with¹⁵N glutamate and¹⁵N alanine were conducted using slices from oat leaves to investigate photorespiratory nitrogen metabolism. It is concluded from the labelling kinetics of glutamine that the refixation of photorespiratory ammonia primarily occurs by glutamine synthetase in the chloroplast. The labelling kinetics of glutamine with¹⁵N glutamate indicate that the chloroplastic and cytoplasmic glutamate pools do not exchange easily in oat leaf cells. Alanine was shown to be an important amino donor for photorespiratory glycine formation. This result is discussed with regard to a possible role of alanine in photorespiration. A modification to the scheme of photorespiratory nitrogen metabolism is proposed.     

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [¹³N]ammonia have shown that about 25?% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [¹³N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: l-Aspartate?+?GTP?+?H₂O?→?Fumarate?+?GDP?+?P_i?+?NH₃) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.     

Ammonia and amino acid metabolism in human skeletal muscle during exercise.

This review focuses on the ammonia and amino acid metabolic responses of active human skeletal muscle, with a particular emphasis on steady-state exercise. Ammonia production in skeletal muscle involves the purine nucleotide cycle and the amino acids glutamate, glutamine, and alanine and probably also includes the branched chain amino acids as well as aspartate. Ammonia production is greatest during prolonged, steady state exercise that requires 60-80% VO2max and is associated with glutamine and alanine metabolism. Under these circumstances it is unresolved whether the purine nucleotide cycle (AMP deamination) is active; if so, it must be cycling with no IMP accumulation. It is proposed that under these circumstances the ammonia is produced from slow twitch fibers by the deamination of the branched chain amino acids. The ammonia response can be suppressed by increasing the carbohydrate availability and this may be mediated by altering the availability of the branched chain amino acids. The fate of the ammonia released into the circulation is unresolved, but there is indirect evidence that a considerable portion may be excreted by the lung in expired air.     

Glitazones regulate glutamine metabolism by inducing a cellular acidosis in MDCK cells

We studied the effect of the antihyperglycemic glitazones, ciglitazone, troglitazone, and rosiglitazone, on glutamine metabolism in renal tubule-derived Madin-Darby canine kidney (MDCK) cells. Troglitazone (25 microM) enhanced glucose uptake and lactate production by 108 and 92% (both P < 0.001). Glutamine utilization was not inhibited, but alanine formation decreased and ammonium formation increased (both P < 0.005). The decrease in net alanine formation occurred with a change in alanine aminotransferase (ALT) reactants, from close to equilibrium to away from equilibrium, consistent with inhibition of ALT activity. A shift of glutamine's amino nitrogen from alanine into ammonium was confirmed by using L-[2-(15)N]glutamine and measuring the [(15)N]alanine and [(15)N]ammonium production. The glitazone-induced shift from alanine to ammonium in glutamate metabolism was dose dependent, with troglitazone being twofold more potent than rosiglitazone and ciglitazone. All three glitazones induced a spontaneous cellular acidosis, reflecting impaired acid extrusion in responding to both an exogenous (NH) and an endogenous (lactic acid) load. Our findings are consistent with glitazones inducing a spontaneous cellular acidosis associated with a shift in glutamine amino nitrogen metabolism from predominantly anabolic into a catabolic pathway.     

Effect of 5-amino-4-imidazolecarboxamide riboside on renal ammoniagenesis. Study with [15N]aspartate

[15N]Aspartate and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) were used to evaluate the contribution of the purine nucleotide cycle to ammonia production in renal tubules isolated from control and chronically acidotic rats. Addition of 1 mM AICAriboside to incubation medium containing 2.5 mM [15N] aspartate significantly stimulated production of 15NH3 and 15N in the 6-amino group of adenine nucleotides during a 30-min incubation. In tubules from both control and acidotic animals, the levels of ATP, AMP, and NH3 were increased. In contrast, 5 mM AICAriboside inhibited 15NH3 production and reduced the total purine nucleotide content. In tubules from acidotic rats, enrichment in 15NH3 exceeded that in the 6-amino group of the adenine nucleotides, indicating that no precursor-product relationship existed between the purine nucleotide cycle and ammonia. Conversely, in tubules from control rats, 15N enrichment in the 6-amino group of the adenine nucleotides exceeded that in NH3. This relationship obtained whether or not AICAriboside was included in the incubation mixture. The current investigations show that the metabolism of aspartate through the purine nucleotide cycle is lower in renal tubules obtained from chronically acidotic rats than in control tubules. The observations indicate that AICAriboside has a biphasic effect on renal ammoniagenesis and adenine nucleotide synthesis, and suggest a possible clinical use of AICAriboside in cases of impaired ammonia formation in renal failure.     

Participation of alpha-amino group of amino acids in ammonia production in heart muscle

The ammoniogenetic capacity of some L-amino acids in rat heart muscle was studied. It was shown that [15N]leucine, [15N]glutamate and [15N]aspartate are involved in ammonia production without being the major source of this compound. The amount of [15N]ammonia produced by [15N]amino acids makes up to 0.25% of its total content. The deamination of L-[15N]leucine catalyzed by 2-oxoglutarate occurred at the highest rate. The [15N]aspartate and [15N]glutamate appeared to be less efficient precursors of ammonia. The bulk of amino nitrogen of L-[15N]amino acids was incorporated into the proteins and free amino acids in heart muscle.     

Physiological Levels of Ammonia Regulate Glutamine Synthesis from Extracellular Glutamate in Astrocyte Cultures

The effect of ammonia on glutamate accumulation and metabolism was examined in astrocyte cultures prepared from neonatal rat cortices. Intact astrocytes were incubated with 70 microM L-[14C(U)]glutamate and varying amounts of ammonium chloride. The media and cells were analyzed separately by HPLC for amino acids and labelled metabolites. Extracellular glutamate was reduced to 8 microM by 60 min. Removal of glutamate from the extracellular space was not altered by addition of ammonia. The rate of glutamine synthesis was increased from 3.6 to 9.3 nmol/mg of protein/min by addition of 100 microM ammonia, and intracellular glutamate was reduced from 262 to 86 nmol/mg of protein after 30 min. The metabolism of accumulated glutamate was matched nearly perfectly by the synthesis of glutamine, and both processes were proportional to the amount of added ammonia. The transamination and deamination products of glutamate were minor metabolites that either decreased or remained unchanged with increasing ammonia. Thus, ammonia addition stimulates the conversion of glutamate to glutamine in intact astrocyte cultures. At physiological concentrations of ammonia, glutamine synthesis appears to be limited by the rate of glutamate accumulation and the activity of competing reactions and not by the activity of glutamine synthetase.     

Metabolism of glutamine and glutamic acid by isolated perfused kidneys of normal and acidotic rats

1. When isolated kidneys from fed rats were perfused with glutamine the rate of ammonia release at pH7.4 (110–360μmol/h per g dry wt.) was one to two times that of glutamine removal. Glucose formation from 5mm-glutamine was 16μmol/h per g. If kidneys were perfused with glutamine at pH7.1 (10–13mm-sodium bicarbonate) there was no increase in glutamine removal or in the formation of ammonia or glucose. 2. When isolated kidneys from fed rats were perfused with glutamate at pH7.4, glucose formation was 59μmol/h per g, glutamine formation was 182μmol/h per g and ammonia release was negligible. At pH7.1 glutamine synthesis was inhibited and formation of ammonia and glucose were increased. 3. In perfused kidneys from acidotic rats, which had received 1.5% (w/v) NH₄Cl to drink for 7–10 days, gluconeogenesis from glutamine was enhanced (101μmol/h per g). Glutamine removal and ammonia formation were also increased, compared with the rates in perfused kidney from normal rats. The extra glutamine consumed was equivalent to the extra glucose formed. 4. When the kidney from the 7–10-day-acidotic rat was perfused with glutamate gluconeogenesis was increased (113μmol/h per g). Synthesis of glutamine was decreased, and ammonia release was approximately equal to the rate of glutamate removal. 5. The time-course of these metabolic alterations was investigated after the rapid induction of acidosis by infusion of 0.25m-HCl into the right side of the heart. The increase in gluconeogenesis from glutamine developed gradually over several hours. When kidneys from 6h-acidotic rats were perfused with glutamate, formation of glucose and glutamine were both rapid. 6. In acidotic rat kidneys perfused with glutamine, tissue concentrations of glutamate and glucose 6-phosphate were increased compared with those in control perfused kidneys from non-acidotic rats. 7. The results are discussed in terms of control of the renal metabolism of glutamine. In particular, it is suggested that in acidotic rats glucose formation is the major fate of the carbon of the extra glutamine utilized by the kidney, and that inhibition of glutamine synthetase could contribute to the increase in intracellular ammonia concentration in the kidney.     

Glutamate metabolism in a human intestinal epithelial cell layer model

Plasma glutamate concentrations are constant despite dynamic changes in diets. Most likely, virtually all the dietary glutamate is metabolized in the gut. The present study investigated permeability and metabolism of dietary glutamate in a Caco-2 intestinal epithelial cell layer model by tracing the fate of [U-¹³C] or [¹⁵N]glutamate added to the apical medium. For comparison, several other labelled essential and non-essential amino acids were tested as well. Almost all the labelled glutamate in the apical medium (98% and 96% at 24 h of the culture, respectively) was incorporated in the cell layer, while it barely appeared at the basolateral side, indicating an almost complete utilization of glutamate. Indeed, the ¹³C was incorporated into alanine, proline, ornithine, and glutamine, and the ¹⁵N was incorporated into alanine, glutamine, ornithine, proline, branched chain amino acids and also found as ammonia indicative of oxidation. In contrast, substantial apical-to-basolateral transport of amino acids (8–85% of uptake) other than glutamate and aspartate was evident in studies using amino acid tracers labelled with ¹³C, ¹⁵N or D. These results suggest that the intestinal epithelial cell monolayer utilizes dietary glutamate which adds to maintaining glutamate homeostasis in the body.

     

Glutamine and glutamate--their central role in cell metabolism and function

Glucose is widely accepted as the primary nutrient for maintenance and promotion of cell function. However, we propose that the 5-carbon amino acids, glutamine and glutamate, should be considered to be equally important for maintenance and promotion of cell function. The functions of glutamine are many and include: substrate for protein synthesis, anabolic precursor for muscle growth, acid-base balance in the kidney, substrate for ureogenesis in the liver, substrate for hepatic and renal gluconeogenesis, an oxidative fuel for intestine and cells of the immune system, inter-organ nitrogen transport, precursor for neurotransmitter synthesis, precursor for nucleotide and nucleic acid synthesis and precursor for glutathione production. Many of these functions are connected to the formation of glutamate from glutamine. We propose that the unique properties regarding concentration and routes of metabolism of these amino acids allow them to be used for a diverse array of processes related to the specialized function of each of the glutamine utilizing cells. In this review we highlight the specialized aspects of glutamine/glutamate metabolism of different glutamine-utilizing cells and in each case relate key aspects of metabolism to cell function.     

Cerebral Ammonia Metabolism in Hyperammonemic Rats

The short-term metabolic fate of blood-borne [13N]ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a "freeze-blowing" technique it was shown that the overwhelming route for metabolism of blood-borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L-[amide-13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.