Renal metabolism and ammoniagenesis during acute respiratory alkalosis in the dog

Acute respiratory alkalosis (blood pH, 7.60; arterial PCO2, 15 mmHg (1 mmHg = 133.322 Pa); plasma bicarbonate, 14 mM) was induced in nine anesthetized dogs by increasing their respiratory rate and depth. Renal glutamine extraction and ammonia production expressed per 100 mL of glomerular filtration rate did not change during acute hypocapnia, whereas arterial glutamine concentration decreased significantly from 0.47 to 0.36 mM. Hypocapnia did not change plasma potassium concentration and its urinary excretion. Acute hypocapnia increased lactate extraction and pyruvate production, whereas citrate extraction and glutamate and alanine production did not change. Citraturia remained minimal. Renal cortical glutamine concentration fell from 0.64 to 0.38 mM during hypocapnia while alpha-ketoglutarate, glutamate, malate, oxaloacetate, and citrate did not change. Lactate concentration rose from 1.1 to 2.0 mM. Glutamine concentration in the liver and muscle decreased following acute hypocapnia. Our data are compatible with the hypothesis that an acute respiratory alkalosis might not result in any change in the hydrogen ion concentration and (or) gradient between the mitochondrial matrix and the cytosol. Consequently, renal glutamine extraction and ammonia production are not reduced, renal cortical concentrations of relevant metabolites in the ammoniagenic pathway are not changed, and renal handling of citrate remains unaffected.     

Renal tissue metabolism in the rat during chronic metabolic alkalosis: importance of glycolysis

Chronic metabolic alkalosis was induced in rats drinking 0.3 M NaHCO3 and receiving 1 mg furosemide/100 g body weight per day intraperitoneally. Another group of animals received a potassium supplement in the form of 0.3 M KHCO3. In this group, hypokalemia did not develop and muscle potassium fell by only 18% versus 50% in those not receiving potassium. In vitro renal production of ammonia and uptake of glutamine fell by 40% with a decrease in the activity of glutaminase I and glutamate dehydrogenase. Activity of phosphofructokinase, a major enzyme of glycolysis, rose only in the kidney of animals receiving a potassium supplement. Fructose-1,6-diphosphatase fell as well as phosphoenolpyruvate carboxykinase. Malate dehydrogenase also fell. The activity of phosphofructokinase also rose in the liver, heart, and leg muscle. The major biochemical changes in the renal cortex were the following: glutamate, alpha-ketoglutarate, malate, lactate, pyruvate, alanine, aspartate, and citrate rose as well as calculated oxaloacetate. The concentration of intermediates like 2-phosphoglycerate, 3-phosphoglycerate, and glucose-6-phosphate fell. The cytosolic redox potential (NAD+/NADH) decreased. In addition to the fall in ammoniagenesis, it could be demonstrated in vitro that the renal tubules incubated with glutamine showed decreased glucose production and increased production of lactate and pyruvate. The concentration of lactate was elevated in all tissues examined including liver, heart, and leg muscle. This study confirms in the rat that decreased renal ammoniagenesis takes place following decreased uptake of glutamine in metabolic alkalosis. All other changes are accounted for by the process of increased glycolysis, which appears to take place in all tissues in metabolic alkalosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of valproate on lactate and glutamine metabolism by rat renal cortical tubules

The metabolic effects of sodium valproate (VPA) on rat renal cortical tubules have been examined. When 1 or 5 mM lactate was used as substrate in the incubation medium, VPA decreased markedly the lactate uptake by the tubules. When 1 or 5 mM glutamine was used, the addition of VPA accelerated glutamine uptake, ammoniagenesis, but also stimulated markedly the accumulation of lactate and pyruvate produced from glutamine. VPA had a dose-dependent inhibitory effect on gluconeogenesis from both glutamine and lactate. With 5 mM glutamine, VPA also induced a significant accumulation of glutamate in the medium. The oxygen consumption by the tubules was diminished by 40% following VPA addition. It is concluded that VPA modifies the metabolism of rat cortical tubules by interfering with the oxidation of natural substrates and stimulates in this fashion the production of ammonia by kidney tubules.     

Metabolic effects of valproate on dog renal cortical tubules

The effect of valproate (0.01-10 mM), an antiepileptic drug inducing hyperammonemia in humans, was studied in vitro on a suspension of renal cortical tubules (greater than 85% proximal tubules) obtained from six normal dogs. When these tubules were incubated with 1 mM glutamine, the addition of valproate accelerated glutamine uptake, ammoniagenesis, and the production of alanine, lactate, and pyruvate. With 5 mM glutamine, a rise in glutamate accumulation, a much greater synthesis of alanine, an important aspartate production, and a striking accumulation of lactate and pyruvate were observed. With 1 or 5 mM lactate, lactate utilization and gluconeogenesis were markedly reduced with increasing concentrations of valproate. Oxygen consumption was reduced by only 15-20% by 10 mM valproate. The accelerated glutamine utilization resulting from valproate could not be prevented by aminooxyacetate, an inhibitor of transamination. Valproate also reduced various enzymatic activities, a finding that could not explain its metabolic effects. Four sites of action may explain these various metabolic changes: (i) a stimulation of mitochondrial glutamine transport, (ii) an increase in the flux of glutamate to malate, and (iii) a reduction in the net oxidation of pyruvate and (iv) in the flux through pyruvate carboxylase.     

Renal metabolism during four types of lactic acidosis in the dog including anoxia

The present study was undertaken to evaluate the metabolic response of the kidney to lactic acidosis. Four types of lactic acidosis were induced in the dog: infusion of lactic acid, infusion of lactic acid with phenformin, administration of phenformin alone, and hypoxia by breathing 95% nitrogen. In all groups of animals, the same degree of acidosis was observed with plasma bicarbonate ranging from 12.8 to 14.9 mM. Plasma lactate concentration ranged from 3.0 to 8.1 mumol/mL. Renal ammoniagenesis failed to be influenced by lactic acidosis. As a matter of fact, it fell during anoxia. The extraction of glutamine by the kidney rose except during anoxia where it fell. The renal production of alanine rose during the infusion of lactic acid with and without phenformin. This coincided with the extraction of glutamine. The renal extraction of lactate rose in all forms of acidosis as well as the production of pyruvate. In the renal cortical tissue, the concentration of malate, pyruvate, and lactate rose. Alanine also rose except during anoxia. An important fall in cytosolic redox potential (NAD+/NADH lactate dehydrogenase) was observed, as well as a fall in mitochondrial redox (NAD+/NADH beta-hydroxybutyrate dehydrogenase). Lactate also accumulated in the liver and in the muscle. We propose that the kidney is unable to respond to lactic acidosis in terms of ammonia production and that this phenomenon is explained by transamination of pyruvate and glutamate into alanine and also by the observed fall in cytosolic redox potential. It is likely that renal gluconeogenesis is also inhibited and this is reflected by the rise in the concentration of malate in the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

Ammonia effects on pyruvate/lactate production in astrocytes--interaction with glutamate

Ammonia exerts a multitude of metabolic and non-metabolic effects on brain tissue. In the present communication we have investigated its effect on lactate production rates, pyruvate production rates and pyruvate/lactate ratios in mouse cerebrocortical astrocytes and neurons in primary cultures. No effects were found in neurons. All three parameters were affected by ammonia in astrocytes, but less potently and to a smaller degree in cells that had been treated with dibutyryl cyclic AMP (morphologically differentiated cells) than in untreated cells (morphologically undifferentiated cells). In the differentiated cells ammonia had virtually no effect up to a concentration of 1.0 mM, but at 3.0 mM it increased lactate production and decreased pyruvate/lactate ratio significantly. In the undifferentiated cells ammonia greatly increased lactate accumulation (by 80% at 3.0 mM) and it inhibited pyruvate accumulation (by 40% at 3.0 mM). It thereby reduced the pyruvate/lactate ratio progressively within the entire range 0.1-3.0 mM ammonia. In support of the hypothesis that the ammonia-induced reduction of pyruvate/lactate ratio is secondary to depletion of cellular glutamate by formation of glutamine (and glutathione) and a resulting interruption of the malate-aspartate shuttle (MAS), the addition of glutamate to the incubation medium significantly diminished the ammonia-induced reduction of pyruvate/lactate ratio, whereas it had no effect on the increased lactate production. It is discussed that MAS interruption may have additional consequences in astrocytes.     

半边结灌注培养中杂交瘤细胞的生长和代谢

考察了半连续灌注培养中ＷｕＴ３杂交瘤细胞在不同灌注速率下细胞生长的动态变化,培养其中主要基质的消耗和代谢物的生成。当灌注速率Ｄ从１．０／升高到２．０／ｄ升高到２．０／ｄ时,乳酸得率系数Ｙｌａｃ／ｇｌｕ降低１８％,氨得率系数Ｙａｍｍ／ｇｌｎ降低４０％,丙氨酸得率系数Ｙａｌａ／ｇｌｎ升高５８％,甘氨酸得率系数Ｙｇｌｙ／ｇｌｎ基本恒定。说明在灌注速率升高的条件下,细胞会调整代谢机制,丙酮酸和过量的谷氨酸     

Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.     

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[1-14C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through alpha-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222, 813-820] and Preuss [(1972) Am. J. Physiol. 222, 1395-1397]. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenous-difference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

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.     

The urea cycle in the liver of arthritic rats

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (–32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.     

The synthesis of glucose and ammonia by kidney tubules isolated from suckling and early-weaned lambs

Glucose and ammonia production were examined in kidney tubules isolated from suckling and early-weaned lambs, on days 10-30 after birth, with abrupt weaning occurring at day 14. There were no differences in the rates of glucose or ammonia production for a given substrate by tubules isolated from any of the lambs, regardless of age or stage of weaning. The preferred substrates for gluconeogenesis were glycerol = lactate greater than propionate = pyruvate = fructose = proline greater than alanine greater than glutamate greater than glutamine greater than aspartate greater than glycine greater than serine, and for ammoniagenesis were glutamine much greater than alanine greater than aspartate much greater than serine greater than glycine = glutamate = proline.     

Glutamine and glucose metabolism in bovine blood lymphocytes.

1. Glutamine and glucose metabolism was studied in bovine blood lymphocytes incubated at 37 degrees C in the presence of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1 mM [U-14C]glutamine and 5 mM [U-14C]glucose, respectively. 2. The major metabolic products from glutamine were ammonia, glutamate, and to a lesser extent, aspartate and CO2. Glucose was metabolized mainly to lactate and, to a lesser extent, pyruvate and CO2. These findings indicate incomplete oxidation of glutamine and glucose carbons in bovine blood lymphocytes. 3. Glucose provided three-fold greater amounts of energy to bovine blood lymphocytes than did glutamine on the basis of their measured end-products. Glycolysis accounted for 50% of glucose-derived ATP production. 4. Our findings suggest similar metabolic patterns of glutamine and glucose in lymphocytes between ruminants and non-ruminant species (e.g. rats). However, in contrast to rat peripheral lymphocytes, glucose, rather than glutamine, was a major energy substrate for bovine blood lymphocytes.     

Ammonia Intoxication: Effects on Cerebral Cortex and Spinal Cord

The effect of an acute systemic ammonia intoxication on the metabolic states of the cerebral cortex and the spinal cord of the same animal was studied in the cat. The intravenous infusion of ammonium acetate (2 and 4 mmol/kg body weight/30 min) increased the gross levels of tissue NH4+, glutamine, glutamine/glutamate ratio, lactate, and the lactate/pyruvate ratio in the cerebral cortex and the spinal cord. Pyruvate increased, but significantly only in the spinal cord; aspartate decreased, but significantly only in the cerebral cortex. The infusion of ammonium acetate did not significantly change the levels of phosphocreatine, ATP, ADP, AMP, total adenine nucleotides, adenylate energy charge, glucose, glutamate, alpha-ketoglutarate, and malate in either tissue. The changes of NH4+, glutamine, and lactate levels as well as glutamine/glutamate and lactate/pyruvate ratios in the spinal cord correlated significantly with the corresponding changes of these metabolites in the cerebral cortex. Thus, cerebral cortex and spinal cord show certain specific and comparable metabolic changes in response to a systemic ammonia intoxication. The effect of ammonia intoxication on the increases of glutamine and lactate levels is discussed.     

Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.     

Ammonia overloading in hepatocytes isolated from liver of fetal and adult rats

Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified by 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals.     

Effects of ketone bodies on amino acid metabolism in isolated rat diaphragm.

1. Diaphragms from 48h-starved rats were incubated in Krebs-Ringer bicarbonate medium at 37degreesC for 30min and then transferred into new medium and incubated for 1, 2 and 3 h. 2. The amount of free amino acids found at the end of each time of incubation was larger than the amount at the beginning of incubation, indicating that in this system proteolysis is prevailing. 3. The diaphragms was releasing mainly alanine and glutamine into the incubation medium. 4. Within the periods of incubation the release and metabolism of free amino acids was proceeding at a constant rate. 5. Addition of sodium DL-3-hydroxybutyrate decreased the tissue content of several amino acids, among which were tyrosine and phenylalanine, suggesting that proteolysis was decreased by ketone bodies. 6. In the presence of glucose (10mM) and branched-chain amino acids (0.5mM), sodium DL-3-hydroxybutyrate at concentrations of 4 or 6 mM resulted in 30% decrease in tissue alanine content and a 20% decline in alanine release. Release of taurine and glutamine was decreased by 19 and 16% respectively with 6 mM-sodium DL-3-hydroxybutyrate. Addition of sodium acetoacetate (1-3mM) also resulted in a 20-35% decrease in tissue content of alanine, glutamine and taurine and in a 15-24% decrease of alanine and glutamine release. Smaller decreases (less than 15%) in the release of glycine, threonine, proline, serine and aspartate were also observed in the presence of sodium DL-3-hydroxybutyrate or sodium acetoacetate. 7. Substitution of pyruvate (1.0mM) for glucose in the presence of acetoacetate restored alanine and glutamine production to control values. In the presence of acetoacetate, pyruvate also increased the tissue content of aspartate by 77% and decreased the tissue content of glutamate by 30%. 8. It is suggested that in diaphragms from starved rats, ketone bodies (a) in the absence of other substrates inhibit protein catabolism and (b) in the presence of glucose and branched-chain amino acids decrease alanine and glutamine production, by inhibiting glycolysis.     

The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium.

Although glutamine is a major carbon source for mammalian cells in culture, its chemical decomposition or cellular metabolism leads to an undesirable excess of ammonia. This limits the shelf-life of glutamine-supplemented media and may reduce the cell yield under certain conditions. We have attempted to develop a less ammoniagenic medium for the growth of BHK-21 cells by a mole-to-mole substitution of glutamine by glutamate. This results in a medium that is thermally stable but unable to support an equivalent growth yield. However, supplementation of the glutamate-based medium with asparagine (3 mM) and a minimal level of glutamine (0.5 mM) restored the original growth capacity of the cultures. Substitution of the low level of glutamine with the glutamine dipeptides, ala-gln (1 mM), or gly-gln (3 mM) resulted in an equivalent cell yield and in a thermally stable medium. The ammonia accumulation in cultures with glutamate-based medium was reduced significantly (>60%). Factors mediating growth and adaptation in medium substituted with glutamate were also investigated. The maximum growth capacity of the BHK-21 cells in glutamate-based medium (without glutamine) was achieved after a period of adaptation of 5 culture passages from growth in glutamine-based cultures. Adaptation was not influenced by increases in glutamate uptake which was constitutively high in BHK cells. Adaptation was associated with changes in the activities of enzymes involved in glutamate or glutamine metabolism. The activities of glutamine synthetase (GS) and alanine aminotransferase (ALT) increased significantly and the activity of phosphate-activated glutaminase (PAG) decreased significantly. The activity of glutamate dehydrogenase (GDH) showed no significant change after adaptation to glutamate. These changes resulted in an altered metabolic profile which included a reduced ammonia production but an increased alanine production. Alanine production is suspected of being an alternative route for removal of excess nitrogen.