Glutamine Production Rate Research Articles

Influence of N-acetylcysteine on hepatic amino acid metabolism in patients undergoing orthotopic liver transplantation

Experimental treatment with the antioxidant and glutathione precursor N-acetylcysteine (NAC) has been performed in orthotopic liver transplantation (OLT) to reduce reperfusion injury. To investigate the effect of NAC on the hepatic and intestinal amino acid metabolism, intraoperative amino acid exchange rates were studied in liver transplant recipients with high dose NAC treatment (n = 10) and in control patients (n = 9). Treatment with NAC was found to cause a loss of amino acids and increased urea nitrogen release from the liver graft. The net balance of most amino acids was shifted to increased hepatic release or decreased hepatic uptake. The initial cumulative splanchnic release of all proteinogenic amino acids in the NAC treated group was significantly higher than in the control group. These findings are tentatively explained by an increased net protein catabolism in the liver. The increased hepatic urea and glutamine production rate of the NAC treated patients is expected to increase the energy and oxygen demand of the liver in this critical situation. Thus, NAC may have caused marked metabolic disturbances in the freshly implanted graft. The dosage of NAC should therefore be modified to avoid these disadvantages.

Mechanisms of Glutamate Metabolic Signaling in Retinal Glial (Müller) Cells

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina. Our results demonstrate that lactate production and release is controlled by the combined action of glutamate and NH(4)(+), both at micromolar concentrations. Transport of glutamate by a high-affinity carrier can produce in Müller cells a rapid rise of glutamate concentration. In our isolated Müller cells, glutamine synthetase (GS) converted transported glutamate to glutamine that was released. This reaction, predominant when enough NH(4)(+) is available, was limited at micromolar concentrations of NH(4)(+), and more glutamate entered then as substrate into the mitochondrial tricarboxylic acid cycle (TCA). Increased production of glutamine by GS leads to increased utilization of ATP, some of which is generated glycolytically. Methionine sulfoximine, a specific inhibitor of GS, suppressed the stimulatory effect of glutamate and NH(4)(+) on glycolysis and induced massive entry of glutamate into the TCA cycle. The rate of glutamine production also determined the amount of pyruvate transaminated by glutamate to alanine. Lactate, alanine, and glutamine can be taken up and metabolized by photoreceptor neurons. We conclude that a major function of Müller glial cells is to nourish retinal neurons and to metabolize the neurotoxic ammonia and glutamate.

KINETICS OF AMMONIUM ASSIMILATION IN TWO SEAWEEDS, ENTEROMORPHA SP. (CHLOROPHYCEAE) AND OSMUNDARIA COLENSOI (RHODOPHYCEAE)

The kinetics of ammonium assimilation were investigated in two seaweeds from northeastern New Zealand, Enteromorpha sp. (Chlorophyceae, Ulvales) and Osmundaria colensoi (Hook. f. et Harvey) R.E. Norris (Rhodophyceae, Ceramiales), with the use of a recently developed method for measuring assimilation. In contrast to ammonium uptake, which was nonsaturable, ammonium assimilation exhibited Michaelis–Menten kinetics in both species. Maximum rates of assimilation (Vmax) were 27 and 12 μmol·(g DW)−1·h−1 for Enteromorpha sp. and O. colensoi, respectively, with half‐saturation (Km) constants for assimilation of 18 and 41 μM. At environmentally relevant concentrations, assimilation accounted for all of the ammonium taken up by both species. The maximum rate of assimilation in Enteromorpha sp. resembled very closely that of the ammonium assimilatory enzyme, glutamine synthetase, when activities of the latter were measured in the presence of subsaturating substrate (glutamate and ATP) concentrations. Moreover, the initial rate of glutamine production (measured with HPLC) following ammonium enrichment was almost identical to the rates determined above. The rate of ammonium assimilation was therefore determined by three independent methods, two of which involve in vivo measurements, and it is suggested that the use of assimilation kinetics may be useful when examining the nutrient relations of seaweeds.

Absence of glutamine isotopic steady state: implications for the assessment of whole-body glutamine production rate

1.During infusion of [5-15N]glutamine in patients with gastrointestinal cancer we unexpectedly observed a gradual decrease in time of the appearance rate (Ra) of glutamine in plasma. Here we investigate whether the failure to achieve a plateau isotopic enrichment in plasma is, among other factors, due to incomplete equilibration of the glutamine tracer with the large intramuscular free glutamine pool. 2.Plasma and intramuscular glutamine enrichment were measured during 6–11 ;h infusions of L-[5-15N]glutamine and L-[1-13C]glutamine in post-absorptive patients admitted to hospital for elective abdominal surgery. L-[1-13C]Leucine and L-[ring-2H5]phenylalanine were infused to measure the proportion of glutamine appearing in plasma directly due to its release from protein. 3.The glutamine tracer entered muscle, but the rise in intramuscular glutamine enrichment was small, presumably as a result of the enormous size of the intramuscular glutamine pool and the limited speed of entry of glutamine into muscle. In each patient the intramuscular glutamine enrichment was lower than that in plasma (P < 0.001), and both increased with tracer infusion time (P < 0.001), indicating incomplete equilibration of the glutamine tracer. 4.A comparison of the results obtained by the two glutamine tracers indicated that recycling of the nitrogen label contributed to about 15% of the decrease in Ra. 5.There was a gradual reduction in the glutamine release from proteolysis, which contributed to 16–21% of the decline in Ra. 6.We conclude that slow equilibration of the glutamine tracer with the large muscle glutamine pool significantly contributes to the absence of isotopic steady state. Consequently, the appearance rate of glutamine in plasma measured during short tracer infusion periods (hours) considerably overestimates the whole-body glutamine flux.

Absence of glutamine isotopic steady state: implications for the assessment of whole-body glutamine production rate

1. During infusion of [5-15N]glutamine in patients with gastrointestinal cancer we unexpectedly observed a gradual decrease in time of the appearance rate (Ra) of glutamine in plasma. Here we investigate whether the failure to achieve a plateau isotopic enrichment in plasma is, among other factors, due to incomplete equilibration of the glutamine tracer with the large intramuscular free glutamine pool.2. Plasma and intramuscular glutamine enrichment were measured during 6-11 h infusions of L-[5-15N]glutamine and L-[1-13C]glutamine in post-absorptive patients admitted to hospital for elective abdominal surgery. L-[1-13C]Leucine and L-[ring-2H5]phenylalanine were infused to measure the proportion of glutamine appearing in plasma directly due to its release from protein.3. The glutamine tracer entered muscle, but the rise in intramuscular glutamine enrichment was small, presumably as a result of the enormous size of the intramuscular glutamine pool and the limited speed of entry of glutamine into muscle. In each patient the intramuscular glutamine enrichment was lower than that in plasma (P<0.001), and both increased with tracer infusion time (P<0.001), indicating incomplete equilibration of the glutamine tracer.4.A comparison of the results obtained by the two glutamine tracers indicated that recycling of the nitrogen label contributed to about 15% of the decrease in Ra.5. There was a gradual reduction in the glutamine release from proteolysis, which contributed to 16-21% of the decline in Ra.6. We conclude that slow equilibration of the glutamine tracer with the large muscle glutamine pool significantly contributes to the absence of isotopic steady state. Consequently, the appearance rate of glutamine in plasma measured during short tracer infusion periods (hours) considerably overestimates the whole-body glutamine flux.

Glutamine and arginine metabolism in tumor-bearing rats receiving total parenteral nutrition

Arginine supplementation increases glutamine levels in muscle and plasma. Since glutamine production is increased in catabolic states, these observations prompted us to investigate whether the flux of arginine to glutamine was increased in tumor-bearing (TB) rats, and we measured the synthesis rate of glutamine from arginine in control versus TB rats receiving standard total parenteral nutrition (TPN) solution. Male Donryu rats (N = 36; body weight, 200 to 225 g) were divided into two groups, control and TB rats. Yoshida sarcoma cells (1 × 10 6) were inoculated into the back of the rats (n = 18) subcutaneously on day 0. The rats were given free access to water and rat chow. On day 5, all animals, including non-TB rats (n = 18), were catheterized at the jugular vein and TPN was begun. On day 10, TPN solution containing either U- 14C-glutamine (2.0 μCi/h) or U- 14C-arginine (2.0 μCi/h) was infused as a 6-hour constant infusion. At the end of the isotope infusion, plasma was collected to determine the glutamine production rate in rats receiving U- 14C-glutamine, and the ratio of specific activity of glutamine to specific activity of arginine was measured in rats receiving U- 14C-arginine. Only 2 g tumor caused a decrease in glutamine levels and an increase in glutamine and arginine production. The low flux rate of arginine to glutamine was observed in control rats (Arg to Gln, 41.0 ± 11.9 μmol/kg/h). On the other hand, TB caused a significant increase in Arg to Gln compared with the control (213.3 ± 66.1 μmol/kg/h, P < .01 v control). An increase in the flux rate of Arg to Gln was associated with an enhancement in the ratio of specific activity of ornithine to specific activity of arginine in TB rats (control 51.5% ± 10.9% v 77.4% ± 8.9%, P < .05). We conclude that (1) glutamine and arginine metabolism is altered with very small tumors, (2) although the flux of Arg to Gln was increased in TB and rats, the small increase in Arg to Gln cannot explain the observed large increase in Gln production.

Ketone bodies promote a rapid rise in glutamate efflux from the isolated perfused rat liver without altering the rate of glutamine production

Livers of starved (48 hr) male Wistar rats were perfused in a non recirculating manner with a near physiological mix of ammonium, lactate, ornithine and pyruvate in Krebs buffer. The addition of ketone bodies (3-DL-hydroxybutyrate [B OHB] 2-30 mM or lithium-acetoacetate (15 mM) to the perfusate resulted in a rapid rise in the efflux of glutamate from the liver (five times above basal). This was not seen with control solutions (sodium chloride or lithium chloride). The increased efflux was sustained for the duration of the addition of the ketone bodies (7 min), was rapidly reversible and dose dependant. Glutamine export rates were not altered, suggesting that either the glutamate originated from cells not responsible for glutamine synthesis or that this glutamate was superfulous to the requirement of glutamine synthesis. There was no evidence that the lactate transporter was involved in the entry of lactate into perivenous hepatocytes for glutamine synthesis; lactate presumably entering the hepatocyte by an alternative pathway, probably nonionic diffusion.

The effect of acidosis on the labelling of urinary ammonia during infusion of [amide-15N]glutamine in human subjects.

In three experiments [amide-15N]glutamine was infused intravenously in male volunteers. After 4-8 h of infusion acidosis was achieved by an oral dose of CaCl2 (1 mmol/kg). In one subject acidosis was maintained for 5 d. The acid load produced an approximately 3-fold increase in urinary NH3 excretion, with a small (approximately 20%) and transient increase in the isotope abundance of urinary NH3. Estimates of glutamine production rate (flux) were obtained in two experiments. There was no evidence that it was increased in acidosis. The extra NH3 production by the kidney represented only a very small part, about 3%, of the total glutamine production rate.

The Capacity of the Malate-Aspartate Shuttle Differs between Periportal and Perivenous Hepatocytes from Rats

The capacity of the malate-aspartate shuttle was evaluated in periportal (PP-H) and perivenous subfraction of rat hepatocytes (PV-H). The rate of glutamine production from alanine was 34-fold higher in PV-H than in PP-H. Statistically significant differences between PP-H and PV-H were found for the activities of lactate dehydrogenase and pyruvate kinase but not for the activities of NAD+-malate dehydrogenase, aspartate aminotransferase, and mitochondrial alanine aminotransferase. The rate of glucose production from sorbitol and the rate of ethanol utilization were higher in PP-H than in PV-H. In the presence of phenazine methosulfate (PMS), the increments in these rates were significantly greater in PV-H than in PP-H. The capacity of malate-aspartate shuttle in the presence of alanine was significantly higher in PP-H than in PV-H but in the presence of asparagine was similar in PP-H and PV-H. The results suggest that the capacity of malate-aspartate shuttle distributes heterogeneously along liver lobules with the dominance in periportal zone and that the difference of the capacity may result from the difference in the transport of aspartate across the mitochondrial membrane.

Glutamine Metabolism in Skeletal Muscle of Glucocorticoid-Treated Rats

1. The effect of dexamethasone (30 micrograms day-1 100 g-1 body weight) on the regulation of glutamine metabolism was studied in skeletal muscles of rats after 9 days of treatment. 2. Dexamethasone resulted in negative nitrogen balance, and produced increases in the plasma concentrations of alanine (23.4%) and insulin (158%) but a decrease in the plasma concentration of glutamine (28.7%). 3. Dexamethasone treatment increased the rate of glutamine production in muscle, skin and adipose tissue preparations, with muscle production accounting for over 90% of total glutamine produced by the hindlimb. 4. Blood flow and arteriovenous concentration difference measurements across the hindlimb showed an increase in the net exchange rates of glutamine (25.3%) and alanine (90.5%) in dexamethasone-treated rats compared with corresponding controls. 5. Dexamethasone treatment produced significant decreases in the concentrations of skeletal muscle glutamine (51.8%) and 2-oxoglutarate (50.8%). The concentrations of alanine (16.2%), pyruvate (45.9%), ammonia (43.3%) and inosine 5'-phosphate (141.8%) were increased. 6. The maximal activity of glutamine synthetase was increased (21-34%), but there was no change in that of glutaminase, in muscles of dexamethasone-treated rats. 7. It is concluded that glucocorticoid administration enhances the rates of release of both glutamine and alanine from skeletal muscle of rats (both in vitro and in vivo). This may be due to changes in efflux and/or increased intracellular formation of glutamine and alanine.

Functional hepatocyte heterogeneity

1. In isolated perfused rat liver maximal rates of 2-[1-14C]oxoglutarate uptake were about 0.4 mumol.g-1 .min-1; half-maximal rates of 2-[14C]oxoglutarate uptake were observed with influent concentrations of about 100 microM. 2-[14C]Oxoglutarate uptake by the liver was not affected by the direction of perfusion, but was decreased by about 80-90% when Na+ in the perfusion fluid was substituted by choline+, suggesting a Na+-dependence of hepatic 2-oxoglutarate uptake. In the absence of added ammonia, [14C]oxoglutarate uptake by the liver was about twice the net oxoglutarate uptake, indicating a simultaneous release of unlabeled oxoglutarate from perfused rat liver. 2. 14C-Labeled metabolites derived from [1-14C]oxoglutarate and recovered in the effluent perfusate were 14CO2 and 14C-labeled glutamate and glutamine; they accounted for 85-100% of the radiolabel taken up by the liver. 14CO2 was the major product (more than 70%) from [1-14C]oxoglutarate taken up the liver, provided glutamine synthesis was either inhibited by methionine sulfoximine or the endogenous rate of glutamine production was below 40 nmol.g-1.min-1. 3. Stimulation of glutamine synthesis by ammonia did not affect [14C]oxoglutarate uptake by the liver, but considerably increased net hepatic oxoglutarate uptake, indicating a decreased release of unlabeled oxoglutarate from the liver. Stepwise stimulation of hepatic glutamine synthesis led to a gradual decrease of 14CO2 production and radiolabel was recovered increasingly as [14C]glutamine in the effluent. At high rates of glutamine formation (i.e. about 0.6 mumol.g-1.min-1), about 60% of the [1-14C]oxoglutarate taken up by the liver was recovered in the effluent as [14C]glutamine. 14CO2 and [14C]glutamine production from added [1-14C]oxoglutarate were dependent on the rate of hepatic glutamine synthesis but not on the direction of perfusion. Extrapolation of 14C incorporation into glutamine to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of the [14C]oxoglutarate taken up by the liver for glutamine synthesis. This was again true for both the antegrade and the retrograde perfusion directions. On the other hand, addition of ammonia did not affect 14CO2 production from labeled oxoglutarate, when glutamine synthetase was inhibited by methionine sulfoximine. 4. The data suggest that vascular oxoglutarate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase, i.e. a cell population comprising only 6-7% of all hepatocytes. Thus, the findings demonstrate the existence of a, to date, uniquely zonally distributed oxoglutarate transport system which is probably Na+-dependent in the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamine production rate and its contribution to urinary ammonia in normal man.

1. Glutamine [15N]amide was infused at a steady rate of 33.34 mumol/h into seven male adult volunteers who were in the fed state and normal acid-base status. 2. Plasma glutamine amide N enrichment and urinary ammonia N enrichment rose to a constant rate within 3 h. 3. The glutamine production rate was 51.8 +/- 7.9 mmol/h. 4. The total ammonia excretion rate was 0.87 mmol/h. Of this excreted ammonia 62.6 +/- 9% was derived from the amide N atom of glutamine. 5. The excreted glutamine amide N (0.53 mmol/h) was only 1% of the glutamine production. If half the ammonia formed by the kidney is excreted in urine and half liberated into the renal vein in subjects with normal acid-base status [E. E. Owen & R. R. Robinson (1963) Journal of Clinical Investigation, 42, 263-276], then the kidney accounts for only 2% of glutamine disposal. 6. Whole body protein turnover, measured from the urinary [15N]ammonia enrichment, was 30.3 +/- 7.7 g of N/day (2.8 g of protein day-1 kg-1).

Control of glutamine synthesis in rat liver

1. On perfusion of livers from fed rats with a semi-synthetic medium containing no added amino acids there is a rapid release of glutamine during the first 15min (15.6+/-0.8mumol/h per g wet wt.; mean+/-s.e.m. of 35 experiments), followed by a low linear rate of production (3.6+/-0.3mumol/h per g wet wt.; mean+/-s.e.m. of three experiments). The rapid initial release can be accounted for as wash-out of preexisting intracellular glutamine. 2. Addition of readily permeating substrates, or substrate combinations, giving rise to intracellular glutamate or ammonia, or both, did not appreciably increase the rate of glutamine production over the endogenous rate. The maximum rate obtained was from proline plus alanine; even then the rate represented less than one-fortieth of the capacity of glutamine synthetase measured in vitro. 3. Complete inhibition of respiration in the perfusions [no erythrocytes in the medium; 1mm-cyanide; N(2)+CO(2) (95:5) in the gas phase] or perfusion with glutamine synthetase inhibitors [l-methionine dl-sulphoximine; dl-(+)-allo-delta-hydroxylysine] abolishes the low linear rate of glutamine synthesis, but not the initial rapid release of glutamine. 4. In livers from 48h-starved rats initial release (0-15min) of glutamine was decreased (10.6+/-1.1mumol/h per g wet wt.; mean+/-s.e.m. of 11 experiments) and the subsequent rate of glutamine production was lower than in livers from fed rats. Again proline plus alanine was the only substrate combination giving an increase significantly above the endogenous rate. 5. The rate of glutamine synthesis de novo by the liver is apparently unrelated to the tissue content of glutamate or ammonia. 6. The blood glutamine concentration is increased by 50% within 1h of elimination of the liver from the circulation of rats in vivo. 7. There is an output of glutamine by the brain under normal conditions; the mean arterio-venous difference for six rats was 0.023mumol/ml. 8. The high potential activity of liver glutamine synthetase appears to be inhibited by some unknown mechanism: the function of the liver under normal conditions is probably the disposal of glutamine produced by extrahepatic tissues.