Glutamine Extraction Research Articles

INTERPHASE DISTRIBUTION OF SOME AMINO ACIDS IN EXTRACTION SYSTEMS BASED ON N-VINYLFORMAMIDE COPOLYMERS

The extraction characteristics of amino acids and mono-amides of acids were studied using copolymers of N-vinylformamide with 1-vinyl- and 1-methacryloyl-3,5-dimethylpyrazole as extractants. Synthesis of water-soluble copolymers was carried out by radical copolymerization in dioxane with thermoinitiation with dinitro-azobisisomoic acid. The distribution coefficients and the degree of extraction of analytes for a single extraction in the presence of a salting out agent are calculated. To determine the representatives of amino acids, the method of capillary electrophoresis was used. The most effective extraction systems for the extraction of amino acids and monoamides of acids are proposed: the intrinsic viscosity of polymers, the concentration of polymer and analyte solution, the ratio of phase volumes, the ratio of the mole fractions of N-vinylformamide to 1-vinyl and 1-methacryloyl-3,5-dimethylpyrazole. The results of the most effective interfacial distribution of lysine between a water-salt solution and an extractant are presented, which is a copolymer of N-vinylformamide with 1-methacryloyl-3,5-dimethylpyrazole. The concentration of the copolymer and the ratio of the volumes of equilibrium phases at which the maximum degree of lysine extraction is reached are established. A copolymer of N-vinylformamide with 1-vinyl-3,5-dimethylpyrazole is used for simultaneous extraction of cystine, asparagine and glutamine. The established extraction characteristics allowed a successful separate determination of the amino acid cystine and monoamides (asparagine and glutamine) in a joint presence in an aqueous solution. When choosing the conditions for separate electrophoretic determination of amino acids, the optimal composition of the buffer solution, the type and concentration of the micelle was found. The dependence of the structure of the analytes on their interphase distribution was established, and the complexing power of the copolymers of N-vinylformamide with 1-vinyl- and 1-methacryloyl-3,5-dimethylpyrazole was revealed with respect to the representatives of amino acids. The extraction systems used on the basis of N-vinylformamide copolymers are distinguished by ecological and economic expediency, good metrological indices.

A Glutamine-Rich Carrier Efficiently Delivers Anti-CD47 siRNA Driven by a "Glutamine Trap" To Inhibit Lung Cancer Cell Growth.

It is not efficient enough using the current approaches for tumor-selective drug delivery based on the EPR effect and ligand-receptor interactions, and they have largely failed to translate into the clinic. Therefore, it is urgent to explore an enhanced strategy for effective delivery of anticancer agents. Clinically, many cancers require large amounts of glutamine for their continued growth and survival, resulting in circulating glutamine extraction by the tumor being much greater than that for any organs, behaving as a "glutamine trap". In the present study, we sought to elucidate whether the glutamine-trap effect could be exploited to deliver therapeutic agents to selectively kill cancer cells. Here, a macromolecular glutamine analogue, glutamine-functionalized branched polyethylenimine (GPI), was constructed as the carrier to deliver anti-CD47 siRNA for the blockage of CD47 "don't eat me" signals on cancer cells. The GPI/siRNA glutamine-rich polyplexes exhibited remarkably high levels of cellular uptake by glutamine-dependent lung cancer cells, wild-type A549 cells (A549WT), and its cisplatin-resistant cells (A549DDP), specifically under glutamine-depleted conditions. It was noted that the glutamine transporter ASCT2 was highly expressed both on A549WT and A549DDP but with almost no expression in normal human lung fibroblasts cells. Inhibition of ASCT2 significantly prevented the internalization of GPI polyplexes. These findings raised the intriguing possibility that the glutamine-rich GPI polyplexes utilize the ASCT2 pathway to selectively facilitate their cellular uptake by cancer cells. GPI further delivered anti-CD47 siRNA efficiently both in vitro and in vivo to downregulate the intratumoral mRNA and protein expression levels of CD47. CD47 functions as a "don't eat me" signal and binds to the immunoreceptor SIRPα inducing evasion of phagocytic clearance. GPI/anti-CD47 siRNA polyplexes achieved significant antitumor activities both on A549WT and A549DDP tumor-bearing nude mice. Notably, it had no adverse effect on CD47-expressing red blood cells and platelets, likely because of selective delivery. Therefore, the glutamine-rich carrier GPI driven by the glutamine-trap effect provides a promising new strategy for designing anticancer drug delivery systems.

Proteomic profiling and pathway analysis of the response of rat renal proximal convoluted tubules to metabolic acidosis.

Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis.

Alterations in glutamine metabolism and its conversion to citrulline in sepsis

In enterocytes, glutamine serves as the major source of energy; another metabolic fate of glutamine is conversion to citrulline. Because sepsis can affect gut function and integrity, alterations in glutamine metabolism may exist and lead to decreased citrulline production. This study aimed to investigate how sepsis affects glutamine metabolism, including its conversion to citrulline, by measuring glutamine and citrulline flux, fractional splanchnic extraction of glutamine and leucine, and the contribution of glutamine nitrogen to citrulline in septic patients and healthy controls. Eight patients with severe sepsis and 10 healthy controls were given primed, constant intravenous infusion of [(2)H2]citrulline and sequential administration of intravenous and enteral [α-(15)N]glutamine and [(13)C]leucine in the postabsorptive state. The results showed that, compared with healthy controls, septic patients had a significantly lower whole body citrulline flux and plasma concentration, higher endogenous leucine flux, and higher glutamine clearance. Fractional splanchnic extraction of leucine was higher in septic patients than in controls, but fractional extraction of glutamine was not different. The majority of the (15)N label transferred from glutamine to citrulline was found at the α-position. These results demonstrate that lower glutamine plasma concentrations in sepsis were a result of increased glutamine clearance. Despite adequate splanchnic uptake of glutamine, there is decreased production of citrulline, suggesting a defect in the metabolic conversion of glutamine to citrulline, decreased uptake of glutamine by the enterocyte but increased uptake by the liver, and/or shunting of glutamine to other metabolic pathways.

Response of the mitochondrial proteome of rat renal proximal convoluted tubules to chronic metabolic acidosis

Metabolic acidosis is a common clinical condition that is caused by a decrease in blood pH and bicarbonate concentration. Increased extraction and mitochondrial catabolism of plasma glutamine within the renal proximal convoluted tubule generates ammonium and bicarbonate ions that facilitate the excretion of acid and partially restore acid-base balance. Previous studies identified only a few mitochondrial proteins, including two key enzymes of glutamine metabolism, which are increased during chronic acidosis. A workflow was developed to characterize the mitochondrial proteome of the proximal convoluted tubule. Based upon the increase in specific activity of cytochrome c oxidase, the isolated mitochondria were enriched eightfold. Two-dimensional liquid chromatography coupled with mass spectrometry was utilized to compare mitochondrial-enriched samples from control and chronic acidotic rats. Proteomic analysis identified 901 proteins in the control and acidotic samples. Further analysis identified 37 peptides that contain an N-ε-acetyl-lysine; of these, 22 are novel sites. Spectral counting analysis revealed 33 proteins that are significantly altered in abundance in response to chronic metabolic acidosis. Western blot analysis was performed to validate the calculated changes in abundance. Thus the current study represents the first comprehensive analysis of the mitochondrial proteome of the rat renal proximal convoluted tubule and its response to metabolic acidosis.

Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH(4)(+) and HCO(3)(-) ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMV(PCT)) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na(+)-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na(+)-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMV(PCT) isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.

Glucagon-like peptide-2 (GLP-2) increases net amino acid utilization by the portal-drained viscera of ruminating calves

Glucagon-like peptide-2 (GLP-2) increases small intestinal mass and blood flow in ruminant calves, but its impact on nutrient metabolism across the portal-drained viscera (PDV) and liver is unknown. Eight Holstein calves with catheters in the carotid artery, mesenteric vein, portal vein and hepatic vein were paired by age and randomly assigned to control (0.5% bovine serum albumin in saline; n = 4) or GLP-2 (100 μg/kg BW per day bovine GLP-2 in bovine serum albumin; n = 4). Treatments were administered subcutaneously every 12 h for 10 days. Blood flow was measured on days 0 and 10 and included 3 periods: baseline (saline infusion), treatment (infusion of bovine serum albumin or 3.76 μg/kg BW per h GLP-2) and recovery (saline infusion). Arterial concentrations and net PDV, hepatic and total splanchnic fluxes of glucose, lactate, glutamate, glutamine, β-hydroxybutyrate and urea-N were measured on days 0 and 10. Arterial concentrations and net fluxes of all amino acids and glucose metabolism using continuous intravenous infusion of [U13-C]glucose were measured on day 10 only. A 1-h infusion of GLP-2 increased blood flow in the portal and hepatic veins when administered to calves not previously exposed to exogenous GLP-2, but after a 10-day administration of GLP-2 the blood flow response to the 1-h GLP-2 infusion was substantially attenuated. The 1-h GLP-2 infusion also did not appreciably alter nutrient fluxes on either day 0 or 10. In contrast, long-term GLP-2 administration reduced arterial concentrations and net PDV flux of many essential and non-essential amino acids. Despite the significant alterations in amino acid metabolism, glucose irreversible loss and utilization by PDV and non-PDV tissues were not affected by GLP-2. Fluxes of amino acids across the PDV were generally reduced by GLP-2, potentially by increased small intestinal epithelial growth and thus energy and amino acid requirements of this tissue. Increased PDV extraction of glutamine and alterations in PDV metabolism of arginine, ornithine and citrulline support the concept that GLP-2 influences intestine-specific amino acid metabolism. Alterations in amino acid metabolism but unchanged glucose metabolism suggests that the growth effects induced by GLP-2 in ruminants increase reliance on amino acids preferentially over glucose. Thus, GLP-2 increases PDV utilization of amino acids, but not glucose, concurrent with stimulated growth of the small intestinal epithelium in post-absorptive ruminant calves.

Corticosteroids increase glutamine utilization in human splanchnic bed

Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups of healthy adults received 5-h intravenous infusions of l-[1-(14)C]leucine and l-[(2)H(5)]glutamine, along with q. 20 min oral sips of tracer doses of l-[1-(13)C]glutamine in the fasting state, either 1) at baseline (control group; n = 6) or 2) after a 6-day course of 0.8 mg.kg(-1).day(-1) prednisone (prednisone group; n = 8). Leucine and glutamine appearance rates (Ra) were determined from plasma [1-(14)C]ketoisocaproate and [(2)H(5)]glutamine, respectively, and leucine and glutamine oxidation from breath (14)CO(2) and (13)CO(2), respectively. Splanchnic glutamine extraction was estimated by the fraction of orally administered [(13)C]glutamine that failed to appear into systemic blood. Prednisone treatment 1) did not affect leucine Ra or leucine oxidation; 2) increased plasma glutamine Ra, mostly owing to enhanced glutamine de novo synthesis (medians +/- interquartiles, 412 +/- 61 vs. 280 +/- 190 mumol.kg(-1).h(-1), P = 0.003); and 3) increased the fraction of orally administered glutamine undergoing extraction in the splanchnic territory (means +/- SE 64 +/- 6 vs. 42 +/- 12%, P < 0.05), without any change in the fraction of glutamine oxidized (means +/- SE, 75 +/- 4 vs. 77 +/- 4%, not significant). We conclude that high-dose glucocorticosteroids increase in splanchnic bed the glutamine requirements. The role of such changes in patients receiving chronic corticoid treatment for inflammatory diseases or suffering from severe illness remains to be determined.

Association between glutamine extraction and release of citrulline and glycine by the human small intestine

Although glutamine is an important fuel for the intestinal epithelium, the metabolic fate of glutamine extracted by the human intestine remains unclear. The aim of this study was to investigate the relationship between glutamine extraction and the release of other amino acids by the human intestine. In 21 patients undergoing major abdominal cancer surgery, differences in the plasma concentrations of 22 amino acids including glutamine across the superior or inferior mesenteric vein draining viscera were measured using a high-performance liquid chromatography. Arterial minus venous ( A − V) or venous minus arterial ( V − A) balances of the amino acids were calculated, and then the correlations between A − V differences of glutamine and V − A differences of amino acids released from the intestine were analyzed. Mean extraction rate of glutamine by the small intestine was 28.45%, approximately 3 times higher than 9.41% in the distal colon. Citrulline, proline, alanine, glycine, and arginine were released by the small intestine into the portal circulation. Positive correlations were found between glutamine uptake and the production of citrulline ( r = 0.814, P = 0.0013) and glycine ( r = 0.734, P = 0.0080). In conclusion, the synthesis of citrulline from glutamine by the small intestine is highly suspected, and the contribution of gut glutamine extraction to the release of glycine into the portal circulation is also supposed.

Glutamine metabolism: Role in acid‐base balance*

The intent of this review is to provide a broad overview of the interorgan metabolism of glutamine and to discuss in more detail its role in acid-base balance. Muscle, adipose tissue, and the lungs are the primary sites of glutamine synthesis and release. During normal acid-base balance, the small intestine and the liver are the major sites of glutamine utilization. The periportal hepatocytes catabolize glutamine and convert ammonium and bicarbonate ions to urea. In contrast, the perivenous hepatocytes are capable of synthesizing glutamine. During metabolic acidosis, the kidney becomes the major site of glutamine extraction and catabolism. This process generates ammonium ions that are excreted in the urine to facilitate the excretion of acids and bicarbonate ions that are transported to the blood to partially compensate the acidosis. The increased renal extraction of glutamine is balanced by an increased release from muscle and liver and by a decreased utilization in the intestine. During chronic acidosis, this adaptation is sustained, in part, by increased renal expression of genes that encode various transport proteins and key enzymes of glutamine metabolism. The increased levels of phosphoenolpyruvate carboxykinase result from increased transcription, while the increase in glutaminase and glutamate dehydrogenase activities result from stabilization of their respective mRNAs. Where feasible, this review draws upon data obtained from studies in humans. Studies conducted in model animals are discussed where available data from humans is either lacking or not firmly established. Because there are quantitative differences in tissue utilization and synthesis of glutamine in different mammals, the review will focus more on common principles than on quantification.

Gut glutamine metabolism at different stages of sepsis in rats.

To investigate gut glutamine metabolism and determine the effects of glutamine supplementation in different stages of sepsis in a rat model. Sepsis was induced by cecal ligation and puncture (CLP), and control rats underwent a sham operation. In the first experiment, a continuous infusion of normal saline was started at the end of the operation. Intestinal blood flow, glutamine concentrations of the abdominal aorta and superior mesenteric vein (SMV) were measured, and gut glutamine extraction and flux were calculated 5 h after the sham operation, and 5 and 20 h after CLP, being groups Ia ( n = 9), Ib ( n = 8), and Ic ( n = 8), respectively. In the second experiment, animals received a continuous infusion of alanyl-glutamine instead of normal saline and were divided into groups IIa ( n = 8), IIb ( n = 8), and IIc ( n = 6). The same parameters were measured in each group and compared with those of the corresponding group in the first experiment. In the first experiment, no significant difference in SMV blood flow was seen among the groups. The arterial glutamine concentration was increased in group Ic ( P < 0.05) compared with that in groups Ia and Ib. Gut glutamine extraction was significantly increased in group Ib ( P < 0.01) and significantly decreased in group Ic ( P < 0.05) compared with that in group Ia. In the second experiment, gut glutamine flux was significantly increased in group Ilb ( P < 0.01) compared with that in group Ib, but the increase did not reach statistical significance between groups Ia and IIa or between groups Ic and IIc. These results indicate that intestinal glutamine uptake is increased and glutamine utilization is enhanced by glutamine supplementation in early sepsis.

The hepatic extraction of plasma free amino acids and response to hepatic portal venous infusion of methionine sources in anesthetized SCWL males ( Gallus domesticus)

This study was conducted to investigate the hepatic extraction of plasma free amino acids in anesthetized Single Comb White Leghorn (SCWL) males ( Gallus domesticus). SCWL males were anesthetized and implanted with cannulae in the carotid artery, hepatic vein, hepatic portal vein and the left hepatic duct. Free amino acids in plasma and bile were determined before, during and after 30-min infusions of Saline (control), dl-Methionine ( dl-Met) or dl-2-hydroxy-4-methylthio-butanoic acid ( dl-HMB) into the hepatic portal vein. Hepatic extraction rates (HER) of amino acids were calculated based on the concentration of amino acids in plasma multiplied by estimations of blood flow in the hepatic portal vein, hepatic artery and hepatic vein. For the non-essential amino acids, alanine had the highest HER (46%). The liver also removed more than 20% of hepatic inflow of tyrosine and asparagine with substantial extraction (14–18%) of serine, glycine and glutamine, also. In contrast, less than 5% of hepatic inflow of glutamate and cystine were removed by liver. For the essential amino acids, HER for methionine, histidine and phenylalanine were 30, 14 and 17%, respectively, with less than 5% for branched-chain amino acids, lysine, arginine and threonine. Biliary secretion of amino acids represented a small percentage (<0.2%) of total hepatic extraction turnover of the amino acids. Infusion of methionine sources, dl-Met and dl-HMB, had no effect on hepatic metabolism of amino acids other than methionine. The results demonstrated for the first time, the hepatic extraction of circulating free amino acids in avian species in vivo.

Net Hepatic Gluconeogenic Amino Acid Uptake in Response to Peripheral versus Portal Amino Acid Infusion in Conscious Dogs

These studies were conducted to determine the effect of route of gluconeogenic amino acid delivery on the hepatic uptake of the amino acids. After a sampling period with no experimental intervention (basal period), conscious dogs deprived of food for 42 h received somatostatin, intraportal infusions of insulin (3-fold basal) and glucagon (basal), and a peripheral infusion of glucose to increase the hepatic glucose load 1.5-fold basal for 240 min. A mixture of alanine, glutamate, glutamine, glycine, serine and threonine was infused intraportally at 7.6 μmol · kg−1 · min−1 (PorAA group, n = 6) or peripherally at 8.1 μmol · kg−1 · min−1 (PerAA, n = 6), to match the hepatic load of gluconeogenic amino acids in PorAA. During the infusion period, there were no differences in PerAA and PorAA, respectively, with regard to arterial plasma insulin (144 ± 18 and 162 ± 18 pmol/L), glucagon (51 ± 8 and 47 ± 11 ng/L), hepatic glucose load (199.8 ± 22.2 and 210.9 ± 16.6 μmol · kg−1 · min−1), net hepatic glucose uptake (2.8 ± 2.2 and 2.2 ± 1.7 μmol · kg−1 · min−1), hepatic load of amino acids (68 ± 14 and 62 ± 7 μmol · kg−1 · min−1), or net hepatic glycogen synthesis (11.1 ± 2.2 and 8.9 ± 2.2 μmol · kg−1 · min−1). The net hepatic uptake of glutamine (2.1 ± 0.4 vs. 0.8 ± 0.3 μmol · kg−1 · min−1) and the net hepatic fractional extractions of glutamine (0.11 ± 0.02 vs. 0.05 ± 0.02) and serine (0.41 ± 0.03 vs. 0.34 ± 0.02) were greater in PorAA than in PerAA (P < 0.05). We speculate that one or more of the amino acids in the mixture causes enhancement of the net hepatic uptake and fractional extraction of glutamine, and perhaps other gluconeogenic amino acids, during intraportal amino acid delivery.

Effects of glucagon on renal and hepatic glutamine gluconeogenesis in normal postabsorptive humans.

Glutamine is an important gluconeogenic amino acid in postabsorptive humans. To assess the effect of glucagon on renal and hepatic glutamine gluconeogenesis, we infused six normal healthy postabsorptive subjects with glucagon at a rate chosen to produce circulating glucagon concentrations found during hypoglycemia and, using a combination of isotopic and net balance techniques, determined the systemic, renal, and hepatic glucose release and renal and hepatic production of glucose from glutamine. Infusion of glucagon increased systemic and hepatic glucose release (both P < .02), but had no effect on renal glucose release (P = .26). Systemic and hepatic glutamine gluconeogenesis increased from 0.45 +/- 0.3 and 0.11 +/- 0.02 micromol x kg(-1) x min(-1), respectively, to 0.61 +/- 0.04 (P = .002) and 0.31 +/- 0.03 micromol x kg(-1) x min(-1) (P = .001), respectively, whereas renal glutamine gluconeogenesis was unchanged (from 0.33 +/- 0.03 to 0.30 +/- 0.04 micromol x kg(-1) x min(-1), P = .20). The hepatic contribution to systemic glutamine gluconeogenesis increased from 25.2% +/- 6.2% to 51.6% +/- 5.5% (P = .002), while that of the kidney decreased from 74.8% +/- 6.2% to 48.4% +/- 5.5% (P = .003). Glucagon had no effect on the renal net balance, fractional extraction, or uptake and release of either glucose or glutamine. We thus conclude that glucagon stimulates glutamine gluconeogenesis in normal postabsorptive humans, predominantly due to an increase in hepatic glutamine conversion to glucose. Thus, under certain conditions such as counterregulation of hypoglycemia, the liver may be an important site of glutamine gluconeogenesis.

Glutamine enhances gut glutathione production.

The gastrointestinal tract is recognized as having important metabolic functions. This study examined gut glutathione (GSH) extraction and the effect of supplemental oral glutamine (GLN) on gut GSH fractional release. Healthy female Fisher-344 rats weighing approximately 150 to 200 g were pair-fed chow and supplemented by gavage with 1 g/kg/d GLN or an isonitrogenous amount of Freamine (McGaw, St. Louis, MO). Rats were sacrificed at 6 weeks. Arterial and portal blood was assayed for GLN and GSH content. The gut GLN and GSH extractions were calculated. The gut GLN fractional uptake was increased by approximately 50%, and there was a near threefold increase in gut GSH fractional release in the GLN-supplemented group. The discovery of gut's role as a major producer of GSH may give insight into why feeding via the gut rather than by the venous route is so important. Supplemental oral GLN further enhances GLN extraction as well as GSH fractional release in the gut.

Hepatic response to increased exogenous supply of plasma amino acids by infusion into the mesenteric vein of Holstein-Friesian cows in late gestation.

The hepatic responses of late gestation, dry dairy cows to acute (6 h) infusions of an amino acid (AA) mixture (Synthamin; 0.0, 1.1, 2.2, 4.4, 8.8 and 17.6 mumol/min) into the mesenteric vein were determined. Neither blood flow nor O2 consumption across the portal-drained viscera (PDV) and liver was significantly altered by infusion. Similarly, there were no effects on net absorption, or hepatic removal, of acetate, propionate, butyrate or NH3. Glucose PDV appearance was unchanged but hepatic glucose production increased (P = 0.032) by 0.2 mumol/min per mumol/min of AA infused. Additional extraction of alanine, glycine (both infused) and glutamine (not infused) by the liver was sufficient to account for most of the extra C required for glucose synthesis. The N that would be liberated from these glucogenic AA would also account for a large proportion of the increase in urea-N produced in response to the AA infusion. This supports the concept of a correlation between gluconeogenesis and ureagenesis. Furthermore, the amide-N liberated from the extracted glutamine would contribute up to 0.17 of hepatic NH3 flux and assist in balancing N inputs into the carbamoyl phosphate and arginosuccinate entry points of the ornithine cycle. Rates of fractional extraction of the various AA by the liver were best fitted by linear equations, indicating that even at the highest rates of administration (approximately twice maximal physiological absorption) the transport systems were not saturated. Hepatic fractional extractions of infused essential AA were highest for methionine (0.83) and phenylalanine (0.87) with the lowest proportion removed observed for valine (0.25), leucine (0.30), lysine (0.31) and isoleucine (0.49). For the non-essential AA, the highest apparent fractional extractions were for glycine (0.73), arginine (0.79) and tyrosine (0.63) followed by alanine (0.54), proline (0.47) and serine (0.37). Hepatic removal of AA-N exceeded the increase in urea-N formation such that, at the highest rate of infusion, approximately 10 mmol/min of the extracted AA was apparently available for hepatic anabolism, more than is required to account for assumed increases in liver mass and export protein synthesis. Similarly, the amount of AA available for peripheral tissue protein gain, when assessed against phenylalanine supply as the limitation, would be the equivalent of a maximum of 0.5 g protein retained/min (6 mmol AA-N/min). This would provide sufficient AA for replenishment of peripheral (muscle) protein stores plus support of the placenta and fetus.

Effects of acid-base alterations and protein depletion on hepatic nitrogen metabolism.

An alteration in acid-base balance has wide-ranging effects on many systems in the body. It has long been recognized that acid-base regulation is linked to nitrogen metabolism. Ammonium (NH4 +), which is a constituent of normal urine, is excreted in greater quantities in metabolic acidosis. Earlier work showed that the rise in NH4 + excretion in acidosis coincided with a decrease in the rate of urea excretion. When it was realized that the enzyme urease was lacking in mammalian tissues, it was concluded that urea is not a direct source of urinary NH4 +. The discovery that the extraction of glutamine by the kidney contributed significantly to urinary NH4 + focused attention on the renal metabolism of glutamine. It is now well established that the extraction of glutamine by the kidney and the renal mitochondrial deamidation of glutamine are accelerated in chronic metabolic acidosis.

Glutamine extraction by the gut is reduced in depleted [corrected] patients with gastrointestinal cancer.

Glutamine is an important fuel for the intestinal mucosa. However, glutamine pools may become depleted in the cancer-bearing host as a result of tumor consumption and diminished production due to nutritional depletion. As human data are lacking, the authors investigated glutamine extraction by different sites of the human intestine, including tumor and the potential relation with the degree of nutritional depletion. Thirty-two patients with gastrointestinal malignancies were studied. Blood from an artery and veins draining jejunum, ileum, colon, or tumor were sampled. Depletion was estimated by the percentage ideal body weight. Fractional glutamine extraction rate in the jejunum was 24%, three times higher than in ileum and colon. Percentage ideal body weight correlated with arterial glutamine levels (r = 0.5275, p = 0.003). In addition, arterial glutamine concentrations were correlated with extraction in the ileum (r = -0.8411, p < 0.001). Colon-containing tumor did not extract more glutamine than did nontumor-containing colon. Glutamine is a quantitatively more important substrate for the proximal intestine than for the distal gut. Nutritional depletion results in decreased arterial glutamine concentration, which in turn results in diminished extraction. Colon cancer does not function as a glutamine trap and does not contribute to glutamine depletion.

Enteral Glutamine Modulates Renal Glutamine Utilization

Enteral glutamine feeding effect on renal glutamine utilization was assessed from the perspective of gamma glutamyltransferase activity-dependent cellular glutamate modulation of phosphate-dependent glutaminase. After 4 d, rats fed an elemental diet supplemented with glutamine exhibited a 21% higher kidney glutamate content and 27% reduction in ammonium excretion, both P < 0.05. Glutamine removal from plasma was depressed 62% in the glutamine-fed group (324 ± 155 vs. 780 ± 154 nmol·min-1·100 g body weight-1, P < 0.05) despite an elevated arterial plasma glutamine load delivered to the kidney. Administration of acivicin, 36 mg/kg body weight, to glutamine-fed rats inhibited gamma glutamyltransferase > 90% and decreased kidney glutamate content 42%. This reduction in kidney glutamate was associated with a 3.3-fold enhancement in both glutamine extraction (474 ± 184 to 1548 ± 255 nmol·min-1·100 g body weight-1) and ammonium excretion (295 ± 30 to 978 ± 96 nmol·min-1·100 g body weight-1), both P < 0.01. These findings are consistent with enteral glutamine regulation of renal glutamine utilization through an elevation of the cellular glutamate level.

Effects of methionine sulphoximine treatment on renal amino acid and ammonia metabolism in rats.

Renal glutamine metabolism in relation to ammoniagenesis has been extensively studied during chronic metabolic acidosis, when arterial glutamine levels are reduced. However, little is known about the effects of reduced glutamine delivery on renal glutamine and ammonia metabolism at physiological systemic pH values. Therefore, a model of decreased arterial glutamine concentrations at normal pH values was developed using methionine sulphoximine (MSO). Renal glutamine and ammonia metabolism was measured by determining fluxes and intracellular concentrations after an overnight fast in ether anaesthetized normal rats, MSO-treated rats and their pair-fed controls. Moreover, fluxes and intracellular concentrations of several other amino acids were determined concomitantly. After 2 and 4 days of MSO treatment, arterial glutamine concentrations were reduced to 55%, while arterial ammonia concentrations increased by 70%. Kidney glutamine uptake reduced, but systemic pH was unchanged. Fractional extraction of glutamine remained unchanged, suggesting that also in vivo net uptake of glutamine by the kidney at subnormal levels is related to arterial glutamine concentrations. As a result, at day 2 but not at day 4, the kidney reduced the net release of ammonia into the renal vein and thus reduced net renal ammonia addition to body ammonia pools. Therefore at day 2, the kidney seems to play an important role in adaptation to both hyperammonaemia and hypoglutaminaemia.