Glutamine Depletion Research Articles

Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease

Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease

Glutamine Increases Autophagy Under Basal and Stressed Conditions in Intestinal Epithelial Cells

Glutamine plays a protective role in intestinal cells during physiologic stress; however, the protection mechanisms are not fully understood. Autophagy functions in bulk degradation of cellular components, but has been recognized recently as an important mechanism for cell survival under conditions of stress. We therefore sought to see if glutamine's actions involve the induction of autophagy in intestinal cells and, if so, the mechanisms that underlie this action. Formation of microtubule-associated protein light chain 3 (LC3)-phospholipid conjugates (LC3-II) in rat intestinal epithelial IEC-18 cells and human colonic epithelial Caco-2(BBE) cells was determined by Western blotting and localized by confocal microscopy. Activation of mammalian target of rapamycin (mTOR) pathway, mitogen-activated protein (MAP) kinases, caspase-3, and poly (ADP-ribose) polymerase were monitored by Western blotting. Glutamine increased LC3-II as well as the number of autophagosomes. Glutamine-induced LC3-II formation was paralleled by inactivation of mTOR and p38 MAP kinase pathways, and inhibition of mTOR and p38 MAP kinase allowed LC3-II induction in glutamine-deprived cells. Under glutamine starvation, LC3-II recovery after heat stress or the increase under oxidative stress was blunted significantly. Glutamine depletion increased caspase-3 and poly (ADP-ribose) polymerase activity after heat stress, which was inhibited by treatment with inhibitors of mTOR and p38 MAP kinase. Glutamine induces autophagy under basal and stressed conditions, and prevents apoptosis under heat stress through its regulation of the mTOR and p38 MAP kinase pathways. We propose that glutamine contributes to cell survival during physiologic stress by induction of autophagy.

Targeting Anaplerotic Pathways That Support Fatty Acid Metabolism as a Therapeutic Strategy for Hematological Malignancies: The Achilles' Heel of the Warburg Effect.

More than half a century ago, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect, i.e. the inhibition of anaerobic fermentation by oxygen. However, permanent and transmissible defects in the respiratory capacity of cancer cells that could broadly support Warburg's hypothesis have not been identified. Notably, we have recently demonstrated that mitochondrial uncoupling – the abrogation of ATP synthesis in response to mitochondrial membrane potential – can promote the Warburg effect in leukemia cells, and may contribute to chemoresistance, via in part, the expression of the highly conserved thermogenic protein UCP2. Here we demonstrate that mitochondrial uncoupling in leukemia cells is supported by the oxidation of fatty acids, and provide evidence that etomoxir (EX) or ranolazine (RAN), pharmacological inhibitors of fatty acid oxidation utilized for the treatment of heart failure, sensitize leukemia cell lines and primary samples to apoptosis induced by the BH3 mimetic ABT-737 and the MDM-2 inhibitor Nutlin 3a. EX and RAN, but not 2-deoxyglucose (2DG), markedly inhibited oxygen consumption in leukemia cell lines and primary samples. In contrast, 2DG, but not EX or RAN, potently depleted ATP levels, suggesting that the oxidation of fatty acids is uncoupled from ATP synthesis – and conversely, the synthesis of ATP primarily depends on the non-oxidative, glycolytic metabolism of glucose. It is noteworthy that albeit EX and RAN inhibited the growth of p53-wild type and -mutant leukemia cells, neither agent induced marked apoptosis. Nonetheless, a pronounced induction of the proapoptotic BH3-only proteins Noxa and Bim was observed regardless of p53 status, suggesting a potential mechanism by which these agents enhance apoptosis by ABT-737. In addition, EX and RAN abrogated the chemoprotective effects of bone marrow-derived stromal feeder layers, and EX provided a survival advantage in combination with ABT-737 in a murine model of leukemia suggesting that inhibition of mitochondrial fatty acid oxidation represents a novel therapeutic strategy for the treatment of leukemia. Intriguingly, C13-NMR analysis, H3–oleate oxidation, and oxymetry experiments revealed that leukemia cells do not oxidize exogenous fatty acids, but rather depend on glucose and glutamine-supported de novo synthesis of fatty acids to maintain mitochondrial function. Accordingly, depletion of glutamine, inhibition of fatty acid synthesis, or reduced pentose phosphate shunt-derived NADPH significantly decreased oxygen consumption and potentiated ABT-737 induced apoptosis. The above results support the hypothesis that glutamine and glucose-dependent anaplerotic reactions sustain fatty acid metabolism and survival of leukemia cells. Our results suggest that the dependence of cancer cells on glycolysis for energy generation indicates a metabolic shift to the ATP-uncoupled oxidation of non-glucose substrates, and most importantly, support the clinical investigation of fatty acid oxidation and synthesis inhibitors as a therapeutic strategy in hematological malignancies.

Glutamine reduces the expression of leukocyte integrins leukocyte function–associated antigen-1 and macrophage antigen-1 in mice exposed to arsenic

Chronic arsenic exposure results in an increased oxidative stress and inflammation in the body. Glutamine (GLN) is an amino acid considered to have immunomodulatory effects and attenuate the inflammatory reaction. This study was designed to examine the effect of GLN supplementation on inflammatory-related leukocyte integrin expression and in vitro splenocyte cytokine production in mice exposed to arsenic. Mice were assigned to the control and experimental groups. The control group drank deionized water, whereas the experimental group drank deionized water containing 50 ppm of sodium arsenite. Each control and experimental group was further divided into 2 subgroups and fed diets for 5 weeks. One subgroup was fed a semipurified diet, whereas the other subgroup was fed a diet where part of the casein was replaced with GLN, which provided 25% of the total amino acid nitrogen. The results showed that plasma GLN levels of mice in the arsenic group were significantly lower than those in the control groups. Glutamine supplementation reversed the depletion of plasma GLN in the arsenic group. beta(2) intergins, including leukocyte function-associated antigen-1 and macrophage antigen-1 expressed by leukocytes, were significantly higher in the arsenic group than the control groups. Glutamine supplementation reduced leukocyte integrin expression in mice exposed to arsenic. There were no differences in interleukin 4, interleukin 6, interferon gamma, and tumor necrosis factor alpha production between the 2 arsenic groups when splenocytes were stimulated with mitogen. These results suggest that arsenic exposure results in depletion of plasma GLN and higher leukocyte integrin expression. Glutamine supplementation normalized the plasma GLN levels and reduced leukocyte leukocyte function-associated antigen-1 and macrophage antigen-1 expression. However, cytokine modulation may not be responsible for reducing leukocyte integrin expression in mice exposed to arsenic.

Treating hyperglycemia improves skeletal muscle protein metabolism in cancer patients after major surgery

Cancer and surgical stress interact to aggravate insulin resistance, protein catabolism, and glutamine depletion in skeletal muscle. We compared the effects of insulin-mediated euglycemia and moderate hyperglycemia on kinetics of protein and selected amino acids in skeletal muscle of female cancer patients after major surgery. In each patient, a 24-hr period of insulin-mediated tight euglycemia (mean blood glucose, 5.8 +/- 0.4 mmol/L) preceded or followed a 24-hr control period of moderate hyperglycemia (mean blood glucose, 9.6 +/- 0.6 mmol/L) on the first and second day after surgery, in randomized order, according to a crossover experimental design. Intensive care unit, cancer hospital. Cancer patients after abdominal radical surgery combined with intraoperative radiation therapy. Intensive (57 +/- 11 units/24 hrs) and conventional (25 +/- 5 units/24 hrs) insulin treatment during total parenteral nutrition. Muscle metabolism was assessed at the end of each 24-hr period of euglycemia and of hyperglycemia by leg arteriovenous catheterization with stable isotopic tracers. We found that euglycemia as compared with hyperglycemia was associated with higher (p < .05) fractional glucose uptake (16% +/- 4% vs. 9% +/- 3%); higher (p < .05) muscle protein synthesis and neutral net protein balance (-3 +/- 3 vs. -11 +/- 3 nmol phenylalanine x 100 mL(-1) x min(-1), respectively); lower (-52% +/- 12%, p < .01) muscle nonprotein leucine disposal (an index of leucine oxidation) and higher (p < .05) plasma leucine concentrations; and higher (3.6 +/- 1.7 times, p < .01) net de novo muscle glutamine synthesis and plasma glutamine concentrations (p < .05). Euglycemia was associated with higher (23% +/- 7%, p < .05) plasma concentrations of arginine but did not affect either arginine release from muscle or plasma concentration and muscle flux of asymmetrical dimethylarginine. Rate of muscle proteolysis correlated (p < .05) with muscle release of asymmetrical dimethylarginine. Treating hyperglycemia improves skeletal muscle protein and amino acid metabolism in cancer patients after major surgery.

Treatment of hyperglycemia for skeletal muscle metabolism in cancer patients after major surgery

20578 Background: Cancer and surgical stress interact to aggravate insulin resistance, protein catabolism and glutamine depletion in skeletal muscle. In the clinical setting, hyperglycemia and glutamine depletion were associated with poor outcome in critically ill patients. The present study was designed to explore the effects of insulin mediated euglycemia and moderate hyperglycemia on kinetics of protein and selected amino acids in skeletal muscle of cancer patients after major surgery. Methods: Adult cancer patients were studied after abdominal radical surgery. In each patient a 24-h period of insulin-mediated tight euglycemia (mean blood glucose: 5.8± 0.4 mmol/l) preceded or followed a 24-h period of moderate hyperglycemia (mean blood glucose: 9.6± 0.6 mmol/l) on the first and the second day after surgery (cross-over design with randomized sequence of treatments). Intensive (57±11 U/24-h) or conventional (25±5 U/24-h) insulin treatments were used to obtain euglycemia or moderate hyperglycemia during total parenteral nutrition. Muscle metabolism was assessed at the end of each 24-h period of tight glycemic control and of hyperglycemia by leg arteriovenous catheterization with stable isotopic tracers. Results in the two different experimental conditions were compared using the Wilcoxon Matched-Pairs Signed-Ranks Test. P values < 0.05 were taken as significantly different. Results: Plasma insulin levels were not significantly different at the end of intensive or conventional insulin treatment period. Strict glycemic control as compared to hyperglycemia was associated with higher (p<0.05) fractional glucose uptake (16±4 vs 9±3 percent); higher (p<0.05) muscle protein synthesis and neutral net protein balance (-3±3 vs - 11±3 mmol PHE · 100cc-1 · min-1, respectively); higher (3.6±1.7 times, p<0.01) net de novo muscle glutamine production. Conclusions: This study shows that, in cancer patients after major surgery, hyperglycemia with relative insulin deficiency promotes muscle wasting and blunted the ability of muscle to provide glutamine to extra-muscular tissues. This latter effect could contribute to increased morbidity and mortality in surgical cancer patients with poor metabolic control. No significant financial relationships to disclose.

Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease

Erythrocyte glutathione depletion has been linked to hemolysis and oxidative stress. Glutamine plays an additional antioxidant role through preservation of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels, required for glutathione recycling. Decreased nitric oxide (NO) bioavailability, which occurs in the setting of increased hemolysis and oxidative stress, contributes to the pathogenesis of pulmonary hypertension (PH) in sickle cell disease (SCD). We hypothesized that altered glutathione and glutamine metabolism play a role in this process. Total glutathione (and its precursors) and glutamine were assayed in plasma and erythrocytes of 40 SCD patients and 9 healthy volunteers. Erythrocyte total glutathione and glutamine levels were significantly lower in SCD patients than in healthy volunteers. Glutamine depletion was independently associated with PH, defined as a tricuspid regurgitant jet velocity (TRV) of at least 2.5 m/s. The ratio of erythrocyte glutamine:glutamate correlated inversely to TRV (r = -0.62, P < .001), plasma arginase concentration (r = -0.45, P = .002), and plasma-free hemoglobin level (r = -0.41, P = .01), linking erythrocyte glutamine depletion to dysregulation of the arginine-NO pathway and increased hemolytic rate. Decreased erythrocyte glutathione and glutamine levels contribute to alterations in the erythrocyte redox environment, which may compromise erythrocyte integrity, contribute to hemolysis, and play a role in the pathogenesis of PH of SCD.

Effect of Glutamine on Gut Glutathione Fractional Release in the Implanted Tumor Model

Cancer and its treatments cause a marked depletion of glutamine (GLN). However, dietary GLN can restore this loss and improve the outcomes of the treatments. The reasons behind this need to be investigated. GLN is suggested to involve in glutathione (GSH) synthesis. Fast-growing tumors alter gut GLN metabolism, but the effect of tumor growth on gut GSH release remains unknown. We hypothesized that gut GSH release would decrease in the tumor-bearing host and this downregulation would be antagonized by supplemental GLN. Female Fisher-344 rats were randomized to the groups: GLN + TUMOR, Freamine (FA) + TUMOR, GLN + SHAM, and FA + SHAM. The rats were implanted with MTF-7 mammary tumors as tumor-bearing groups, whereas the rats were sham operated as control groups. The rats were pair fed chow, gavaged with 1 g/kg/day GLN or an isonitrogenous FA. Tumor growth, blood and gut mucosa GLN, glutamate, and/or GSH were measured. The gut extractions, defined as the difference of concentrations across the gut, were calculated. Supplemental GLN enhanced the gut GLN uptake and GSH release with tumor growth and significantly increased blood and gut mucosa GLN and/or GSH concentrations. Our results demonstrate the important antioxidant role of GLN and thus may have significant implications in nutritional immune modulation in cancer patients.

Manipulation of citrulline availability in humans

To determine whether circulating citrulline can be manipulated in vivo in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate (0.36 g.kg(-1).day(-1)), a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g.kg(-1).day(-1)), in randomized order. Plasma, erythrocyte (RBC), and urinary citrulline concentrations were determined by gas chromatography-mass spectrometry at 3-h intervals between 1100 and 2000 on each study day. Regardless of treatment, RBC citrulline was lower than plasma citrulline, with an RBC-to-plasma ratio of 0.60 +/- 0.04, and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an approximately 7% drop in plasma glutamine (P = 0.013), and 18 +/- 14% (P < 0.0001) and 19 +/- 17% (P < 0.01) declines in plasma and urine citrulline, respectively, with no alteration in RBC citrulline. Oral L-citrulline administration was associated with 1) a rise in plasma, urine, and RBC citrulline (39 +/- 4 vs. 225 +/- 44 micromol/l, 0.9 +/- 0.3 vs. 6.2 +/- 3.8 micromol/mmol creatinine, and 23 +/- 1 vs. 52 +/- 9 micromol/l, respectively); and 2) a doubling in plasma arginine level, without altering blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, because citrulline is bioavailable and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.

Exogenous glutamine—Compensating a shortage?

There is still insufficient knowledge about in vivo glutamine metabolism and the regulation of glutamine homeostasis, particularly during metabolic stress. A shortage of glutamine is associated with a poor outcome, whereas for septic patients in the intensive care unit an increased availability of glutamine can prevent mortality and morbidity. Cellular defense mechanisms depend on normal glutamine availability to respond adequately to challenges presented. In clinical practice, treatment of plasma glutamine depletion improves outcome for the critically ill patient. An increased metabolic need for glutamine must be met with an increased consumption of glutamine. Ordinary food is not a sufficient supply of glutamine for the patient with multiple organ failure in the intensive care unit, but that is also true for several other nutrients. It is, therefore, debatable whether an exogenous supply of glutamine should be regarded as a pharmacologic treatment or whether this just represents physiology in stressed states. If a glutamine shortage requires substitution, supplementation to the normal concentration is compensation of a shortage, and the effect is physiological.

Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells

The idea that conversion of glucose to ATP is an attractive target for cancer therapy has been supported in part by the observation that glucose deprivation induces apoptosis in rodent cells transduced with the proto-oncogene MYC, but not in the parental line. Here, we found that depletion of glucose killed normal human cells irrespective of induced MYC activity and by a mechanism different from apoptosis. However, depletion of glutamine, another major nutrient consumed by cancer cells, induced apoptosis depending on MYC activity. This apoptosis was preceded by depletion of the Krebs cycle intermediates, was prevented by two Krebs cycle substrates, but was unrelated to ATP synthesis or several other reported consequences of glutamine starvation. Our results suggest that the fate of normal human cells should be considered in evaluating nutrient deprivation as a strategy for cancer therapy, and that understanding how glutamine metabolism is linked to cell viability might provide new approaches for treatment of cancer.

Acute depletion of plasma glutamine increases leucine oxidation in prednisone-treated humans

To determine whether depletion in plasma glutamine worsens the catabolic response to corticosteroids, seven healthy volunteers received oral prednisone for 6 days on two separate occasions, at least 2 weeks apart, and in random order. On the sixth day of each treatment course, they received 5 h intravenous infusions of L-[1-(14)C]-leucine and L-[1-(13)C]-glutamine in the postabsorptive state (1) under baseline conditions (prednisone only day) and (2) after 24h of treatment with phenylbutyrate (prednisone+phenylbutyrate day), a glutamine chelating agent. Phenylbutyrate treatment was associated with (1) an approximately 15% decline in plasma glutamine concentration (627+/-39 vs. 530+/-31 micromol l(-1); P<0.05), (2) no change in leucine appearance rate, an index of protein breakdown (124+/-9 vs. 128+/-9 micromol kg(-1) h(-1); NS) nor in non-oxidative leucine disposal, an index of whole body protein synthesis (94+/-9 vs. 91+/-7 micromol kg(-1) h(-1); NS), and (3) a approximately 25% rise in leucine oxidation (30+/-1 vs. 38+/-2 micromol kg(-1) h(-1), P<0.05), despite an approximately 25% decline (P<0.05) in leucine concentration. In a model of mild, stress-induced protein catabolism, depletion of plasma glutamine per se may worsen branched chain amino acid and protein wasting.

Selective protection of nuclear thioredoxin-1 and glutathione redox systems against oxidation during glucose and glutamine deficiency in human colonic epithelial cells

Little is known about the relative sensitivities of antioxidant systems in nuclei, mitochondria, and cytoplasm. The present study examined the oxidation of the thiol-dependent antioxidant systems in these subcellular compartments under conditions of limited energy supply of human colonic epithelial HT-29 cells induced by depletion of glucose (Glc) and glutamine (Gln) from the culture medium. Increased oxidation of dichlorofluoroscein (DCF) indicated an increased level of reactive oxygen species (ROS). Redox Western blot analysis showed oxidation of cytosolic thioredoxin-1 (Trx1) and mitochondrial thioredoxin-2 (Trx2) by 24 h, but little oxidation of nuclear Trx1. The Trx1 substrate, redox factor-1 (Ref-1), was also oxidized in cytosol but was reduced in nuclei. Protein S-glutathionylation (PrSSG), expressed as a ratio of protein thiol (PrSH), was also increased in the cytosol, while nuclear PrSSG/PrSH was not. Taken together, the data show that oxidative stress induced by depletion of Glc and Gln affects the redox states of proteins in the cytoplasm and mitochondria more than those in the nucleus. These results indicate that the nuclear compartment has better protection against oxidative stress than cytoplasm or mitochondria. These results further suggest that energy and/or substrate supply may contribute to sensitivity of mitochondrial and cytoplasmic systems to oxidative damage.

Glutamine Supplementation in Critical Illness: Evidence, Recommendations, and Implications for Clinical Practice in Burn Care

Glutamine is considered a conditionally essential amino acid in metabolic stress. Depletion of plasma and muscle glutamine is observed in acute burn injury and contributes to muscle wasting, weight loss, and infection. In critical illness, supplementation has been shown in patients to minimize these effects and reduce the rate of mortality and length of stay. The evidence for glutamine use and its implications for burn care practice are considered here. Work published to February 2006, which investigated enteral and parenteral glutamine supplementation in burns and critical care, is reviewed. Randomized controlled trials in burns, systematic reviews, and nutrition support practice guidelines are considered. Randomized controlled trials in burns suggest significant clinical benefit in terms of morbidity, mortality, and length of stay but are limited by sample size. Parenteral glutamine studies are under-represented. Systematic reviews and practice guidelines generally support glutamine supplementation in critical illness but vary in the level of recommendations for its use in burns. There also are features unique to burn injury that require consideration. Patients with severe burns or inhalation injury may have a prolonged critical illness phase. In large burns, inflammation and hypermetabolism may persist well beyond 4 weeks of injury. The justification and safety of long-term glutamine supplementation is yet to be established. The outlook for glutamine therapy in burns is promising. However, to strengthen recommendations for routine therapy in burns, further research focusing on larger-scale enteral glutamine studies, parenteral glutamine supplementation, and long-term use of the substrate is necessary.

158 POSTER Targeting bladder tumor cells in vivo and in the urine by a peptide identified using phage display

A number of clinical trials have shown that KRAS mutations of colorectal cancer (CRC) can predict a lack of responses to anti-epidermal growth factor receptor–based therapy. Recently, there have been several studies to elucidate metabolism reprogramming in cancer. However, it remains to be investigated how mutated KRAS can coordinate the metabolic shift to sustain CRC tumor growth. In this study, we found that KRAS mutation in CRC caused alteration in amino acid metabolism. KRAS mutation causes a marked decrease in aspartate level and an increase in asparagine level in CRC. Using several human CRC cell lines and clinical specimens of primary CRC, we demonstrated that the expression of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate, was upregulated by mutated KRAS and that ASNS expression was induced by KRAS-activated signaling pathway, in particular PI3K-AKT-mTOR pathway. Importantly, we demonstrated that KRAS-mutant CRC cells could become adaptive to glutamine depletion through asparagine biosynthesis by ASNS and that asparagine addition could rescue the inhibited growth and viability of cells grown under the glutamine-free condition in vitro. Notably, a pronounced growth suppression of KRAS-mutant CRC was observed upon ASNS knockdown in vivo. Furthermore, combination of L-asparaginase plus rapamycin markedly suppressed the growth of KRAS-mutant CRC xenografts in vivo, whereas either L-asparaginase or rapamycin alone was not effective. These results indicate ASNS might be a novel therapeutic target against CRCs with mutated KRAS.

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

The Effect of Glutamine‐Enriched Enteral Nutrition on Intestinal Permeability in Very‐Low‐Birth‐Weight Infants: A Randomized Controlled Trial

Very-low-birth-weight (VLBW) infants are susceptible to glutamine depletion. Glutamine depletion has negative effects on intestinal integrity. The lower infection rate in VLBW infants receiving glutamine-enriched enteral nutrition may originate from improved intestinal integrity, as reflected by decreased intestinal permeability. The aim of our study was to investigate whether glutamine-enriched enteral nutrition in VLBW infants enhances the normal decrease in intestinal permeability, as measured by the sugar absorption test (SAT). In a double-blind, randomized, placebo-controlled trial, VLBW infants (gestational age <32 weeks or birth weight <1,500 g) received enteral glutamine supplementation (0.3 g/kg/d) or an isonitrogenous placebo supplementation (alanine) between days 3 and 30 of life. Intestinal permeability, determined from the urinary lactulose/mannitol (L/M) ratio after an oral dose of lactulose and mannitol, was assessed at 4 time points: before the start of the study, and at days 7, 14, and 30 of life. At least 2 SATs were performed in 45/52 (86%) and 45/50 (90%) infants in the glutamine-supplemented and control groups, respectively. Baseline patient and nutrition characteristics were not different between the groups. There was no effect of glutamine-enriched enteral nutrition on the decrease of the L/M ratio between the start and end of the study (p = .78). In both treatment groups, median urinary lactulose concentrations decreased (p < .001), whereas median urinary mannitol concentrations increased (p = .003). Glutamine-enriched enteral nutrition does not enhance the postnatal decrease in intestinal permeability in VLBW infants. Any beneficial effect of glutamine may involve other aspects of intestinal integrity; for example, modulation of the intestinal inflammatory response.

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.

Arginine-enriched diet limits plasma and muscle glutamine depletion in head-injured rats

Objective Injury is associated with a depletion in glutamine (GLN) pools, which may contribute to impairment of immune and nutritional statuses. Total parenteral nutrition enriched with arginine (ARG) is able to generate GLN in surgical patients. We hypothesized that this same concept may be applicable to enteral administration and could be extended to muscle GLN reserves. This study investigated the ability of an enteral formula enriched with ARG to restore the GLN pools in an experimental model of head injury. Methods Twenty-five male Sprague-Dawley rats were randomized into 4 groups: ad libitum access to food, head injury plus free access to nutrition, head injury plus standard enteral nutrition (Sondalis), and an immune-enhancing diet (IED). The two enteral diets were adjusted to be isocaloric (290 kcal · kg −1 · d −1) and isonitrogenous (3.29 g · kg −1 · d −1) and were delivered for 4 d (24 h/24 h). After sacrifice, plasma and muscle amino acids were determined. Results Head injury was associated with a large depletion of muscle and plasma GLN pools that were restored by IED administration but not by the standard diet. Moreover, the IED but not the standard diet improved or normalized ornithine and glutamate pools, suggesting that the modification of GLN pools is related to ARG administration. Conclusion In our model of head injury, our IED, a diet without free GLN, is efficient in restoring the plasma and muscle pools of GLN, probably due to its high ARG content.

Pharmacokinetics and pharmacodynamics of asparaginases in antibody-negative pediatric patients with higher risk acute lymphoblastic leukemia (ALL): A report from CCG-1961

9027 Background: Asparaginase (ASNase) induces apoptosis in ALL lymphoblasts by depleting cells of extracellular asparagine (ASN) and possibly glutamine (GLN). On CCG-1961 [Seibel NL et al. Blood 2003; 102(11): 224a (abstract #787)], all patients (pts) received 9 doses of native E.coli-ASNase in induction (IND). Pts with rapid early response (marrow blasts &lt; 25% on Day 7, RER) were randomized to standard intensity therapy and received native ASNase thereafter or to stronger intensity therapy and received PEG-ASNase post induction. All pts with slow early response (marrow blasts ≥ 25% on Day 7, SER) received PEG-ASNase after induction. Pts with clinical allergy to E.coli-ASNase were switched to Erwinase. Methods: Anti-ASNase Antibody (Ab) titers were assayed at the end of IND, prior to Delayed Intensification #1 and prior to starting Maintenance. Amino acid levels were measured in 430 sera from 187 patients with measurable ASNase activity. Results: Population pharmacokinetics (PK) and pharmacodynamics (PD) of the ASNase formulations demonstrated half-lives of native ASNase (6000 IU/m2): 30 hours, PEG-ASNase (2500 IU/m2): 156 hours, and Erwinase (6000 IU/m2): 18 hours. Subsequent exposures to either formulation resulted in increased activity with moderate prolongation of the elimination half-lives. One-hundred forty-two sera from 70 patients with ASNase activity between 0.03 and 1.11 IU/ml, had a mean ± SDEV ASNase activity, ASN levels, and GLN levels of 0.21 ± 0.23 IU/ml, 14.6 ± 17.7 μM, and 300.4 ± 233.8 μM, respectively; with median values of 0.11 IU/ml (range: 0.03–1.114 IU/ml), 4.7 μM (range: 0.38 - 81.34 μΜ), and 270.2 μM (range: 5.3 - 1189.1 μM), respectively. TTEST analyses linked higher ASNase activity with lower ASN levels and greater % ASN deamination. Higher ASNase activity did not assure greater GLN depletion. Conclusions: A sigmoid relationship between ASNase levels and % ASN deamination showed that activity of 0.41 ± 0.24 IU/ml (mean ± SDEV) provided 93% ± 13% ASN deamination. Therefore, &gt;90% ASN deamination required ASNase &gt;0.4 IU/ml. Kaplan-Meier analyses are pending. [Table: see text]