Plasma Gln Levels Research Articles

Overcoming Venetoclax (Ven) Resistance through Glutamine (Gln) Depletion: Final Analysis of the Phase 1 Trial of Ven and Pegcrisantaspase (PegC) Combination in Relapsed and Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Ven-based therapies have been an advancement in the front-line treatment of AML; however development of resistance is common and the efficacy of Ven-containing regimens in R/R AML has been far less promising. We reported in vitro and in vivo depletion of Gln induced by long-acting Erwinia asparaginase, PegC, inhibited proliferation of complex karyotype (CK) AML and synergistically enhanced the antiapoptotic activity of Ven-mediated antagonism of Bcl-2 by decreasing the expression of proteins such as Mcl-1, whose translation is cap-dependent. Here we report the final results of our phase 1 clinical trial (NCT04666649) of VenPegC for treatment of R/R AML including patients (pts) previously treated with Ven. Methods: Pts received Ven 400 mg on D1-28 and PegC (500, 750, 1000 IU/m 2 in cohorts 1-3, IV) D1 & D15 every 4 weeks (wk). Primary study endpoints were incidences of regimen limiting toxicities (RLT) and RP2D. Secondary endpoints included rate of composite complete remissions. Correlative studies included monitoring plasma amino acid levels and asparaginase activity, and expression of proteins regulating cap-dependent mRNA translation in bone marrow (BM) mononuclear cells pre- and post-VenPegC treatment. Results: As of July 31, 2023, 26 pts were enrolled of whom 24 received at least 1 dose of PegC and 17 completed at least 1 cycle of PegC (2 doses). The median age was 61 (range 24-79), 16 (62%) were female; 9 (35%) had CK-AML and 5 (19%) had TP53 mutations; 18 (69%) were previously treated with Ven. Median prior lines of treatment were 2 (range 1-5). No RLT was observed in cohort 1 (n=3). Cohort 2 was expanded to 6 pts due to transient asymptomatic hypertriglyceridemia. Two pts in cohort 3 (n=3) developed grade 3 hyperbilirubinemia determined as RLT. Expansion cohort included 11 pts who were treated at MTD/RP2D (PegC 750 IU/m 2 plus Ven 400 mg) given as out-pt without a need for hospitalization. No pancreatitis, thrombohemorrhagic events, or silent inactivation were observed in the entire study population. Overall response rate (ORR) for pts who received ≥1 cycle of PegC was 47% (8/17), Fig 1. CR+ CRh+CRi rate was 29% (5/17); 1 pt had a partial remission with full count recovery and is currently receiving cycle 6; 2 pts had stable disease. Four pts showed hematologic improvement. Twelve (71%) pts who received ≥1 cycle of PegC were previously treated with Ven and were Ven-refractory. ORR in Ven-refractory pts was 42% (5/12); of the 5 pts who harbored a RAS or PTPN11 mutation and previously treated with Ven, 60% responded. Remarkably, all pts (3/3) who had a RUNX1 mutation achieved a CR; Pt 6 (relapsed AML with NRAS/cKIT/RUNX1 mutations) achieved an MRD-negative CRh and underwent an allogeneic stem cell transplant (alloHSCT); Pt 12 (primary refractory AML with TP53/NRAS/RUNX1 mutations) achieved a clearance of BM blasts after 1 dose of PegC and achieved MRD-negative CR at the end of cycle 1, and Pt 15 (post-alloHSCT relapsed AML with NRAS/PTPN11/CSF3R/RUNX1 mutations) achieved a CRi and received 10 cycles of VenPegC. Pt 31 (post-alloHSCT relapsed AML with FLT3-ITD) who had persistent disease after gilteritinib achieved an MRD-negative CRi, pending 2 nd alloHSCT. Nadir plasma asparaginase activity of >0.1 IU/mL was achieved in cycle 1 for all pts who received ≥1 cycle of VenPegC. All pts with available results achieved undetectable asparagine (Asn) level by day 7 and Asn remained undetectable throughout the study. Plasma Gln reduction from baseline occurred in all pts (except 1) within cycle 1. Pts in cohort 1 reached nadir Gln after initial PegC exposure in an average of 22 days compared to 16 days in cohort 2. In general, pts who achieved CR had the lowest absolute plasma Gln levels. Available Glu and Gln are shown in Fig.1 for pts who achieved a CR/PR. Out of the 8 assessed pts (4 responders & 4 non-responders), VenPegC inhibited cap-dependent translation after 1 cycle in responders, as evidenced by decreased phosphorylated eIF4E (active, pheIF4E) in 4/4 pts and decreased phosphorylated 4EBP1 (inactive, ph4EBP1), a negative regulator of eIF4E, in 2/4 pts. Conversely, pheIF4E and ph4EBP1 expression was increased in 3/4 and 4/4 of the non-responders, respectively (Fig.2). Conclusion: VenPegC is a novel regimen that can induce complete remission in some heavily pretreated R/R AML patients, even with previous exposure to Ven. Pts with RUNX1 mutation, whose translation depends on pheIF4E, may benefit further from this regimen.

Glutamine and leucine administration attenuates muscle atrophy in sepsis

AimsThis study investigated whether l-glutamine (Gln) and/or l-leucine (Leu) administration could attenuate muscle atrophy in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. Materials and methodsSeptic mice were given a daily intraperitoneal injection of Gln, Leu, or Gln plus Leu, and mice were sacrificed on either day 1 or 4 after CLP. Blood and muscles were collected for analysis of amino acid contents and markers related to protein degradation, muscle regeneration, and protein synthesis. Key findingsLeu treatment alone increased both muscle mass and total muscle protein content on day 4 after CLP. Gln administration reduced muscular Gln contents on day 1 and enhanced plasma Gln levels on day 4. Higher plasma branched-chain amino acid (BCAA) abundances and lower muscular BCAA levels were observed in Leu-treated mice on day 4. Gln and Leu individually suppressed muscle expressions of the E3 ubiquitin ligase genes, Trim63 and Fbxo32, on day 4 after CLP. As to muscle expressions of myogenic genes, both Gln and Leu upregulated Myog expression on day 1, but Leu alone enhanced Myf5 gene expression, whereas Gln plus Leu increased MyoD and Myog expression levels on day 4. Akt/mammalian target of rapamycin (mTOR) signaling was only activated by Gln and Leu when individually administered. SignificanceGln and/or Leu administration reduces sepsis-induced muscle degradation and promotes myogenic gene expressions. Leu treatment alone had more-pronounced effects on maintaining muscle mass during sepsis. A combination of Gln and Leu failed to show synergistic effects on alleviating sepsis-induced muscle atrophy.

Phase 1 Dose Escalation Trial of Pegcrisantaspase in Combination with Venetoclax in Adults with Relapsed or Refractory Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) cells are sensitive to depletion of extracellular glutamine (Gln) or manipulation of Gln metabolism. We recently reported the synergistic anti-leukemic activity of the combination of the Bcl-2 inhibitor Venetoclax (Ven) and pegcrisantaspase (PegC), a pegylated Erwinia asparaginase, in complex karyotype AML (CK-AML). The potent in vitro and in vivo activity of Ven-PegC regimen is mediated through inhibition of 4EBP1 phosphorylation resulting in decreased mRNA translation and protein synthesis. We hypothesized that safe and tolerable doses of Ven-PegC will result in anti-AML activity in patients (pts) with relapsed/refractory (R/R) AML. Study Design and Methods: The study is an open-label phase 1b clinical trial (NCT04666649) with 3+3 design with 4 cohorts of Ven (400 mg daily in the cohorts 1-3, 600 mg daily in cohort 4) in combination with outpatient intravenous PegC (500, 750, 1000, 1000 IU/m2 in cohorts 1-4, respectively) biweekly in a 28-day cycle. Key eligibility criteria include AML in an adult that has relapsed after, or is refractory to first-line therapy including Ven-containing regimens. The primary endpoints are the incidences of regimen limiting toxicities (RLT) and treatment emergent adverse events of Ven-PegC. Secondary endpoints include rate of composite complete remissions. Exploratory endpoints include the evaluation of plasma amino acid levels of Gln, asparagine (Asn), glutamate (Glu), and serine (Ser), and plasma asparaginase activity post-PegC administration. Results: As of August 1, 2022, 11 pts who received at least 1 dose of PegC were enrolled into the first two cohorts. The median age was 60 (range 24-76) years; 6 (55%) were female; 10 (91%) had adverse-risk AML by European Leukemia Net (ELN) criteria; 7 (64%) had CK-AML, and median prior treatments were 2. Overall response rate was 64% (7 pts) including 3 pts (27%) with CR/CRh/CRi [2 previously Ven-refractory]. In the first cohort, pt2 cleared peripheral blood (PB) blasts within 3 days after the first dose of PegC. Pt5 bone marrow (BM) blasts significantly decreased (78% to 9%) after cycle 1, and pt6 achieved CR with partial hematologic recovery (CRh) with MRD-negativity after 1 cycle and proceeded to alloHSCT after 3 cycles of Ven-PegC. In cohort 2, pt9 cleared PB blasts within 3 days after the first dose of PegC, and pt12 (primary refractory AML with two TP53 mutations) achieved a rapid clearance of PB blasts (41% to 0% after 2 days) and BM blasts after 1 dose of PegC and achieved MRD-negative CR at the end of cycle 1. Pt15 achieved CR with incomplete hematologic recovery (CRi) after 1 cycle of Ven-PegC and is currently receiving cycle 3, and in pt16 absolute PB blasts reduced from 4876/mcL to 192/mcL on Cycle 1 Day 3 and is currently receiving cycle 1 (Fig 1). Eight patients were evaluable for RLT assessment. No RLT was observed in cohort 1. In cohort 2, one transient (<48h) asymptomatic hypertriglyceridemia was observed and resulted in the expansion of cohort 2 to 6 patients. No serious asparaginase-related adverse events of interest have been observed. Pharmacokinetic and pharmacodynamic data are available for 8 patients. All pts achieved a nadir plasma asparaginase activity >0.1 IU/mL during cycle 1 and repetitive doses of PegC did not result in silent inactivation. Mean (range) plasma amino acid levels (µmol/L) at baseline were: Asn 31 (24-38), Gln 412 (323-465), Glu 58 (22-103). All pts achieved undetectable Asn level by day 7 and Asn remained undetectable throughout the study. Plasma Gln reduction from baseline occurred in all pts within cycle 1, with pts in cohort 2 demonstrating a more rapid decrease in Gln levels after initial exposure, confirming a dose-response correlation. Of note, in both cohorts, the pts that responded to Ven-PegC achieved the lowest absolute plasma Gln levels. A concomitant increase in plasma Glu levels was observed in all pts, indicative of the PegC-induced conversion of Gln to Glu. Remarkably, plasma Ser increased ~2-fold at the nadir of plasma Gln, from 75 ± 21 µmol/L at baseline to 114 ± 30 µmol/L after the first dose of PegC (Fig 2). Conclusion: Ven-PegC is a well-tolerated regimen with a novel metabolically-targeted mechanism of action. Early efficacy results in patients with poor prognosis R/R AML, a population with very limited treatment options, has been very promising. A clear correlation between the dose of PegC administered and the reduction in plasma Gln was observed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P527: PHASE 1 TRIAL OF PEGCRISANTASPASE IN COMBINATION WITH VENETOCLAX IN ADULTS WITH RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA (R/R AML) - SAFETY, EFFICACY AND PK/PD IN THE FIRST TWO COHORTS

Background: Glutamine depletion induced by Erwinia asparaginases including pegcrisantaspase (PegC) downregulates the mTOR/p70S6K pathway and inhibits 4EBP1 phosphorylation. We recently found that the combination of PegC with the Bcl-2 inhibitor venetoclax (Ven) has strong in vitro and in vivo activity in poor-prognosis AML, mediated by enhancement of eIF4E-4EBP1 interaction on the cap-binding complex and decreasing protein synthesis. We hypothesized that Ven-PegC treatment will be safe and effective in the treatment of R/R AML. Here we report the results from the first two cohorts of a phase 1 trial of Ven-PegC in adults with R/R AML. Aims: The primary objective is to estimate the maximum tolerated dose and to determine the regimen-limiting toxicities (RLT) of Ven-PegC. The exploratory endpoints include plasma amino acids levels 1- and 2-weeks post-PegC administration. Methods: This is an open-label phase 1b clinical trial (NCT04666649) of daily oral Ven in combination with biweekly out-patient IV PegC in 28-day cycles that will be administered to 4 cohorts (Table 1A) based on a standard 3 + 3 design. Results: Eight patients (pts) were enrolled into the first two cohorts; 7 received ≥ 1 dose of PegC and one dropped out before receiving study drugs. Pts’ characteristics are shown in Table 1B. Median age was 66 (59-76) years; two (29%) were female; six (86%) had adverse-risk AML by ELN classification; and four (57%) had complex karyotypes. Pharmacokinetic (PK) and pharmacodynamic (PD) data are available for 5 pts; all pts achieved a nadir plasma asparaginase activity > 0.1 IU/mL during cycle 1 (3/3 in cohort 1 within 3 weeks and 2/2 in cohort 2 within 10 days). Mean plasma amino acid levels (µmol/L) at baseline were: asparagine (Asn) 29 (17-38), glutamine (Gln) 421 (384-465), glutamate (Glu) 59 (22-103), serine (Ser) 66 (49-80) and glycine (Gly) 203 (166-207). All pts achieved an Asn level of 0 by day 7; Asn remained undetectable throughout the study. Plasma Gln (µmol/L) reduction from baseline occurred in 4/5 (80%) pts within cycle 1: pt4 423→273 (Day15), pt5 446→86 (Day28), pt6 467→216 (Day22), pt7 388→156 (Day8), pt10 420→395 (Day10). Plasma Glu levels increased in all pts, indicating conversion of Gln to Glu by PegC. Interestingly, plasma levels of Ser increased 1.5-2-fold at the nadir of plasma Gln (Figure 1A-C, maximum amino acid changes after Ven-PegC). Seven pts from cohorts 1 (4 pts) and 2 (3 pts) who received at least one dose of Ven-PegC were included in analysis of toxicity and efficacy, with data cutoff February 26, 2022 (Table 1B). No RLT has been observed. In cohort 1, pt2 died before completing cycle 1 due to septic shock and delay in seeking medical care, pt4 died of AML progression. In cohort 2, pt7 had progressive disease after cycle 1, pt9 opted for comfort care before evaluation of response, and pt10 died of AML progression, In cohort 1, pt6 achieved measurable residual disease (MRD)-negative complete remission with partial hematologic recovery (CRh) and proceeded to allogeneic hematopoietic stem cell transplant (allo-HSCT) after 3 cycles of Ven-PegC, and bone marrow blasts in pt5 decreased from 78% to 9% after cycle 1. These two pts had the lowest plasma Gln levels (103 [pt6, cycle2 day29] and 86 [pt5, cycle1 day28]). Image:Summary/Conclusion: No RLT has been observed following Ven-PegC in cohorts 1 or 2. The Ven-PegC combination at the lowest dose of PegC administered, produced an MRD negative CRh in one patient and a transient substantial reduction in bone marrow blast count in another, both associated with decreases in plasma Gln levels. The study is ongoing.

Glutamine depletion in patients with Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a viral disease. There is not enough knowledge about plasma amino acid levels in CCHF. Therefore, we investigated plasma amino acid levels in patients with CCHF and the association between the levels of these amino acids and disease severity. The plasma amino acid levels (including glutamate [Glu], aspartate [Asp], glutamine [Gln], asparagine [Asn] and gamma-aminobutyric acid [GABA]) in CCHF patients and controls were measured by using liquid chromatography-mass spectrometry. Plasma levels of Gln were lower while Asp, Glu, and GABA levels were higher in patients. In fatal CCHF patients, we found the plasma level of Asn was increased whereas the plasma level of GABA was decreased. This study is the first in the literature to evaluate the plasma Gln, Glu, Asn, Asp, and GABA levels in CCHF patients. We found that the plasma Gln levels were significantly lower in CCHF patients while Asp, Glu, and GABA levels were elevated. Considering that these amino acids are important for immune cells, the plasma amino acid levels of CCHF patients may contribute to the understanding of the pathophysiology of disease and it can be important for supportive treatment of CCHF.

Oral glutamine supplementation attenuates inflammation and oxidative stress-mediated skeletal muscle protein content degradation in immobilized rats: Role of 70 kDa heat shock protein

Skeletal muscle disuse results in myofibrillar atrophy and protein degradation, via inflammatory and oxidative stress-mediated NF-kB signaling pathway activation. Nutritional interventions, such as l-glutamine (GLN) supplementation have shown antioxidant properties and cytoprotective effects through the modulation on the 70-kDa heat shock protein (HSP70) expression. However, these GLN-mediated effects on cell signaling pathways and biochemical mechanisms that control the myofibrillar protein content degradation in muscle disuse situations are poorly known yet. This study investigated the effects of oral GLN plus l-alanine (ALA; GLN ​+ ​ALA-solution) supplementation, either in their free or dipeptide (L-alanyl-l-glutamine-DIP) form, on GLN-glutathione (GSH) axis and cytoprotection mediated by HSP70 protein expression in the slow-twitch soleus and fast-twitch gastrocnemius skeletal muscle of rats submitted to 14-days of hindlimb immobilization-induced disuse muscle atrophy. Forty-eight Wistar rats were distributed into 6 groups: hindlimb immobilized (IMOB group) and hindlimb immobilized orally supplemented with either GLN (1 g kg−1) plus ALA (0.61 g kg−1) ​(GLN ​+ ​ALA-IMOB group) or 1.49 ​g ​kg−1 of DIP (DIP-IMOB group) and; no-immobilized (CTRL) and no-immobilized supplemented GLN ​+ ​ALA and DIP baselines groups. All animals, including CTRL and IMOB rats (water), were supplemented via intragastric gavage for 14 days, concomitantly to immobilization period. Plasma and muscle GLN levels, lipid (thiobarbituric acid reactive substances-TBARS) and protein (carbonyl) peroxidation, erythrocyte concentration of reduced GSH and GSH disulfide (GSSG), plasma and muscle pro-inflammatory TNF-α levels, muscle IKKα/β-NF-kB signaling pathway and, the myofibrillar protein content (MPC) were measured. The MPC was significantly lower in IMOB rats, compared to CTRL, GLN ​+ ​ALA, and DIP animals (p ​< ​0.05). This finding was associated with reduced plasma and muscle GLN concentration, equally in IMOB animals. Conversely, both GLN ​+ ​ALA and DIP supplementation restored plasma and muscle GLN levels, which equilibrated GSH and intracellular redox status (GSSG/GSH ratio) in erythrocytes and skeletal muscle even as, increased muscle HSP70 protein expression; attenuating oxidative stress and TNF-α-mediated NF-kB pathway activation, fact that reverberated on reduction of MPC degradation in GLN ​+ ​ALA-IMOB and DIP-IMOB animals (p ​< ​0.05). In conclusion, the findings shown herein support the oral GLN ​+ ​ALA and DIP supplementations as a therapeutic and effective nutritional alternative to attenuate the deleterious effects of the skeletal muscle protein degradation induced by muscle disuse.

Glutamine and Glutamate Supplementation Increases the Levels of These Amino Acids in the Milk of Pasture-fed Mares

Background: L-Glutamine (Gln), the most abundant free alpha amino acid in the body, plays a major role in the transport of nitrogen and carbon between tissues, and is an important source of respiratory energy for intestinal and immune system cells. Mares lose lean body mass during lactation, when plasma and milk Gln levels change significantly. However, supplementation with Gln combined with other amino acids may not alter equine plasma Gln levels. The work reported here was designed to test the hypothesis that supplementation with a mixture of glutamine and glutamate (AminoGut) alters blood and milk free glutamine and glutamate levels in pasture-fed lactating mares.Materials, Methods &amp; Results: This study involved 31 multiparous Quarter Horse mares, which were divided into three groups immediately postpartum, as follows: G-CON (n = 19); G-50 g supplemented with 50 g of Gln + Glu plus 200 g of concentrate (n = 6); and G-100 g, supplemented with 100 g of Gln + Glu plus 200 g of concentrate (n = 6). Blood and milk samples were collected on the day of parturition prior to supplementation, and monthly until weaning. The milk samples were used to analyze the Gln, Glu composition and levels, while the blood samples were used for further analysis of blood biomarkers. The results were analyzed by ANOVA and by Tukey’s test and the P value was set at 5%. The G-CON group showed a significant reduction of 11-35% in the mean blood glutamine levels from the first month postpartum and throughout lactation. In contrast, blood glutamine levels in groups G-50 g and G-100 g did not change significantly from parturition through 5 months of lactation. The supplemented groups showed no significant differences in blood variables such as protein, albumin, urea, creatinine, cholesterol, triglycerides and minerals. Free glutamine levels in milk did not change from parturition through the end of lactation in the G-CON group, but groups G-50 g and the G-100 g showed a marked rise in milk glutamine levels throughout the first three months of lactation (~3x), which remained high (~2x) until the foals were weaned (P &gt; 0.05).Discussion: The results of this study indicate that Gln + Glu supplementation successfully increased Gln levels in mare milk in the first three months of lactation, and Glu levels in G-100 g in the first four months, without affecting the levels of these amino acids in the animals’ blood, which remained similar to data obtained at parturition. In fact, the Gln levels in both supplemented groups exceeded 1,000 mmol/mL throughout lactation, unlike those of the control group and of the samples obtained at parturition. Moreover, supplementation did not produce significant changes in blood biomarkers, including those pertaining to protein metabolism (urea, creatinine, uric acid, albumin and total proteins), indicating that the product used for supplementation did not interfere in these biomarkers, which remained within the normal physiological variations for the species. It was concluded that daily dietary supplementation with 50 g of a mixture of glutamine and glutamate produced an effect similar to supplementation with 100 g/day. Both supplementation protocols succeeded in raising glutamine levels in mare milk in the first three months of lactation, without interfering in blood biomarkers or milk composition. In view of the cost of the product, we recommend that the daily diet of mares during lactation be supplemented with 50 g of a mixture of Gln + Glu in order to produce the desired nutritional effects.

Role of glutamine administration on T-cell derived inflammatory response after cardiopulmonary bypass

Cardiac surgery provokes an inflammatory response for which the endothelium, the myocardium, and monocytes/macrophages are primarily responsible. T cells are altered in a different way whereby the pro-inflammatory pathway is suppressed. From the results of experimental studies it was concluded that glutamine (Gln) enhances the production of T-cell cytokines in conditions of Gln deprivation. The aim of this clinical study was to evaluate the role of a perioperative Gln infusion on intracellular inflammatory T-cell cytokine expression in patients undergoing elective cardiac surgery and to evaluate the effects on systemic inflammation, organ dysfunction and ICU length of stay. In this prospective, randomized, double-blind study, we included 78 patients (age level older than 70 years, ejection fraction less than 40%, or mitral valve replacement) undergoing elective cardiosurgery with cardiopulmonary bypass. We randomly assigned each subject to receive an infusion with either Gln (0.5 g/kg/day, group A) or an isonitrogenous, isocaloric, isovolemic nutritional solution (group B) or physiological NaCl 0.9% (group C, to eliminate an unspecific nutritional effect). We started the infusion after the induction of anesthesia with 1000 ml/24 h and maintained this state for 3 days. On the first postoperative day plasma Gln levels in group A were significantly increased (958 +/- 331 microM) compared to group B (527 +/- 105 microM) and group C (489 +/- 104 microM), and remained higher until the third postoperative day. At the beginning and after surgery intracellular interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor-alpha levels in T cells showed no differences between the groups. Also, no differences could be observed with regard to C-reactive protein, SOFA score, heart and circulation support, postoperative ventilation time, and ICU length of stay. The elevation of Gln plasma levels as a result of 0.5 g/kg/day perioperative Gln infusion has no influence on the T-cell derived inflammatory response, indicating a sufficient supply of Gln. A Gln supplementation in cardiac surgery patients without a clear Gln deficiency seems not to affect the intracellular inflammatory T-cell cytokine expression.

Adaptive cellular mechanisms in response to Glutamine-starvation

Glutamine (Gln) utilising cells suffer from Gln-starvation during critical illness when plasma Gln levels are decreased. This study investigates whether such cells activate adaptive mechanisms. Monocytic U937 cells were cultured at 0.6 and 0.2 mM Gln for up to four days. Within the first day a decrease of ATP (78% of control), intracellular free Gln (13%), Hsp70 (74%) and proliferation rate (79%) was observed. A prolonged culture at 0.6 mM Gln for additional three days led to a recovery of ATP (97%), Hsp70 (91%) and proliferation (92%). The intracellular free Gln increased only to 41%. At 0.2 mM Gln, however, all levels remained decreased. The activation of the metabolic sensor AMP activated kinase (AMPK) increased immediately in Gln-starving cells but regained normal values only in cells cultured at 0.6 mM. A proteomic analysis identified 23 proteins, which were affected by Gln starvation including metabolic enzymes, proteins involved in synthesis and degradation of RNA and proteins, and stress proteins. These data show that Gln-utilising cells activate adaptive mechanisms in response to Gln-starvation, which enable them to overcome a Gln shortage. At very low Gln concentrations, these adaptive mechanisms are not sufficient to countervail the lack of the amino acid.

Increased Susceptibility of Glutamine-Depleted Monocytes to Fever-Range Hyperthermia

This study investigates the effect of fever-range hyperthermia on Gln-starving monocytes and the role of the 70-kDa heat shock protein Hsp70. Fever is a protective acute-phase response to infection. However, in critically ill patients, the harmful effects of fever seem to be predominant. Critical illness is frequently associated with reduced plasma glutamine (Gln) levels, which contribute to the immune suppression in these patients due to impaired monocyte function. Isolated monocytes were suspended in Gln-depleted medium and exposed to 41 degrees C. Cell survival was determined by an MTT-based assay, and phagocytosis of Escherichia coli was measured by flow cytometry. Expression of Hsp70 was determined by Western blot. Hyperthermia for 300 minutes strongly decreased the viability of Gln-depleted monocytes (85%), whereas it had only a moderate effect on Gln-supplied cells (45%, P < 0.05). Shorter treatments (45 minutes) of Gln-starving monocytes had almost no effects on viability but decreased the phagocytosis activity by 30.8%. In addition, the expression of Hsp70 was inhibited almost completely. These data show that Gln-starving monocytes have a reduced thermoresistance. This suggests that elevated body temperature damages monocytes in critically ill patients with reduced plasma Gln-levels possibly via an inhibition of the cytoprotective protein Hsp70.

Impact of oral l-glutamine on glutathione, glutamine, and glutamate blood levels in volunteers

OBJECTIVE: We investigated the effect of glutamine supplementation on plasma glutamine (Gln), glutamate (Glu), and whole-blood glutathione (GSH) concentrations in human volunteers. METHODS: Subjects first adapted to a standard diet with known intakes of protein, total GSH, cysteine, methionine, and total Glu (Glu values include Glu and Gln) for 3 d. Plasma Gln, Glu, and whole-blood GSH levels were then measured at 4-h intervals over 24 h. Supplemental oral Gln (0.3 g · kg −1 · d −1) was ingested for 10 d and then 24-h plasma levels of Gln, Glu, and whole-blood GSH were measured. RESULTS: The plasma concentrations of Glu (116%; P = 0.006) and Gln (20%; P = 0.046) were significantly higher, whereas concentrations of GSH were significantly lower (37%; P = 0.00091) after oral Gln supplementation. CONCLUSION: Oral Gln increases Glu and Gln levels in plasma of healthy subjects but does not increase GSH red cell (whole-blood) levels. Thus, GSH biosynthesis and preservation of GHS stores in red blood cells may involve rate-limiting substrates other than Gln.

Glutamine depletion impairs cellular stress response in human leucocytes.

During sepsis and major trauma the blood glutamine (Gln) level is reduced. The administration of Gln can improve the outcome of these patients. However, the mechanism of this beneficial effect of Gln is poorly understood. In the course of critical illness leucocytes are confronted with cytotoxic inflammatory mediators. To protect themselves against these factors, cells express heat shock proteins (HSP). Previous studies have shown that the expression of the major inducible HSP (HSP70) is improved by high Gln concentrations above 4 mM. In this study we investigated whether Gln depletion, such as observed during critical illness, has an effect on HSP70 expression. Human lymphocytes exposed for 2 h to 42 degrees C showed a 3-fold increase in HSP70 expression (P<0.01). A preceding Gln starvation period over 3 days had no influence on this increase. However, when Gln is reduced during the stress response, HSP70 expression is impaired. A reduction of Gln from 0.5 mM (physiological) to 0.125 mM (pathological) led to a 40% lower HSP70 level (P<0.002). In contrast, increasing Gln concentrations (up to 2 mM) had only minor stimulatory effects (about 15%). This Gln-dependency of heat mediated HSP70 expression was observed in resting as well as proliferating lymphocytes. Our data indicate that during periods of reduced plasma Gln levels the stress response of human lymphocytes is impaired. Thus, Gln may be essential to minimize the susceptibility of leucocytes to cytotoxic inflammatory mediators. This is a new aspect of the protective effect of Gln supplementation in critically ill patients.

HSP70 expression in granulocytes and lymphocytes ofpatients with polytrauma: comparison with plasma glutamine

Heat shock proteins (HSP's) are a set of conserved proteins which confer tolerance to stress. These proteins play a major role in the pathophysiology of infection and inflammation. Induction of HSP's before onset of sepsis is able to reduce or prevent organ damage and death. GLN is known to influence the expression of HSP70 in different cell types. In this work we tried to find out if there is an association between plasma GLN levels and HSP70 expression in immune cells. We investigated six polytraumatized patients and a control group of six healthy donors. HSP70 expression was investigated by western blot analysis and immune-histochemistry. We demonstrated that granulocytes and lymphocytes behave differently in the expression of HSP70 in polytraumatized patients. In healthy donors both lymphocytes and granulocytes showed a pronounced expression of HSP70. In contrast, most of the polytraumatized patients showed no HSP70 expression in granulocytes. In lymphocytes of these patients, however, a pronounced expression similar to that of healthy volunteers was observed. Plasma glutamine levels were reduced in all patients and at normal range in healthy donors. These results suggest that lymphocytes and granulocytes behave different when confronted with a reduction of plasma GLN levels.

Discussion

The intestinal hypomotility associated with purulent peritonitis is generally regarded as a contraindication to enteral nutrition. However, enteral nutrition may be feasible in suppurative peritonitis if administered with great caution, i.e., assuring the appropriate amount, delivery speed, and osmolality of the enteral formulation. Glutamine (Gln) increases muscle protein synthesis and decreases muscle protein degradation in sepsis, regardless of the route of administration. Therefore, administering small amounts of supplemental Gln via the enteral route to peritonitis patients may be beneficial. Two purulent peritonitis patients received L-Gln through a jejunostomy tube. The average amount of supplemental Gln was 16 g/d. Systemic inflammatory responses, i.e., high temperature and a high serum C-reactive protein level, persisted throughout the treatment period. Femoral arterial and venous blood samples were drawn simultaneously for determination of amino acid levels before and after 7 d of Gln supplementation. Enterally administered Gln was well-tolerated by both patients. There was an increase in plasma Gln levels after Gln supplementation. Moreover, the release of Gln, alanine, and phenylalanine from the lower extremities was lower after as compared to before Gln supplementation. Enteral administration of Gln may be feasible even in purulent peritonitis.

Glutamine synthetase expression in rat lung is regulated by protein stability.

During physiological stress, the lung increases production of the amino acid glutamine (Gln) using the enzyme Gln synthetase (GS) to maintain Gln homeostasis. Glucocorticoid hormones are considered the principal mediators of GS expression during stress. However, whereas animal studies have shown that glucocorticoids increase lung GS mRNA levels 500-700%, GS activity levels rise only 20-45%. This discrepancy suggests that a posttranscriptional control mechanism(s) ultimately determines GS expression. We hypothesized that the level of GS protein in the lung is governed by the intracellular Gln concentration through a mechanism of protein destabilization, a feedback regulatory mechanism that has been observed in vitro. To test this hypothesis, Sprague-Dawley rats were treated with a Gln-free diet and the GS inhibitor methionine sulfoximine (MSO) to deplete tissue Gln levels and prevent this feedback regulation. Exposure to Gln-free chow and MSO (100 mg/kg body wt) for 6 days decreased plasma Gln levels 50% (P < 0.01) and decreased lung tissue Gln levels by 70% (P < 0.01). Although lung GS mRNA levels were not influenced by Gln depletion, there was a sevenfold (P < 0.01) increase in GS protein. A parenteral Gln infusion (200 mM, 1.5 ml/h) for the last 2 days of MSO treatment replenished lung Gln levels to 65% of control level and blunted the increase in GS protein levels by 33% (P < 0.05) compared with rats receiving an isomolar glycine solution. The acute effects of glucocorticoid and MSO administration on lung GS expression were also measured. Whereas dexamethasone (0.5 mg/kg) and MSO injections individually augmented lung GS protein levels twofold and fourfold (P < 0.05), respectively, the combination of dexamethasone and MSO produced a synergistic, 12-fold induction (P < 0.01) in lung GS protein over 8 h. The data suggest that, whereas glucocorticoids are potent mediators of GS transcriptional activity, protein stability greatly influences the ultimate expression of GS in the lung.

Glutamine: from basic science to clinical applications.

Glutamine (Gln) has been one of the most intensively studied nutrients in the field of nutrition support in recent years. Interest in provision of Gln derives from animal studies in models of catabolic stress, primarily in rats. Enteral or parenteral Gln supplementation improved organ function and/or survival in most of these investigations. These studies have also supported the concept that Gln is a critical nutrient for the gut mucosa and immune cells. Recent molecular and protein chemistry studies are beginning to define the basic mechanism involved in Gln action in the gut, liver and other cells and organs. Double-blind prospective clinical investigations to date suggest that Gln-enriched parenteral or enteral feedings are generally safe and effective in catabolic patients. Intravenous Gln (either as the L-amino acid or as Gln-dipeptides) has been shown to increase plasma Gln levels, exert protein anabolic effects, improve gut structure and/or function and reduce important indices of morbidity, including infection rates and length of hospital stay in selected patients subgroups. Additional blinded studies of Gln administration in catabolic patients and increasing clinical experience with Gln-enriched nutrient products will determine whether routine Gln supplementation should be given in nutrition support, and to whom. Taken together, the data obtained over the past decade or so of intensive research on Gln nutrition demonstrate that this amino acid is an important dietary nutrient and is probably conditionally essential in humans in certain catabolic conditions.

Glutamine Does Not Increase Ammonia in Systemically Drained Small Bowel Transplants

Glutamine (Gln) enhances small bowel function and ameliorates acute injury, but its metabolism generates portal ammonia (NH 4), which normally is detoxified by the liver. Its beneficial use in small bowel transplantation (SBTx) therefore, may be offset by hyperammonemia, since such grafts may be systemically drained. We tested the hypothesis that oral glutamine supplementation increases plasma NH 4 in rats with systemically drained SBTx. Lewis rats with isologous SBTx had plasma NH 4 and Gln assayed during isonitrogenous, isocaloric Gln dietary supplementation and were compared to controls. Plasma NH 4 levels were higher in the SBTx group during all dietary manipulations, consistent with previous studies. A Gln-deficient diet (0%) caused plasma Gln levels to fall in both experimental and control animals, but had no consistent effect on NH 4 levels. With Gln supplementation (12.5 and 25% of total protein) Gln levels returned to baseline but again, plasma NH 4 levels did not significantly change. We conclude that oral glutamine supplementation given in an isonitrogenous manner does not increase ammonia beyond that which is usually seen in animals with systemically drained SBTx. This suggests that Gln-enriched diets are not specifically contraindicated in patients with systemically drained SBTx and may be beneficial.