Inhibition of tumor cell glutamine uptake by isolated neutrophils.

Antitumor activity of phorbol myristate acetate-(PMA) stimulated neutrophils was measured against CCRF-CEM cells. Neutrophils and tumor cells were incubated (a) as a suspension with continuous mixing to maximize the availability of oxygen or (b) after centrifugation as a pellet to maximize cell-cell contact. The cells were then incubated briefly as a suspension with [14C]glutamine under conditions that blocked further damage to the tumor cells. When cells were incubated as a suspension, inhibition of tumor-cell glutamine uptake was mediated by the myeloperoxidase/hydrogen peroxide/chloride system of stimulated neutrophils. Inhibition was blocked by adding catalase, an inhibitor of myeloperoxidase, or compounds that scavenge hypochlorous acid or chloramines. When cells were incubated as a pellet, a portion of the inhibition could not be blocked in this way, indicating that a nonoxidative mechanism contributed to inhibition. In both systems, inhibition of glutamine uptake was rapid and was obtained at effector-cell/target-cell ratios as low as 0.5:1. This inhibition was obtained under conditions that did not result in 51Cr release from cells labeled with [51Cr]-chromate, indicating that inhibition of glutamine uptake measured cytotoxicity rather than cytolysis. 51Cr release was observed only when cells were incubated together for an hour or more as a pellet at high E/T ratios. This cytolysis was mediated by the myeloperoxidase system, and a nonoxidative contribution to cytolysis was not observed. The results indicate that stimulated neutrophils are potent antitumor effectors cells when cytotoxicity rather than cytolysis is the measure of activity. Because glutamine is required for growth of many tumor cells, inhibition of glutamine uptake may represent a significant tumoristatic or tumoricidal effect.     

Effects on skeletal muscle of intravenous glutamine supplementation to ICU patients

Objective: To evaluate the effect of four doses of intravenous glutamine supplementation on skeletal muscle metabolism. Design: A prospective, blinded, randomized study. Setting: The general Intensive Care Unit (ICU) of a university hospital. Patients: ICU patients with multiple organ failure (n=40), who were expected to stay in the unit for more than five days. Intervention: Patients received 0, 0.28, 0.57 or 0.86 g of glutamine per kg bodyweight per day intravenously for five days as part of an isocaloric, isonitrogenous and isovolumetric diet. Results: Plasma glutamine concentration responded to glutamine supplementation with normalization of plasma levels in a dose-dependent way, while free muscle glutamine concentration, as well as muscle protein synthesis and muscle protein content, did not change significantly. Conclusion: Intravenous glutamine supplementation to ICU patients for a period of five days resulted in normalization of plasma glutamine concentrations in a dose-dependent way whereas muscle glutamine concentrations were unaffected.     

Skeletal muscle glutamine production in thermally injured rats

1. The effect of thermal injury (33-35% of body surface area) on the regulation of glutamine metabolism was studied in skeletal muscles of rats 7 days after injury. 2. Injury increased the rates of glutamine production in muscle, skin and adipose tissue preparations, with muscle production accounting for over 90% of total glutamine produced by the hindlimb. 3. Injury produced decreases in the concentrations of skeletal muscle glutamine (36%, P less than 0.001), glutamate (39%, P less than 0.001), alanine (24%, P less than 0.001), pyruvate (35%, P less than 0.001), 2-oxoglutarate (51%, P less than 0.001) and adenosine 5'-triphosphate (38%, P less than 0.001). The concentrations of ammonia (42%, P less than 0.001) and inosine 5'-phosphate (430%, P less than 0.001) were increased. 4. The maximal activity of glutamine synthetase was increased (22-40%, P less than 0.001) in muscles of injured rats, whereas that of glutaminase was unchanged. 5. Hindlimb blood flow decreased by approximately 15% in injured rats, which was accompanied by an enhanced net release of glutamine (80%, P less than 0.001) and alanine (44%, P less than 0.001). 6. It is concluded that there is an enhanced rate of release of both glutamine and alanine from skeletal muscle of thermally injured rats. This may be due to changes in efflux and/or increased intracellular formation of glutamine and alanine.     

Leucine meal increases glutamine and total nitrogen release from forearm muscle.

To assess the consequences of elevated branched chain amino acid levels on alanine, glutamine, and ammonia metabolism in muscle, L-leucine meals (14.7 g) were consumed by six normal postabsorptive individuals. Bilateral forearm studies were performed, and the dominant arm was subjected to 15 min of light exercise, using a calibrated dynamometer, beginning 45 min after the ingestion of the meal. Large uptakes of leucine were seen across both forearm muscle beds within 30 min of the meal. After exercise, blood flow in the dominant arm increased from 3.1 +/- 0.4 to 5.2 +/- 0.9 ml/100 ml forearm per minute (mean +/- SEM, P less than 0.005). Glutamine flux out of the dominant forearm increased threefold after the ingestion of the leucine meal and increased eightfold over base line after exercise. Less marked changes (significant only at 90 min) in the nonexercised, nondominant arm were also seen. Alanine flux out of the dominant forearm muscle bed increased modestly at 75 and 90 min. No significant change in ammonia flux across either forearm muscle bed was noted. Unexpectedly, large and significant net nitrogen loss from both forearm muscle beds was documented. Thus, following the ingestion of a leucine meal and light exercise, the primary means by which excess nitrogen is routed out of muscle is via glutamine formation and release with alanine and ammonia pathways playing relatively minor roles. More importantly, the ingestion of significant amounts of leucine by normal subjects, presumably in optimal nitrogen balance, results in a net loss of nitrogen from muscle.     

Familial hypercatabolic hypoproteinemia. A disorder of endogenous catabolism of albumin and immunoglobulin.

The metabolism of albumin and IgG was investigated in two siblings, products of a first-cousin marriage, a female aged 34 yr and a male aged 17, who had a marked reduction in their respective serum concentrations of IgG (1.3 and 3.1 mg/ml) and albumin (19 and 21 mg/ml). The metabolism of radioiodinated IgG and albumin was studied in the two patients. The total circulating and body pools of IgG were less than 28% of normal. The IgG synthetic rates were within the normal range. However, the IgG survival was short, with their respective fractional catabolic rates increased fivefold to 31% and 36% of the intravenous pool per day (normal, 6.7 +/- 2%/d). Furthermore, the patients had reduced total body pools, normal synthetic rates, and increased fractional catabolic rates for albumin. There was no proteinuria or abnormality of renal or liver function. In addition, the patients did not have circulating antibodies directed toward IgG, IgA, or albumin. Furthermore, both patients had normal fecal 51Cr-labeled albumin tests, thus excluding excessive gastrointestinal protein loss. We propose that these siblings have a previously unrecognized familial disorder characterized by reduced serum concentrations of IgG and albumin caused by a defect in endogenous catabolism, leading to a short survival of these proteins that is associated in this family with chemical diabetes and a skeletal deformity.     

Inhibition of CFU-E colony formation in uremic patients with inflammatory disease: role of IFN-gamma and TNF-alpha.

BACKGROUND: There is evidence for the role of inflammatory cytokines in the inhibition of erythropoiesis in the anemia of chronic disease, but the extent to which they contribute to resistance to erythropoietin (EPO) in patients with chronic renal failure is not clear. The purpose of the present study was to assess the effect of sera from patients with end-stage renal failure with and without infection or inflammatory disease on CFU-E colony formation in vitro. METHODS: Bone marrow was obtained from uremic patients with inflammatory disease and from healthy controls. Standard colony assays were used to assess erythroid colony formation (CFU-E) in response to EPO in the presence or absence of 5% autologous serum. Normal bone marrow mononuclear cells were cultured with 5% v/v sera from three groups of patients: healthy volunteers, uremic controls, and uremic patients with inflammatory disease. RESULTS: There was no difference between normal and uremic bone marrow response to EPO. However, when uremic/inflammatory bone marrow was cultured with autologous serum the optimal response to EPO was significantly inhibited. Optimal CFU-E colony formation was suppressed significantly by sera from either uremic group when compared with cultures containing sera from controls. Treatment of parallel cultures with a combination of antibodies to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) almost completely restored the response to EPO. Additionally, bone marrow from healthy controls incubated with uremic sera showed an increased production of interleukin-1 alpha (IL-1 alpha) and IFN-gamma, and TNF-alpha was present in uremic sera. CONCLUSIONS: CFU-E colony formation is inhibited by soluble factors present in the sera of uremic patients with or without inflammatory disease. These soluble factors stimulate the production of IFN-gamma and TNF-alpha, which directly inhibit erythropoiesis at a local level in the bone marrow.     

Endogenous glutamine production in critically ill patients: the effect of exogenous glutamine supplementation

Introduction

Glutamine rate of appearance (R_a) may be used as an estimate of endogenous glutamine production. Recently a technique employing a bolus injection of isotopically labeled glutamine was introduced, with the potential to allow for multiple assessments of the glutamine R_a over time in critically ill patients, who may not be as metabolically stable as healthy individuals. Here the technique was used to evaluate the endogenous glutamine production in critically ill patients in the fed state with and without exogenous glutamine supplementation intravenously.

Methods

Mechanically ventilated patients (n = 11) in the intensive care unit (ICU) were studied on two consecutive days during continuous parenteral feeding. To allow the patients to be used as their own controls, they were randomized for the reference measurement during basal feeding without supplementation, before or after the supplementation period. Glutamine R_a was determined by a bolus injection of ¹³C-glutamine followed by a period of frequent sampling to establish the decay-curve for the glutamine tracer. Exogenous glutamine supplementation was given by intravenous infusion of a glutamine containing dipeptide, L-alanyl-L-glutamine, 0.28 g/kg during 20 hours.

Results

A 14% increase of endogenous glutamine R_a was seen at the end of the intravenous supplementation period as compared to the basal measurements (P = 0.009).

Conclusions

The bolus injection technique to measure glutamine R_a to estimate the endogenous production of glutamine in critically ill patients was demonstrated to be useful for repetitive measurements. The hypothesized attenuation of endogenous glutamine production during L-alanyl-L-glutamine infusion given as a part of full nutrition was not seen.     

Inhibition of apatite formation by phosphorylated metabolites and macromolecules.

Apatite formation from synthetic extracellular fluids is rate-limited both at the initial amorphous precursor deposition step and at the amorphous-crystalline transformation reaction. Nucleotide diphosphates and triphosphates and low molecular weight metabolites containing two attached ester phosphate groups all inhibited amorphous-crystalline conversion at concentrations of 10(-5) to 10(-6)M. Both native and synthetic polynucleotides as well as the phosphoproteins from rat dentin or egg yolk also inhibited crystal formation from amorphous calcium phosphate. In all cases, substantial amounts of inhibitor molecules were incorporated into the stabilized amorphous precipitates. Treatment of isolated, inhibitor-stabilized amorphous precipitates with hydrolytic enzymes such as alkaline phosphatase or papain reversed the inhibitory effect and permitted crystallization to proceed normally.     

Rosette formation between human lymphocytes and sheep erythrocytes. Inhibition of rosette formation by specific glycopeptides.

Rosette formation with unsensitized sheep erythrocytes is a characteristic of human thymus dependent lymphocytes. Release of glycopeptides from the sheep erythrocyte by trypsin reduces rosette formation. These tryptic glycopeptides inhibit rosette formation by untrypsinized sheep erythrocytes; this suggests that rosetting is mediated by erythrocyte surface glycopeptides. To investigate the molecular nature of this interaction, we examined the abilities of various model compounds to act as haptenic inhibitors of rosette formation. Inhibition is given by glycopeptides bearing oligosaccharide units rich in sialic acid, galactose, N-acetylglucosamine, and mannose linked to asparagine residues through glycosylamine bonds. Among compounds tested, fetuin glycopeptide is most effective, but human transferrin glycopeptide and human erythrocyte glycopeptide I also inhibit rosette formation. Other compounds including human erythrocyte glycopeptide II, human IgG glycopeptide, lacto-N-neotetraose, 3'- and 6'-sialyllactose show no significant inhibition. Neither sialic acid, galactose, manose, nor N-acetyl-glucosamine alone inhibits rosette formation. Stepwise degradation of fetuin glycopeptide established the galactose residues as important determinants of inhibitory activity. Fetuin glycopeptide blocks rosette formation when added to a suspension of human lymphocytes and sheep erythrocytes or when preincubated with human lymphocytes, but not when preincubated with sheep erythrocytes. Studies of the binding of [3H] fetuin glycopeptide to normal lymphocytes demonstrate 7.5 x 10(6) saturable binding sites per cell. No saturable binding of this compound to sheep erythrocyte membranes is observed. Compared to normals, lymphocytes from patients with chronic lymphatic leukemia demonstrate decreased fetuin glycopeptide binding with a mean of 0.9 x 10(6) sites per cell. This decreased binding correlates with the impaired ability of these cells to form rosettes. The data suggest that fetuin glycopeptide inhibits rosette formation by binding to the thymus-dependent cell where competition occurs with sheep erythrocytes for specific lymphocyte surface receptors.     

Alanylglutamine reduces muscle loss of alanine and glutamine in post-operative anaesthetized dogs

1. The present study examined the effect of an infusion of the dipeptide alanylglutamine or of the corresponding amino acids alanine and glutamine in equimolar amounts (10 mumol min-1 kg-1) on the canine hindlimb exchange of alanine and glutamine in the post-operative anaesthetized dog. In contrast to glutamine, the dipeptide alanylglutamine is stable in aqueous solution and therefore would be a suitable substrate for parenteral nutrition. 2. The infusion of alanylglutamine increased (a) the arterial concentration of alanylglutamine to a plateau level (120 +/- 9.5 mumol/l, mean +/- SEM) 20 min after start of the infusion, (b) the mean arterial alanine concentration from 761 +/- 42 to a plateau of 1500-1700 mumol/l (P greater than 0.01) and (c) the arterial glutamine concentration from 407 +/- 51 to a plateau of 1050-1500 mumol/l (P greater than 0.01). Alanine and glutamine levels were slightly higher (14% and 26%, respectively, NS) in the group receiving the equimolar amount of alanine and glutamine. 3. Infusion of alanylglutamine for 1 h abolished the net efflux of glutamine (from -0.80 +/- 0.1 to -0.03 +/- 0.2 mumol min-1 kg-1; P greater than 0.05) and invoked a net influx of alanine (from -0.50 +/- 0.19 to +0.27 +/- 0.14 mumol min-1 kg-1; P greater than 0.01). These changes were similar to those achieved when the two amino acids were infused. 4. This study demonstrates that during short-term administration of alanylglutamine or of the corresponding amino acids the nitrogen release from the hindlimb of the anaesthetized post-operative dog via alanine and glutamine is reduced.     

Inhibition of serotonin-induced vascular smooth muscle cell proliferation by sarpogrelate.

Antiproliferative behavior of sarpogrelate (Anplag, MCI-9042, (+/-)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-pro pyl hydrogen succinate hydrochloride), a serotonin 2A (5-HT2A) receptor antagonist, was established using radioactive incorporation of [(3)H]thymidine, [(3)H]uridine, and [(3)H]phenylalanine in cultured rat aortic smooth muscle cells in response to a 5-HT-induced cytokine trigger. Fluorescence-activated cell sorting was used to confirm these observations. 5-HT-induced DNA, RNA, and protein synthesis were inhibited maximally at a concentration of 1 microM sarpogrelate. Although other cytokines such as platelet-derived growth factor and endothelin also induced DNA, RNA, and protein synthesis in rat aortic smooth muscle cells, cell proliferation was not influenced by sarpogrelate, even at large pharmacological concentrations (10 microM). Sarpogrelate's antiproliferative actions were found to be more potent than ketanserin. These data indicate that sarpogrelate operates as a specific inhibitor of 5-HT-mediated cell proliferation and is a good candidate for preventing serotonin-induced neointimal hyperplasia.     

肝硬化患者血清谷氨酰胺合成酶活性检测

目的 探讨肝硬化患者血清谷氨酰胺合成酶(GS)的活性变化及其临床意义.方法 采用终点法测定48例肝硬化患者血清GS的活性;计算GS诊断肝硬化的阳性率、阴性率、灵敏度和特异性;并分析血清GS与清蛋白(ALB)、丙氨酸氨基转移酶(ALT)、总胆红素(TB)的相关性.结果 肝硬化组血清GS活性为(13.28±3.62)U/mgprot,正常组血清GS活性为(6.62±3.23)U/mgprot,二者差异有统计学意义(P<0.001),但GS活性与ALB、ALT、TB均无相关性.结论 肝硬化患者GS活性增高,可作为肝硬化诊断和鉴别诊断的参考指标.     

Effects in vivo of decreased plasma and intracellular muscle glutamine concentration on whole-body and hindquarter protein kinetics in rats.

Glutamine is considered to be a 'conditionally' essential amino acid. During situations of severe stress like sepsis or after trauma there is a fall in plasma glutamine levels, enhanced glutamine turnover and intracellular muscle glutamine depletion. Under these conditions, decreased intramuscular glutamine concentration correlates with reduced rates of protein synthesis. It has therefore been hypothesized that intracellular muscle glutamine levels have a regulatory role in muscle protein turnover rates. Administration of the glutamine synthetase inhibitor methionine sulphoximine (MSO) was used to decrease glutamine levels in male Wistar rats. Immediately after the MSO treatment (t=0 h), and at t=6 h and t=12 h, rats received intraperitoneal injections (10 ml/100 g body weight) with glutamine (200 mM) to test whether this attenuated the fall in plasma and intracellular muscle glutamine. Control animals received alanine and saline after MSO treatment, while saline was also given to a group of normal rats. At t=18 h rats received a primed constant infusion of L-[2,6-3H]phenylalanine. A three-pool compartment tracer model was used to measure whole-body protein turnover and muscle protein kinetics. Administration of MSO resulted in a 40% decrease in plasma glutamine and a 60% decrease in intracellular muscle glutamine, both of which were successfully attenuated by glutamine infusions. The decreased intracellular muscle glutamine levels had no effect on whole-body protein turnover or muscle protein kinetics. Also, glutamine supplementation did not alter these parameters. Alanine supplementation increased both hindquarter protein synthesis and breakdown but the net balance of phenylalanine remained unchanged. In conclusion, our results show that decreased plasma and muscle glutamine levels have no effect on whole-body protein turnover or muscle protein kinetics. Therefore, it is unlikely that, in vivo, the intracellular muscle concentration of glutamine is a major regulating factor in muscle protein kinetics.     

Inhibition of hepatic triglyceride formation by clofibrate

The effect of clofibrate (CPIB) on hepatic glycerolipid formation has been studied in vivo and in vitro in the rat. Feeding 0.25% CPIB in laboratory chow significantly reduced serum triglyceride levels by 6 hr and concomitantly decreased the rate of glycerol-(14)C incorporation into hepatic and serum glycerides, in vivo. These changes persisted for at least 14 days. A similar decrease in serum triglyceride and glycerol incorporation into hepatic glycerides was observed in rats fed high glucose diets containing 0.25% CPIB. Serum glycerol was reduced by feeding CPIB for 14 days. The formation of diglyceride and triglyceride from (14)C-sn-glycerol-3-P by rat liver homogenates was inhibited by addition of 1-40 mM CPIB to the reaction mixture. These results suggest that CPIB reduces hepatic glycerolipid synthesis, possibly by inhibition of one or more reactions in the esterification of sn-glycerol-3-P. This change may account for the early fall in serum triglyceride. At later time periods, serum glycerol levels fall and in some experiments, hepatic triglyceride content increases. Therefore, it is likely that additional metabolic alterations may contribute to the sustained hypotriglyceridemic effects of CPIB.     

Inhibition of stress ulcer formation with somatostatin in rats

Prophylactitc effect of somatostatin (Growth hormone inhibiting hormone) on restraint stress ulcer formation was studied in rats. Rats treated with somatostatin before and during stress had only the fifth part of the ulcers of the untreated animals after 9 hours of immobilisation. Pathophysiologic mechanism for stress ulcer production is as well discussed as the prohibiting effect of somatostatin on ulcer formation. Prophylactic clinical use has to be considered.     

Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow.

Bisphosphonates inhibit bone resorption in vivo and in vitro by unknown mechanisms. The effect of bisphosphonates on the formation of osteoclasts from their mononuclear hematopoietic precursors was investigated using human long-term marrow cultures in which multinucleated cells form that express most of the known features of the osteoclast phenotype (e.g., bone resorption, tartrate-resistant acid phosphatase, calcitonin responsiveness, and reactivity with specific MAbs). The five bisphosphonates that were tested strongly inhibited 1,25-dihydroxyvitamin D3-stimulated formation of these cells with the same relative potencies as they inhibit bone resorption in vivo. Two representative compounds (3-amino-1-hydroxypropylidene-1,1-bisphosphonate and dichloromethylene bisphosphonate) failed to inhibit the proliferation of precursors of the osteoclast-like cells. However, these compounds decreased the proportion of mononuclear and multinucleated cells expressing an osteoclast antigen, thus suggesting a degree of specificity for cells of the osteoclast lineage. We conclude that bisphosphonates are potent inhibitors of osteoclast-like cell formation in long-term human marrow cultures, and that this may be related to their ability to inhibit bone resorption in vivo.     

Deficiency in peripheral glutamine production in pediatric patients with burns

Plasma glutamine levels decrease in association with severe injury, which suggests that the consumption of glutamine exceeds the production of glutamine or possibly represents a deficit in the release of glutamine from skeletal muscle. The goal of this study was to assess the peripheral glutamine kinetic response to prolonged stress in children with critical injuries. To accomplish this purpose, we quantitated peripheral glutamine kinetics in vivo with the use of 5N15 glutamine in 5 children with severe burns (total body surface area, 74%+/-14%; mean +/- SEM) and 3 children who underwent elective scar reconstruction. In the children with severe burns, leg blood flow was significantly elevated (16.2+/-2.1 vs 7.5 +/-0.3 mL/min/100 mL leg volume, P < .02) and the arterial concentration of glutamine was significantly reduced (0.31+/-0.04 vs 0.84+/-0.05 mmol/L, P < .001). The rate of glutamine turnover within the leg was significantly reduced in the patients with acute burns, whereas the net efflux of glutamine was similar between the 2 groups. These findings suggest that plasma glutamine concentrations decrease during severe stress as a result of a deficit in peripheral glutamine release in conjunction with an increased central consumption. This preliminary study supports the notion that exogenous glutamine supplementation in pediatric patients with severe injuries may be needed because of this inadequate skeletal muscle response.     

Inhibition of diet-induced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall.

Apolipoprotein E (apoE) plays a crucial role in lipoprotein metabolism both in plasma and in peripheral tissues. To test whether apoE in the vascular wall has a direct and local effect on atherogenesis, we established transgenic mice expressing human apoE under control of H2 Ld promoter. Studies on mRNA levels and immunohistochemistry demonstrated that this line was characterized by high expression of human apoE in the arterial wall while its expression was relatively low in other tissues as compared with the respective endogenous expression of mouse apoE. They showed no difference in plasma cholesterol levels and lipoprotein profile from controls when fed both normal and atherogenic diets. However, after 24 wk of an atherogenic diet, the formation of fatty streak lesions in proximal aorta was markedly inhibited in transgenic mice as compared with controls. Both lesion area and esterified cholesterol content were < 30% of those in controls. In a tissue cholesterol labeling study with 3H-cholesterol, the specific activity of aorta cholesterol was much less in transgenic mice, suggesting that apoE enhances cholesterol efflux from the aortic wall into plasma. Thus, apoE has anti-atherogenic action which is mediated via enhancing reverse cholesterol transport from arterial wall.