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We examined the interaction between Alimta and ionizing radiation (IR) as a potential strategy to enhance the therapeutic ratio of combined-modality cancer treatment. Mice bearing human esophageal adenocarcinoma xenografts (Seg-1) or squamous cell carcinoma xenografts (SQ-20B) were treated with Alimta and IR employing a fractionated treatment schedule. Treatment with Alimta alone slowed the growth of Seg-1 but not SQ-20B tumors compared with control tumors. In Seg-1 xenografts combined treatment with Alimta and IR produced significant tumor growth inhibition compared with Alimta alone or IR alone. In SQ-20B xenografts, treatment with Alimta did not enhance IR-mediated tumor growth inhibition suggesting that sensitivity to Alimta is necessary for an interactive cytotoxic effect with IR. The present data suggest the potential clinical efficacy of combining Alimta administration with radiotherapy for Alimta-sensitive cells and indicate that further testing needs to be conducted to optimize the dosing schedule to enhance the interaction between the therapeutic agents.

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.

Purpose: The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR). Endostatin was injected to coincide with fractionated radiotherapy., Methods: Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice. Lewis lung carcinoma cells were injected into C57BI/6 mice. Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose). Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days. Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy. Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells. Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy., Results: In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21). By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone. Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors. Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone. Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone. Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells. Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone., Discussion: The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors. Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent. The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction.

Background: Although ionizing radiation (IR) has been demonstrated to attenuate vessel wall restenosis and intimal hyperplasia (IH), dose-related mural injury and atrophy are possible deleterious side effects. We tested the hypothesis that a radiosensitizing strategy may improve IR-induced inhibition of in vivo vascular smooth muscle cells (VSMCs) without influencing apoptotic cell death., Methods: In 28 New Zealand White rabbits, the right common carotid artery (CCA) was injured and subjected to low-flow conditions to promote IH. The CCA was transfected with an adenoviral vector incorporating the cytosine deaminase (CD) gene (1 x 10(9) PFU/ml). 5-Fluorocytosine (5-FC), a prodrug that is converted to the radiosensitizing agent 5-fluorouracil (5-FU) by CD, was thereafter administered intravenously. The CCA was exposed to 5 Gy IR at 24 h. Intimal/medial (I/M) area and thickness ratios were determined in the harvested CCAs at 14 days. VSMC proliferative and apoptotic indices were assessed with immunohistochemistry., Results: A 50% reduction in I/M area was found in rabbits treated with IR and IR + CD/5-FC (0.19 +/- 0.03 and 0.18 +/- 0.02) when compared with untreated controls (UC) (0.37 +/- 0.06) (P = 0.005). This finding was substantiated by attenuation of I/M thickness in the IR groups [0.47 +/- 0.13 (IR), 0.41 +/- 0.11 (IR + CD/5-FC), 0.61 +/- 0.17 (UC)] (P = 0.007). The number of proliferating VSMCs was notably smaller when IR was combined with CD/5-FC (4.17 +/- 1.16 vs 2.97 +/- 1.09 log transformed cells/mm(2), P < 0.07). Apoptosis was similar in all groups., Conclusions: Both IR alone and IR combined with a radiosensitizing agent are effective in attenuating experimental IH. However, combination therapy is synergistic and achieves greater inhibition of VSMC proliferation and may involve selective killing of radioresistant S-phase VSMCs. IR + CD/5-FC represents a novel therapeutic strategy that offers potential for long-term control of IH., (Copyright 2000 Academic Press.)

The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the lation of angiogenesis Inhibition of VEGF-induced angiogenesis either by neutralizing antibodies or dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect. In vitro, the addition of VEGF decreases IR-induced killing of human umbilical vein endothelial cells, and the anti-VEGF treatment potentiates IR-induced lethality of human umbilical vein endothelial cells. Neither recombinant VEGF protein nor neutralizing antibody to VEGF affects the radiosensitivity of tumor cells These findings support a model in which induction of VEGF by IR contributes to the protection of tumor blood vessels from radiation-mediated cytotoxicity and thereby to tumor radioresistance.

Angiostatin, a proteolytic fragment of plasminogen, inhibits the growth of primary and metastatic tumors by suppressing angiogenesis. When used in combination with ionizing radiation (IR), angiostatin demonstrates potent antitumor synergism, largely caused by inhibition of the tumor microvasculature. We report here the temporal interaction of angiostatin and IR in Lewis lung carcinoma (LLC) tumors growing in the hind limbs of syngeneic mice. Tumors with an initial mean volume of 510 +/- 151 mm3 were treated with IR alone (20 Gy x 2 doses on days 0 and 1), angiostatin alone (25 mg/kg/day divided twice daily) on days 0 through 13, or a combination of the two as follows: (a) IR plus angiostatin (days 0 through 13); (b) IR plus angiostatin (days 0 and 1); and (c) IR followed by angiostatin beginning on the day after IR completion and given daily thereafter (days 2 through 13). By day 14, tumors in untreated control mice had grown to 6110 +/- 582 mm3, whereas in mice treated with: (a) IR alone, tumors had grown to 2854 +/- 338 mm3 (P < 0.05 compared with untreated controls); and (b) angiostatin alone, tumors had grown to 3666 +/- 453 mm3 (P < 0.05 compared with untreated controls). In combined-treatment groups, in mice treated with: (a) IR plus longer-course angiostatin, tumors reached 2022 +/- 282 mm3 (P = 0.036 compared with IR alone); (b) IR followed by angiostatin, tumors reached 2677 +/- 469 mm3 (P > 0.05 compared with IR alone); and (c) IR plus short-course angiostatin, tumors reached 1032 +/- 78 mm3 (P < 0.001 compared with IR alone). These findings demonstrate that the efficacy of experimental radiation therapy is potentiated by brief concomitant exposure of the tumor vasculature to angiostatin.

Background: Epidermal growth factor (EGF) stimulates cell replication and increases DNA content of the small intestine, but its effects on mucosal amino acid transport are unknown., Methods: To investigate these effects, we treated adult rats with vehicle or EGF (10 micrograms/100 gm body weight subcutaneously every 8 hours for three doses). Jejunal brush border membrane vesicles (BBMVs) from each group were prepared by Mg++ aggregation/differential centrifugation. BBMVs were enriched fifteen-fold in alkaline phosphatase, indicating BBMV purity. Transport of 3H-glutamine and 3H-alanine was studied by a rapid mixing filtration technique. Uptakes were primarily Na+ dependent, occurred in an osmotically active space, exhibited classic overshoots, and had similar 2-hour equilibrium values., Results: Glutamine transport by BBMVs more than doubled in rats treated with EGF (16.4 +/- 0.1 pmol glutamine/mg protein/10 sec in EGF vs 7.1 +/- 0.5 pmol glutamine/mg protein/10 sec in controls; p < 0.001). Kinetic studies of the glutamine transporter showed that the increase in transport was the result of a 70% increase in maximal transport velocity (total maximum glutamine uptake = 193 +/- 8 pmol glutamine/mg protein/10 sec in EGF vs 114 +/- 7 pmol glutamine/mg protein/10 sec in controls; p < 0.0001 with no change in transporter affinity (transporter affinity = 224 +/- 6 mumol/L in EGF vs 242 +/- 37 mumol/L in controls; difference, not significant). Alanine uptake by BBMVs was also increased with EGF administration (10.2 +/- 2.0 pmol alanine/mg protein/10 sec in EGF vs 4.5 +/- 0.5 pmol alanine/mg protein/10 sec in controls; p < 0.005). Simultaneously, glucose transport was decreased by 50% in EGF-treated rats, indicating that the Na(+)-dependent glucose cotransporter is regulated independently from and opposite to amino acid transporters., Conclusions: We conclude that EGF up-regulates amino acid transport activity in jejunal BBMVs, an event that is most likely caused by an increase in de novo biosynthesis of transporter protein. The increase in amino acid uptake not only may support de novo protein synthesis but, in the case of glutamine, also may be required for energy production and nucleotide biosynthesis.

The sodium-dependent transporter system responsible for L-glutamine uptake by brush border membrane vesicles prepared from equine jejunum was characterized. Vesicle purity was ascertained by a 14- to 17-fold increase in activity of the brush border enzyme markers. Glutamine uptake was found to occur into an osmotically active space with negligible membrane binding. The sodium-dependent velocity represented approximately 80% of total uptake and demonstrated overshoots. Kinetic studies of sodium-dependent glutamine transport at concentrations between 5 microM and 5 mM revealed a single saturable high-affinity carrier with a Michaelis constant of 519 +/- 90 microM and a maximal transport velocity of 3.08 +/- 0.97 nmol/mg of protein/10 s. Glutamine uptake was not affected by changes in environmental pH. Lithium could not substitute for sodium as a contransporter ion. 2-Methylaminoisobutyric acid inhibited the sodium-dependent carrier only minimally, but marked inhibition (> 90%) was observed in the presence of histidine, alanine, cysteine, and nonradioactive glutamine. Kinetic analysis of the sodium-independent transporter revealed it to have a Michaelis constant = 260 +/- 47 microM and a maximal transport velocity of 0.32 +/- 0.06 nmol/mg of protein/10 s. We conclude that glutamine transport in equine jejunal brush border membrane vesicles occurs primarily via the system B transporter and, to a lesser extent, by a sodium-independent carrier.

The early effects of endotoxin (4 hr after a single dose of Escherichia coli LPS, 7.5 mg/kg) on L-glutamine (GLN) transport across the jejunal brush border of rats were studied. Jejunal brush border membrane vesicles (BBMVs) were prepared by a Mg2+ aggregation/differential centrifugation technique. Vesicle purity and integrity were confirmed by a 15-fold enrichment of brush border marker enzymes, osmotic activity, transport overshoots in the presence of sodium, and similar 1- and 2-hr equilibrium values. L-[3H]GLN transport in jejunal BBMVs was measured by a millipore filtration technique. Na(+)-dependent glutamine transport, which accounted for greater than 80% of total transport, was increased twofold in BBMVs from endotoxin-treated rats (67 +/- 5 pmole/mg protein/15 sec vs 38 +/- 3, P less than 0.01). Endotoxin treatment did not alter the activity of the Na(+)-independent carrier. Simultaneously, intestinal extraction of glutamine from the bloodstream fell by 56% (15.1 +/- 2.3% in controls vs 6.6 +/- 1.3% in endotoxin-treated rats, P less than 0.01). This reduction in the uptake of circulating glutamine could not be accounted for by a fall in the arterial concentration. Thus, soon after endotoxemia brush border glutamine uptake is increased while consumption of glutamine across the basolateral membrane is decreased. This increased uptake may support protein synthesis and may provide a biochemical rationale for the use of early enteral nutrition after the onset of critical illness.

The alterations in lung glutamine (GLN) metabolism that occurs in the endotoxin-injured lung were studied in rats and subsequently correlated with flux changes that occur in patients with the adult respiratory distress syndrome (ARDS). Measurements in animals were made at various time-points following the administration of endotoxin, while studies in surgical patients were done in a group of healthy controls, in patients with "early" sepsis who had normal chest x-ray films, and in patients with radiographic and physiologic evidence of ARDS. In healthy control rats, net amounts of GLN are released by the lungs into the systemic circulation. This release rate doubled 30 minutes after intravenous endotoxin (1,580 +/- 320 nmol GLN/100 g BW/min vs. 736 +/- 179 in controls, p less than 0.01) but glutamine synthetase activity was unchanged, suggesting an outpouring of cellular glutamine stores. Two hours after endotoxin treatment, this accelerated fractional release of glutamine by the lungs was no longer detected. By the 12-hour time-point, the lungs reversed to an organ of net glutamine balance (234 +/- 248 nmol/100 g BW/min, p less than 0.05 vs. controls and ENDO30 min) despite a more than two-fold increase in glutamine synthetase activity (p less than 0.01). Simultaneously, lung weights were increased by 21% (p less than 0.01) and histologic examination showed an interstitial infiltrate and pulmonary edema. Similar observations were made in humans; patients with "early" sepsis exhibited a marked increase in lung glutamine release, while patients with ARDS demonstrated glutamine balance across the lungs (4,030 +/- 910 nmol GLN/kg BW/min vs. 637 +/- 496 in ARDS, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of tumor necrosis factor and interleukin-1 on sodium-dependent glutamine transport by cultured pulmonary artery endothelial cells (PAECs) were studied. Incubation of PAECs with cytokines (10 to 1000 units/ml) resulted in a significant increase in System ASC-mediated glutamine transport that was dose-dependent, first observable after 8 hours, and maximal after 12 hours of exposure. Kinetic studies indicated that the increase in carrier-mediated activity was not due to a change in Km (transporter affinity) but instead to a 45% to 75% increase in maximal transport rate (Vmax). The cytokine-stimulated increase in glutamine uptake by PAECs was completely blocked by actinomycin D and cycloheximide, indicating that the accelerated glutamine transport was dependent on de novo RNA and protein synthesis, perhaps of the transporter itself. The data indicate that these cytokines accelerate glutamine uptake by PAECs, either directly or indirectly, a response which may be required to support endothelial metabolism, structure, and function during infection and inflammation. The results of this study represent, to our knowledge, the first reports of cytokine-mediated modulation of System ASC activity, a carrier that has historically been unresponsive to hormonal regulation in other tissues.

The effects of severe infection on luminal transport of amino acids and glucose by the small intestine were investigated. Studies were done in endotoxin-treated rats and in septic patients who underwent resection of otherwise normal small bowel. In rats the kinetics of the brush border glutamine transporter and the glutaminase enzyme were examined. In patients the effects of severe infection on the transport of glutamine, alanine, leucine, and glucose were studied. Transport was measured using small intestinal brush border membrane vesicles that were prepared by Mg++ aggregation/differential centrifugation. Uptake of radiolabeled substrate was measured using a rapid mixing/filtration technique. Vesicles demonstrated 15-fold enrichments of enzyme markers, classic overshoots, transport into an osmotically active space, and similar 2-hour equilibrium values. The sodium-dependent pathway accounted for nearly 90% of total carrier-mediated transport. Kinetic studies on rat jejunal glutaminase indicated a decrease in activity as early as 2 hours after endotoxin secondary to a decrease in enzyme affinity for glutamine (Km = 2.23 +/- 0.20 mmol/L [millimolar] in controls versus 4.55 +/- 0.67 in endotoxin, p less than 0.03), rather than a change in Vmax. By 12 hours the decrease in glutaminase activity was due to a decrease in Vmax (222 +/- 36 nmol/mg protein/min in controls versus 96 +/- 16 in endotoxin, p less than 0.03) rather than a significant change in Km. Transport data indicated a decrease in sodium-dependent jejunal glutamine uptake 12 hours after endotoxin secondary to a 35% reduction in maximal transport velocity (Vmax = 325 +/- 12 pmol/mg protein/10 sec in controls versus 214 +/- 8 in endotoxin, p less than 0.0001) with no change in Km (carrier affinity). Sodium-dependent glutamine transport was also decreased in septic patients, both in the jejunum (Vmax for control jejunum = 786 +/- 96 pmol/mg protein/10 sec versus 417 +/- 43 for septic jejunum, p less than 0.01) and in the ileum (Vmax of control ileum = 1126 +/- 66 pmol/mg protein/10 sec versus 415 +/- 24 in septic ileum, p less than 0.001) The rate of jejunal transport of alanine, leucine, and glucose was also decreased in septic patients by 30% to 50% (p less than 0.01). These data suggest that there is a generalized down-regulation of sodium-dependent carrier-mediated substrate transport across the brush border during severe infection, which probably occurs secondary to a decrease in transporter synthesis or an increase in the rate of carrier degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Intestinal extraction of circulating glutamine across the basolateral membrane is diminished in the tumor-bearing rat (TBR). This study was designed to investigate the effects of progressive malignant growth on brush border glutamine transport in order to gain further insight into the adaptive/regulatory changes in intestinal glutamine metabolism that occur in the tumor-bearing rat. Fischer 344 rats (225 +/- 5 g) were implanted with fibrosarcoma cells and were studied at various time points after implantation when the tumors comprised 7%, 20%, and 29% of total body weight. Control and tumor-bearing rats were pair-fed throughout the study. Jejunal brush border membrane vesicles (BBMVs) were prepared by magnesium aggregation/differential centrifugation and transport of radioactively labeled L-glutamine, L-leucine, L-alanine, and D-glucose by BBMVs was measured using a Millipore filtration technique. BBMVs were enriched 15-fold in alkaline phosphatase, indicating brush border vesicle purity. Uptake of all substrates occurred into an osmotically active space, exhibited overshoots, and had similar 1-hr equilibrium values. The rate of glutamine uptake by BBMVs from all tumor-bearing rats was significantly greater than controls, regardless of tumor size. The increase in transport activity was not due to a change in carrier affinity but rather to an increase in maximal transport velocity. In rats with small tumors (7% of body weight), the Vmax was 431 +/- 40 pmole/mg protein/10 sec compared to 259 +/- 30 in control animals (P less than 0.01). In marked contrast, the mean transport of alanine was diminished in BBMVs from TBR (31 +/- 3 pmole/mg protein/10 sec in TBR vs 23 +/- 2 in controls, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

In critically ill surgical patients, nutritional therapy is an important component of overall care. As our knowledge increases, "tailor-made" diets designed to meet specific nutritional requirements in specific patients may play an important role. Although the data reviewed here suggest that glutamine-supplemented diets may have a significant impact in some clinical settings, it should be emphasized that additional carefully designed studies are necessary before the use of glutamine-enriched nutrition in critically ill patients should be advocated. It is clear, however, that the concept that the intestine is an organ of inactivity during critical illness merits reconsideration.

The healing effects of glutamine given orally for 8 days as a single amino acid nutrient after treatment with whole abdominal radiation (10 Gy) were studied. Rats received isonitrogenous and isovolumic diets containing 3% glutamine or 3% glycine. Control rats were not irradiated but were given identical diets. In irradiated animals, survival was 100% in animals receiving glutamine compared with 45% in animals receiving glycine. Glutamine ingestion diminished bloody diarrhea and the incidence of bowel perforation. Arterial glutamine level was higher in animals receiving glutamine in the diet, as were gut glutamine extraction (35% +/- 8% vs 12% +/- 7%) and intestinal glutaminase activity. These metabolic improvements were associated with a marked increase in villous height, villous number, and the number of mitoses per crypt in rats receiving glutamine. Glutamine was not beneficial in control nonirradiated animals. The data demonstrated that provision of oral glutamine after abdominal radiation supported gut glutamine metabolism, improved mucosal morphometrics, and decreased the morbidity and mortality associated with this abdominal radiation model.

The role of the glucocorticoid hormones as possible mediators of the accelerated lung glutamine and alanine release that occurs during critical illness was investigated. Studies were done in adult rats receiving dexamethasone (0.6 mg intramuscularly/100 gm body weight/day for 2 consecutive days; n = 24) or saline solution (controls; n = 20). Measurements were made in the postabsorptive state and amino acid flux was calculated by multiplying pulmonary blood flow by the right ventricular-arterial concentration difference for glutamine and alanine. Lung glutamine release was 703 +/- 184 nmol/100 gm body weight/min in control rats. This release rate doubled in the dexamethasone-treated rats (1476 +/- 256; p less than 0.05). The activity of the glutamine synthetase enzyme increased by 33% in the dexamethasone-treated animals and there was a 50% decrease in lung glutamine content (p less than 0.01). Likewise, dexamethasone accelerated the release of alanine by the lungs twofold (559 +/- 173 nmol/100 gm body weight/min in controls vs 1113 +/- 184 nmol/100 gm body weight/min in dexamethasone-treated rats; p less than 0.05). The increased release of both amino acids was caused by a significant increase in the concentration difference across the lungs and not a change in pulmonary blood flow. Glucocorticoids appear to be key mediators of the accelerated lung amino acid release that characterizes catabolic diseases.

The gut plays a key role in interorgan glutamine metabolism in normal and catabolic states. During critical illness, the gut responds to prevailing metabolic pressures that may result in a temporary "reset" in interorgan glutamine flow. As the body recovers from the disease process, the alterations in gut glutamine metabolism revert to normal, which helps restore glutamine homeostasis. Overwhelming stresses, such as severe systemic infection, may lead to permanent organ dysfunction and further adaptive changes in glutamine metabolism.

The effects of a glutamine-enriched diet on the transport of glutamine across brush border membrane vesicles (BBMV) from the rat jejunum were studied to gain further insight into the effects of diet on regulating gut glutamine utilization. Following fasting, rats were randomized to one of three nutritionally complete elemental diets supplemented with glutamine, glutamate, or glycine (control). Brush border membrane vesicles were prepared by a Mg2+ aggregation/differential centrifugation technique and uptake of radioactive [3H]glutamine by the BBMV was studied using a rapid mixing/filtration technique. BBMVs from all test diet groups were enriched in alkaline phosphatase 14-fold. [3H]Glutamine uptake courses for all groups demonstrated sodium dependency, overshoots, and similar 2-hr equilibrium values. Vesicles from animals fed the glutamine-enriched diet had a 75% increase in glutamine uptake compared to those of the control diet and a 250% increase compared to those of the glutamate-enriched diet (P less than 0.05). alpha-Methylamino isobutyric acid and glycine did not significantly inhibit total [3H]glutamine uptake, whereas asparagine and glutamine inhibited total [3H]glutamine uptake compared to the mannitol control. The brush border appears to possess the glutamine selective System N transporter, the activity of which can be stimulated by providing dietary glutamine.

The effects of sepsis on gut glutamine (GLN) metabolism were studied to gain further insight into the regulation of the altered glutamine metabolism that characterizes critical illnesses. Studies were done in laboratory rats and in hospitalized patients. The human studies were done in seven healthy surgical patients (controls) and six septic patients who underwent laparotomy. Radial artery and portal vein samples were obtained during operation and were analyzed for GLN and oxygen content. Despite no reduction in arterial glutamine concentration in the septic patients, gut glutamine extraction was diminished by 75% (12.0% +/- 1.6% in controls vs. 2.8% +/- 0.8% in septic patients, p less than 0.01). Similarly gut oxygen extraction was diminished by nearly 50% in the septic patients (p less than 0.05). To further investigate these abnormalities, endotoxin (10 mg/kg intraperitoneally) or saline (controls) was administered to adult rats 12 hours before cannulation of the carotid artery and portal vein. The arterial GLN concentration was increased by 13% in the endotoxin-treated animals (p less than 0.05) but gut glutamine uptake was diminished by 46% (526 +/- 82 nmol/100 g BW/minute in controls vs. 282 +/- 45 in endotoxin, p less than 0.01). Simultaneously gut glutaminase activity was diminished by 30% (p less than 0.01) and intestinal glutamate release fell by two thirds. Blood cultures were negative in control animals (0 of 20), but were positive in 25% of endotoxemic animals (6 of 24) for gram-negative rods (p = 0.019). Sepsis and endotoxemia impair gut glutamine metabolism. This impairment may be etiologic in the breakdown of the gut mucosal barrier and in the development of bacterial translocation.

Glutamine is a principal fuel utilized by rapidly growing tumors. Advanced malignant disease results in muscle glutamine depletion and weight loss. Concern exists about providing dietary glutamine to the host with cancer since it may stimulate tumor growth. This study examined the effects of oral glutamine on muscle glutamine metabolism and tumor growth. Twenty-four rats with large sarcomas were pair fed a glutamine-enriched or glutamine-free elemental diet. Diets were isonitrogenous and isocaloric. After 6 days of feeding, the animals were anesthetized and arterial glutamine, hindquarter glutamine flux, muscle glutamine content, tumor weight, tumor DNA content, tumor glutaminase activity, and number of metaphase mitoses/high power field (HPF) in the tumor were determined. There was no difference in arterial glutamine between the two groups, but provision of a glutamine-enriched diet increased muscle glutamine content by 60% (2.31 +/- 0.21 mumole/g tissue vs 1.44 +/- 0.22 mumol/g tissue, P less than 0.05), which supported muscle glutamine release. There were no differences among tumor DNA content, tumor glutaminase activity, or tumor weight and there was no difference histologically in the number of metaphase mitoses/HPF. Glutamine-enriched oral diets may replete host glutamine stores and support muscle glutamine metabolism without stimulating tumor growth.

In the critically ill surgical patient a variety of therapeutic maneuvers is required to maintain a "healthy gut." Provision of adequate amounts of glutamine to the gastrointestinal mucosa appears to be just one of these maneuvers. Other methods utilized to protect the gut from becoming a wound include: (a) minimizing additional systemic insults (such as hypotension, sepsis, multiple operative procedures); (b) aggressive pulmonary care; (c) the judicious use of antibiotics; and (d) aggressive enteral or parenteral feedings. The concept that the gut is an organ of quiescence following surgical stress merits reconsideration. The intestinal tract plays a central role in interorgan glutamine metabolism and is a key regulator of nitrogen handling following surgical stress. Critically ill patients are susceptible to developing gut-origin sepsis, the incidence of which will be diminished by instituting measures and providing treatments which support intestinal structure, function, and metabolism. Provision of glutamine-enriched diets to such patients may be one of these therapies.

Gut glutamine utilization after massive small bowel resection was studied to gain further insight into the alterations and adaptations in intestinal glutamine metabolism that occur during the development of post-resectional hyperplasia. After resection of the middle 60% of the small intestine in the rat, gut glutamine metabolism was studied immediately and 1, 2, and 3 weeks later. Whole gut glutamine extraction was 22% in sham controls and it acutely declined to 12% (p less than 0.01) after bowel resection. Extraction increased to 31% 1 week later (p less than 0.05) and then returned to normal by week 2. Gut ammonia release decreased after massive small bowel resection, whereas intestinal alanine release increased. The increase in gut glutamine extraction at 1 week occurred at a time when jejunal and ileal DNA and protein content were markedly increased (p less than 0.01). Intestinal glutaminase content declined initially and then increased by the third week after bowel resection (p less than 0.01). With time, increases in gut cellularity and glutaminase content are associated with gut glutamine utilization in the shortened small bowel that is equal to that of the intact unresected intestine.