Do we have unrealistic expectations of the potential of immuno-nutrition?

Heavy sports training schedules and competition is often associated with immuno-suppression, and so there is a theoretical justification for providing athletes with nutrients that display immuno-regulatory properties. Among such immuno-nutrients, considerable attention has been paid in recent years to two amino acids, arginine (ARG) and glutamine (GLN). ARG and GLN availability regulate the function of T lymphocytes, macrophages and polymorphonuclear cells. ARG acts through nitric oxide and polyamine synthesis. The mechanism of action of GLN in immune cells remains unclear. Experience in clinical nutrition suggests that an ARG-enriched diet may limit infectious morbidity in critically ill patients. Data concerning oral/enteral GLN supplementation are more controversial. There have been few trials of supplementation in sports medicine, but results are promising, justifying further studies in which dosages and administration schedules should be taken into account.

Cold preservation injury in rat liver: effect of liposomally-entrapped adenosine triphosphate.

BACKGROUND/AIMS: Energy charge and capacity for adenosine triphosphate (ATP) synthesis have been demonstrated to play a major role in the maintenance of organ function after liver preservation for transplantation. The aim of this study was to evaluate whether a supply of liposomally-entrapped ATP during preservation could improve the energy state and metabolism of cold-stored rat liver. METHODS: In the first set of experiments, the uptake of ATP-containing liposomes and their effects on hepatic viability were determined in isolated perfused unstored rat liver. In the second set of experiments, rat livers were preserved for 18 h at 4 degrees C in UW solution in the presence of these liposomes, and effects on energy state, cell volume and metabolism were evaluated. In each part, data were compared with adequate control, unloaded liposome-treated, and free ATP-treated groups (n=6 in each group). RESULTS: In non-stored livers, ATP-containing liposomes were taken up by the liver; they did not alter hepatic viability and induced a decrease in energy substrate consumption (glucose and amino acids), and an improvement in intrahepatic ATP content (+23% vs. Control). Addition of liposomally-entrapped ATP during cold storage produced a significant attenuation of the decrease in hepatic ATP content (Lip ATP 2: 524+/-45 vs. Control 2: 364+/-106 nmol/g; p<0.05), and induced, during reperfusion, a decrease in proteolysis associated with an increase in cell volume compared with the other groups (Lip ATP 2: 633+/-63 vs. Control 2: 532+/-38, Unloaded Lip 2: 483+/-55 and Free ATP 2: 500+/-29 microl/g; p<0.01). CONCLUSIONS: These data indicate that liposomally-entrapped ATP represents an effective means to improve liver graft energy state and function. The decrease in protein degradation may be related to the modification of cell volume.

Amino acid nutrition and immune function in tumour-bearing rats: a comparison of glutamine-, arginine- and ornithine 2-oxoglutarate-supplemented diets.

Dietary supplementation with glutamine (Gln), arginine (Arg) or ornithine 2-oxoglutarate (alpha-ketoglutarate; OKG) has attracted recent attention for the potential to improve anti-cancer immune function. However, since these compounds have not been compared systematically in an internally controlled study, their relative efficacy is difficult to estimate. Buffalo rats were fed on nutritionally complete semi-purified diets supplemented with Gln, Arg or OKG for 14 days after implantation of the Morris hepatoma 7777 (n>/=7 per diet). The control diet was made isonitrogenous and isoenergetic by addition of a mixture of non-essential amino acids. After 14 days, peritoneal macrophages and splenocytes were isolated to determine cell phenotypes, macrophage cytostatic activity and natural killer (NK) cell cytotoxicity, as well as nitric oxide (NO) and cytokine production. Diet had no effect on tumour weight (1.6+/-0.2 g; n=59). However, rats fed OKG had increased macrophage cytostatic activity and NK cell cytotoxicity (P<0.05). Although enhanced killing ability by NK cells was associated with higher splenocyte NO production (P<0.04), increased cytotoxicity was not inhibited by a specific inhibitor of inducible NO synthase. The proportion of interleukin-2-receptor-positive T cells after stimulation increased in rats fed OKG (P<0.05); however, cytokine production was not affected by diet. None of OKG, Gln or Arg altered tumour growth compared with a control mixture of non-essential amino acids. These results suggest no net advantage for anti-cancer immunity, but do not preclude benefits in immune responses to disease recurrence or metastasis, therapy or secondary infection.

Comparative study of glycine, alanine or casein as inert nitrogen sources in endotoxemic rats.

Pharmacological effects of dietary amino acids (AA) and peptides must be compared to an isonitrogenous control that is as inert as possible. To establish a rationale for the choice of such a control, potential metabolic and nutritional effects of three currently used nitrogenous controls (glycine, alanine, and casein) were evaluated in an endotoxemic rat model that has well-defined alterations in AA and protein metabolism. Five-week-old male Sprague-Dawley rats (113 +/- 1 g) were randomly assigned to four groups and received at d 0 an intraperitoneal injection of endotoxin (3 mg/kg). After withdrawal of food for 24 h, the rats were enterally refed for 48 h with a liquid diet (Osmolite((R))) supplemented with 0.19 g N. kg(-1). d(-1) in the form of glycine [lipopolysaccharide (LPS)-GLY group], alanine (LPS-ALA group) or casein (LPS-CAS group). One group (LPS group) received only Osmolite((R)). Plasma, two skeletal muscles, the liver and the intestine were then removed. Body and tissue weights and tissue protein contents did not differ among the four groups. Intestine histomorphometry showed no significant difference among groups. Jejunal hydrolase activities were significantly affected by the nitrogenous supplementations, but no effect was observed in the ileum. Only limited significant effects were observed on plasma and tissue-free AA concentrations, except for an accumulation of glycine in the plasma and tissues from the LPS-GLY group, compared to other groups. Overall, whereas glycine as a nitrogenous control should be used with care, either alanine or casein may be used as the "placebo," with the choice depending on the study to be performed.

Is alpha-ketoisocaproyl-glutamine a suitable glutamine precursor to sustain fibroblast growth?

BACKGROUND: Glutamine is considered an essential nutrient for cellular growth. AIM: To test the suitability of alpha-ketoisocaproyl-Gln (Kic-Gln) as a new glutamine (Gln) precursor to sustain human fibroblast growth. METHODS: [3H] thymidine uptake into cellular DNA of human fibroblasts. Extracellular and intracellular amino acid patterns were determined with peptides and acylated compounds. RESULTS: L-alanyl-L-glutamine (used here as a recognized Gln precursor) promoted DNA synthesis, while N-acetyl-L-glutamine (used here as a negative control since it is known to be a poor Gln precursor) and alpha-ketoisocaproyl-glutamine had no effect. Alanyl-glutamine progressively gave rise to free glutamine in the growth medium. In contrast, glutamine supplied in acylated form was poorly available and did not appear in free form in the medium. In addition, only alanyl-glutamine increased intracellular glutamine and glutamate levels. In contrast, Kic-Gln was able to sustain net protein synthesis as judged by total protein content and reduced intracellular levels of most essential amino acids. CONCLUSION: Kic-Gln appears to be a poor extra-cellular precursor of Gln to sustain cell growth.

The use of alpha-ketoglutarate salts in clinical nutrition and metabolic care.

Theoretically, alpha-ketoglutarate is a precursor of glutamine, a fact that may be of importance given the key regulatory properties of this amino acid. Although the literature suggests that glutamine synthesis accounts only for a marginal part of the disposal of exogenously supplied alpha-ketoglutarate, administered alpha-ketoglutarate has a potent 'sparing' effect on endogenous glutamine pools. When alpha-ketoglutarate is supplied as an ornithine salt, a synergistic effect of the two parts of the molecule increases the synthesis of glutamine or the 'sparing' of endogenous glutamine pools. In addition, alpha-ketoglutarate in combination with ornithine dramatically increases the synthesis of arginine, proline and polyamines, which also play key roles in metabolic adaptation to trauma. The recent literature suggests that the administration of alpha-ketoglutarate in combination with ornithine improves gut morphology and functions, counteracts trauma-induced dysimmunity and exerts anabolic/anticatabolic actions on protein metabolism.

Effects of amino acids on bile acid-dependent and independent bile flow in the isolated perfused rat liver.

BACKGROUND/AIMS: Conflicting data on the effects of amino acids on biliary function led us to investigate their interaction with taurocholate in the perfused rat liver model. METHODS: To investigate the influence of amino acids on the bile acid-independent component of bile flow, 12 livers were perfused with (n = 6) and without (n = 6) amino acid addition from t30 min. For the study of bile acid-dependent bile flow, 24 livers were perfused under 8 experimental conditions according to the perfusate taurocholate concentration (12.5, 25, 37.5 or 50 microM) and whether amino acids were or were not added from t30 min. RESULTS: In the absence of taurocholate, amino acids induced a 40% (p<0.01) decrease in bile flow together with an increase in hepatic water content (17.8%, p< 0.05). Thus, amino acids exert an inhibitory effect on bile acid-independent bile flow despite the postulated cell swelling-dependent increase in bile flow. When livers were perfused at various taurocholate concentrations, amino acids induced, in addition to their inhibitory effect on bile acid-independent bile flow, a significant increase in taurocholate apparent choleretic activity (13.2 microl/micromol vs. 10.6 microl/micromol; p = 0.05), while taurocholate intrinsic clearance was significantly decreased (4.5+/-1.2 ml x min(-1) x g(-1) vs. 6.1+/-1.3 ml x min(-1) x g(-1); p<0.01). CONCLUSIONS: These data suggest that at physiological bile acid concentrations amino acids exert an inhibitory effect on both bile acid-dependent and- independent bile flow, whereas at higher taurocholate concentrations this inhibitory effect disappears, probably because of cell swelling-dependent mechanisms.

Phagocyte functions in stressed rats: comparison of modulation by glutamine, arginine and ornithine 2-oxoglutarate.

The effects of diets supplemented with 6.8 mmol.day-1.kg-1 glutamine, arginine or ornithine 2-oxoglutarate [ornithine alpha-ketoglutarate (OKG), a precursor of both glutamine and arginine] on phagocyte functions [i.e. H2O2 production by leucocytes and secretion of tumour necrosis factor alpha (TNFalpha) by stimulated macrophages] of stressed rats were studied. The relationship between the immunological effects of these amino acids and their plasma and tissue (muscle and intestine) concentrations was also explored. The catabolic model used consisted of injections of dexamethasone (DEX; 1.5 mg.day-1.kg-1) for 5 days. As previously described, DEX suppressed TNFalpha secretion in stimulated macrophages. Supplementation with arginine or OKG, but not glutamine, was able to counteract the DEX effect on TNFalpha secretion. Glutamine, arginine and OKG supplementation increased H2O2 production by monocytes and polymorphonuclear neutrophils from DEX-treated rats. All DEX-treated rats showed plasma and muscle glutamine depletion and also a decrease in the concentration of arginine in the gastrocnemius. Supplementation with glutamine, arginine or OKG was not able to counteract these depletions. It was concluded that glutamine, arginine and OKG improve phagocyte responses during stress, and that glutamine depletion is not necessarily associated with dysimmunity, since no correlation between glutamine tissue pools and the immune state was observed.

Amino acids with anabolic properties.

Experimental studies have clearly demonstrated both the indispensability in stress situations of amino acids, previously considered to be non-essential, and the importance of the specific properties of these same amino acids. Glutamine, arginine and their precursors/metabolites, ornithine and alpha-ketoglutarate, exert anabolic or anticatabolic effects through their involvement in protein metabolism, in the immune response and in cell proliferation. Clinical studies suggest that the supplementation of nutritional therapy with these amino acids can be of significant benefit for injured patients.

Deletion of hydroxyethylstarch from University of Wisconsin solution induces cell shrinkage and proteolysis during and after cold storage of rat liver.

Among the numerous components of the University of Wisconsin (UW) solution used for organ preservation, the usefulness of hydroxyethylstarch (HES), the colloido-osmotic support of this solution, is controversial. The aim of our study was to determine the influence of HES on hepatic metabolism and intracellular hydration state during hypothermic preservation and after reperfusion in a model of isolated perfused rat liver. Three groups of eight livers were perfused either immediately or after 18 hours of cold storage in a UW-based preservation solution with or without HES. Omission of HES results in 1) a stimulation of protein degradation shown by the marked increase in branched-chain amino acid (BCAA) release (211 +/- 55 vs. 87 +/- 28 nmol/min/g; P < .05, modified UW group vs. UW group), 2) an increase in oxygen consumption (81.7 +/- 4.8 vs. 61.5 +/- 5.0 micromol/h/g; P < .05), 3) a decrease in glucose production (2.3 +/- 0.6 vs. 5.0 +/- 0.6 micromol/min/g; P < .05), and 4) a reduction in intracellular volume (414 +/- 36 vs. 557 +/- 41 microL/g; P < .05). We conclude that HES plays an important role in liver preservation by limiting proteolysis, possibly through the observed preservation of cell volume.

Enteral administration of ornithine alpha-ketoglutarate or arginine alpha-ketoglutarate: a comparative study of their effects on glutamine pools in burn-injured rats.

OBJECTIVES: Ornithine alpha-ketoglutarate has proved to be an efficient nutritional support in trauma situations, especially after burn injury. To determine whether the action of ornithine alpha-ketoglutarate is due to its alpha-ketoglutarate moiety (as a glutamine precursor), we studied the effects of alpha-ketoglutarate administered to rats as ornithine alpha-ketoglutarate, or in combination with arginine salt (arginine alpha-ketoglutarate), as the two closely related amino acids have similar metabolic behavior. DESIGN: Prospective, randomized trial. SETTING: Animal laboratory. SUBJECTS: Forty-six male Wistar rats, weighing approximately 90 g. INTERVENTIONS: Rats were burned over 20% of their body surface area, starved for 24 hrs, with water ad libitum, and then enterally refed for 48 hrs using Osmolite (210 kcal/kg/day, 1.2 g of nitrogen/kg/day), supplemented with one of the following: a) an amount of glycine isonitrogenous to ornithine alpha-ketoglutarate (group 1); b) 5 g of monohydrated ornithine alpha-ketoglutarate/kg/day (group 2); c) an amount of arginine alpha-ketoglutarate isonitrogenous to ornithine alpha-ketoglutarate (group 3); or d) an amount of arginine alpha-ketoglutarate isomolar to ornithine alpha-ketoglutarate (group 4). MEASUREMENTS AND MAIN RESULTS: We measured amino acid concentrations in plasma, muscle, and liver, and plasma urea concentration. At refeeding, ornithine alpha-ketoglutarate increased plasma glutamine concentration (p < .05 vs. the three other groups), and counteracted the increase in plasma phenylalanine concentration. In muscle, although the three alpha-ketoglutarate combinations induced similar increases in the glutamate pool, ornithine alpha-ketoglutarate induced the highest increase in glutamine (7.0 +/- 0.3 vs. 5.4 +/- 0.3 micromol/g in group 3, 6.3 +/- 0.3 in group 4, and 4.6 +/- 0.2 in group 1, p < .01 between group 2 and groups 3 or 1). Also, only ornithine alpha-ketoglutarate increased liver glutamine concentration. Finally, isomolar arginine alpha-ketoglutarate increased plasma urea concentration (+50% vs. the three other groups, p < .01). CONCLUSIONS: Our results demonstrate, for the first time, the following: a) the action of ornithine alpha-ketoglutarate as a glutamine precursor cannot solely be ascribed to alpha-ketoglutarate since arginine alpha-ketoglutarate combinations did not exhibit this effect to the same extent; and b) the action of ornithine alpha-ketoglutarate is not due to its nitrogen content since isonitrogenous arginine alpha-ketoglutarate did not reproduce the effects of ornithine alpha-ketoglutarate.