Immunonutrition: the role of arginine

Nutrition Vol. 14, Nos. 7/8, 1998

Immunonutrition: The Role of Arginine DENIS EVOY, FRCSI, MICHAEL D. LIEBERMAN, MD, THOMAS J. FAHEY III, MD, AND JOHN M. DALY, MD, FACS From the Department of Surgery, The New York Hospital, Cornell Medical Center, New York, New York, USA ABSTRACT

Arginine, a non-essential amino acid, has a role in stimulating the host’s immune system. Animal and human studies suggest outcome benefit to the use of supplemental dietary arginine. Nutrition 1998;14:611– 617. ©Elsevier Science Inc. 1998 Key words: arginine, macrophage, nitric oxide, immunity, infection

INTRODUCTION

ARGININE BIOCHEMISTRY

Immunonutrition may be defined as the effect of the provision of specific nutrients on immune function. The amino acid arginine is of particular interest in this regard as it has been demonstrated to have an array of desirable biological properties. Arginine stimulates anabolic hormone release, improves nitrogen balance, and has been demonstrated to be immunostimulatory and thymotrophic.1–3 Arginine has fundamental roles in nitrogen metabolism, creatine, and polyamine synthesis, and is the major substrate for the production of nitric oxide. Arginine was first isolated in crystalline form and named in 1886. Its presence in animal protein was documented in 1895.4 In 1930, Scull and Rose5 demonstrated that arginine was synthesized by the growing rat. They showed that arginine in the carcasses of rats fed a low arginine diet contained two to three times as much arginine as had been consumed.5 In both children and adults arginine is regarded as a nonessential amino acid.6 A deficiency of any indispensable amino acid leads to a decrease in food intake, reduced growth, and negative nitrogen balance. Rats deprived of arginine do not lose weight, but gain weight more slowly than rats allowed arginine in the diet. Rose stated that ‘‘arginine can be synthesized by the rat but not at a sufficiently high rate to meet maximum growth. Its classification, therefore, as essential or nonessential is purely a matter of definition.’’7 However, rats fed diets without arginine develop fatty livers and altered glucose tolerance as well as abnormalities of nitrogen metabolism.8,9 In rats, mice, hamsters, guinea pigs, and rabbits, the excretion of citric and orotic acid is a sensitive indicator of arginine availability. Orotic acid production is reduced or prevented by inclusion of the urea cycle intermediates, arginine, citrulline, or ornithine, in the diet.10 For many mammals, arginine is essential for normal growth and development of the young and is regarded as nonessential only in the fully grown adult.11

The biochemistry of arginine is complex and involves many key metabolic pathways and organ systems (Fig. 1). Glutamate and glutamine are the amino acids that allow the incorporation of ammonia into amino acids and other organic biomolecules in the eukaryotic cell. In most cell types one or both of these amino acids are present at elevated concentrations, often a half log higher than other amino acid levels. In animals, glutamate is synthesized from the substrates of a-ketoglutarate and NH41 by the action of L-glutamate dehydrogenase, and plasma glutamate levels are maintained by the transamination of a-ketoglutarate during amino acid metabolism. Glutamine is a nonessential amino acid that can be synthesized by the addition of NH3 to glutamate, and is a major constituent of normal dietary protein intake. Glutamine is the preferred energy source of gastrointestinal mucosal cells. Much of the glutamine utilized by the intestine is metabolized to citrulline and released into the portal circulation. Citrulline is taken up by the kidney and converted into arginine. The production of citrulline by the intestine, arginine by the kidney, and glucose and urea by the liver are metabolically linked and this cycle (Fig. 2) is regulated by differential activity of enzymes present in each of these tissues.12 Arginine is an integral constituent of the urea cycle. In 1932, Krebs4 identified the urea cycle and noted that urea production from ammonia by the liver was greatly accelerated by adding any one of three amino acids; arginine, citrulline, or ornithine. The addition of ammonia to ornithine synthesizes citrulline; the addition of ammonia to citrulline synthesizes arginine; the loss of these two NH groups in the form of urea from arginine is used to synthesize ornithine. Arginase activity in the liver is very high; therefore, hepatic arginine levels are very low, and there is very little arginine released into the circulating amino acid pool by the liver. Tissue arginase activity and tissue arginine content are inversely related. Kidney and muscle have 1% the arginase content of the liver and 10 times the arginine content. Most of the arginine formed in the liver is cleaved to form urea.

Correspondence to: John M. Daly, MD, FACS, Lewis Atterbury Stimson Professor and Chairman, Department of Surgery, The New York Hospital, Cornell Medical Center, 525 East 68th Street, Room F-739, New York, NY 10021, USA.

Nutrition 14:611– 617, 1998 ©Elsevier Science Inc. 1998 Printed in the USA. All rights reserved.

0899-9007/98/$19.00 PII S0899-9007(98)00005-7

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FIG. 1. The biochemistry of arginine.

The urea cycle straddles the mitochondrial-cytoplasmic divide (Fig. 3). Ammonia is transported in the blood stream to the liver from the periphery in the form of glutamine (transaminated glutamate) or alanine (transaminated pyruvate).13 It is then delivered to the mitochondria of the hepatocyte and is immediately utilized with bicarbonate to form carbamoyl phosphate. The enzyme catalyzing this reaction is a mitochondrial enzyme, carbamoyl phosphate synthetase I. This is a key regulatory step in the urea cycle that is ultimately controlled by the concentration of arginine. Arginine may be regarded as a urea cycle intermediate that accumulates when urea production is insufficient to accommodate the ammonia produced by amino acid catabolism. The accumulation of arginine upregulates the urea cycle. Citrulline is a major end product of intestinal glutamine metabolism and is released by the intestine into the circulating amino acid pool. It is estimated that more than 25% of intestinal glutamine is released as citrulline. Renal citrulline uptake is approximately 80% of the amount released by the intestine.14 The kidney

FIG. 2. The intestinal production of citrulline, the kidney production of arginine, and the liver production of glucose and urea are regulated by the differential activities of an array of enzymes present in each tissue.

then synthesizes arginine from citrulline,15 and releases it into the blood stream allowing peripheral uptake of arginine and protein synthesis. The kidney, therefore, is the primary organ responsible for maintenance of the plasma arginine level. In our laboratory the standard fasting plasma arginine level approximates 75 mM/L (preoperative patients) and fluctuates according to the fasting or fed state.3 The average intake of arginine in the diet is estimated to be 5 g daily.11 ARGININE AND IMMUNE FUNCTION

Two pathways of arginine metabolism have been identified as being critical to the immunomodulatory actions of arginine in vivo. First, the ‘‘arginase’’ pathway, in which arginine is converted to urea and ornithine, generates polyamines by the action of ornithine decarboxylase. This route of polyamine synthesis may be the mechanism whereby arginine augments lymphocyte mitogenesis.16 Induction of arginase has also been proposed as the effector pathway in arginine-dependent macrophage-mediated tumor cell cytotoxicity.17 Second, arginine is the sole substrate for nitric oxide synthesis in biologic systems. Nitric oxide is synthesized from arginine by the action of nitric oxide synthase resulting in the formation of nitric oxide and citrulline. Nitric oxide is a ubiquitous molecule with important roles in the maintenance of vascular tone, coagulation, the immune system, and the gastrointestinal tract, and has been implicated as a factor in disease states as diverse as sepsis, hypertension, and cirrhosis.18,19 Arginine is the starting substance in the synthesis of polyamines, which include spermine. Polyamines appear to play a key role in cell division, DNA replication, and regulation of the cell cycle.20,21 Arginine is also the only amino acid that provides the amidino group for synthesis of creatine, a major reserve of high energy phosphate for regeneration of ATP in muscle. The pathways for de novo biosynthesis of pyrmidines and arginine are interrelated by the common substrate, carbamoyl phosphate. In

IMMUNONUTRITION: THE ROLE OF ARGININE

613

FIG. 3. The urea cycle straddles the mitochondrial-cytoplasmic divide.

addition, the urea cycle intermediate, arginosuccinate, has important links with the citric acid cycle.22 Arginine, therefore, plays an important role in protein synthesis, urea cycle metabolism, the synthesis of the high-energy compounds, creatine and creatine phosphate, polyamine synthesis, and the production of nitric oxide. The use of arginine, therefore, is of particular interest in the field of immunonutrition and many studies have been undertaken attempting to exploit the desirable biological properties of arginine. Studies by Barbul et al.23 have demonstrated that administration of dietary supplemental arginine to injured rats results in accelerated wound healing. Increased wound tensile strength and collagen deposition were demonstrated. Wound healing was improved in the arginine parenterally supplemented group as assessed by fresh wound strip breaking strength, fixed breaking strength, and the amount of reparative collagen deposition indexed by the hydroxyproline content of the implanted sponges.23 These findings are explained by the fact that in the growing animal, arginine is used for protein synthesis; in the injured animal there is an increased requirement for arginine to synthesize reparative connective tissue. Arginine-supplemented rats also had improved thymic function as assessed by thymic weight, the total number of thymic lymphocytes in each thymus, and the mitogenic reactivity of thymic lymphocytes to phytohemagglutinin and concanavalin A. Thymic involution is usually associated with injury in the rat. Of note, arginine supplementation in uninjured rats is associated with an increase in thymic weight. This response was accompanied by an increase in total lymphocyte counts and enhanced blastogenic response in the noninjured rats. Only rarely has any substance been identified that augments a normal cellular function. The effect of arginine on wound healing in humans has also been assessed. Barbul et al.2 implanted polytetrafluoroethylene tubing into the subcutaneous tissue of volunteers. After 2 wk of

arginine supplementation (25 g/d), the amount of hydroxyproline in the tubing was measured as an index of collagen synthesis. Hydroxyproline levels doubled in arginine-supplemented subjects. Arginine supplementation also increased peripheral lymphocyte mitogenesis in response to phytohemagglutinin and concanavalin A in humans.2 Other studies have documented similar results in a healthy elderly human population.24 The beneficial effect of arginine on the immune system appears distinct from its more moderate effect on nitrogen metabolism as only minor improvements in postoperative nitrogen balance in patients are observed compared to the more robust upregulation of immune parameters.3 The effect of arginine supplementation in the postoperative period has been investigated.3 Thirty patients with gastrointestinal cancer were randomized to receive either arginine 25 g/d or isonitrogenous glycine 43 g/d for 7 d after surgery as part of an enteral diet. Arginine supplementation resulted in an enhanced response by peripheral blood lymphocytes to mitogens on the 7th d after operation compared with the 1st d, and was also associated with an increased number of circulating CD41 T cells. Arginine supplementation increased plasma arginine levels from a base line level of 75 mM/L to 200 mM/L after 7 d of arginine supplementation. There was a similar magnitude of increase in plasma ornithine levels. There was no difference in clinical outcome detected between groups in this study. There has been a trend to supplement enteral diets with arginine, RNA, and v-3 fatty acids, in order to determine if these agents in combination will be of greater benefit to the patient than any single agent. Sixty adult patients with gastrointestinal cancers were randomized to receive supplemental or standard diet via jejunostomy beginning on the first postoperative day (goal 5 25 kcal z kg21 z d21) until hospital discharge. Patients were also randomized to receive or not receive enteral jejunostomy feedings in the 12- to 16-wk recovery and radiation/chemotherapy periods.

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IMMUNONUTRITION: THE ROLE OF ARGININE TABLE I.

THE INCIDENCE OF SEPTIC COMPLICATIONS IN PROSPECTIVE RANDOMIZED TRIALS COMPARING ARGININE-SUPPLEMENTED PATIENTS TO NONSUPPLEMENTED CONTROLS Infectious complications Study cohort

Author

Year

N

Experimental diet

Control diet

Experimental

Control

11%** 10%** 10%NS per patient mean 6 SD 0.74 6 0.97NS 16%** 6%** 0%** Intra-abdominal abcess per patient mean 5 0.4**

37% 43% 15% per patient mean 6 SD 0.98 6 1.27 56% 41% 11% Intra-abdominal abcess per patient mean 5 1.1

Postop cancer Postop cancer Postop cancer Mixed

Daly25 Daly25 Braga27 Bower26

1992 1995 1996 1995

85 60 60 296

Ent* Ent* Ent* Ent*

Ent Ent Ent Ent

Trauma patients Trauma patients Trauma patients

Brown29 Kudsk28 Moore42

1994 1996 1994

37 33 98

Ent Ent* Ent*

Ent Ent Ent

Burn patients

Gottschlich43

1990

50

Ent*

Ent

* Ent: Arginine, v-3 fatty acids and nucleotide supplementation. ** P , 0.05 (References: 25–29, 42– 43). NS, not significant.

Infectious wound complications occurred in 10% of the supplemented group compared with 43% of the standard group. We conclude that enteral feeding, supplemented with immune modulating agents including arginine, decreases postoperative infectious wound complications compared with standard enteral feeding.25 A multicenter, prospective, randomized clinical trial26 using early enteral administration of a standard formula supplemented with arginine, nucleotides, and fish oil in intensive care unit

patients has been completed. From intensive care units in eight different hospitals, 296 trauma, surgery, or septic patients were eligible for analysis. Patients receiving the supplemented formula had a significant (P 5 0.0001) increase in plasma arginine and ornithine concentrations by day 7. The mortality rate was not different between groups. However, for patients stratified as septic, the median length of hospital stay was reduced by 10 d (P , 0.05). In this study there was no significant difference in infectious

FIG. 4. In A/J mice, bearing C1300 murine neuroblastoma, arginine supplementation significantly (P , 0.05) retarded tumor growth.

IMMUNONUTRITION: THE ROLE OF ARGININE

615

FIG. 5. IL-2 treated A/J mice, bearing C1300 murine neuroblastoma receiving supplemental dietary arginine compared with those receiving glycine results in prolonged host survival.36 GLY, glycerine; PBS, phosphate-buffered saline; ARG, arginine; IL-2, interleukin-2.

complications between the total numbers of supplemented and non-supplemented patients (2001). However, a subgroup of patients who were able to complete the total planned intake of supplemental diet in the first 7 d of enteral supplementation were compared to patients who received the same quantity of standard enteral formula; a total of 100 patients. There was a significant reduction in infectious complications (mean 6 SD 0.54 6 0.78 versus 0.94 6 0.87) in this supplemented subgroup.26 Braga et al.27 demonstrated no difference in the incidence of infectious complications in patients undergoing surgery for malignancy supplemented postoperatively with arginine, RNA, and v-3 fatty acids compared to control patients receiving standard enteral nutrition. However, the infectious complications were assessed as being significantly less severe in the supplemented group.27 Kudsk et al.28 prospectively randomized 35 severely injured trauma patients to an enteral diet containing glutamine, arginine, v-3 fatty acids, and nucleotides or to an isonitrogenous, isocaloric diet to investigate the effect of septic outcome. A third group of patients, without enteral access but eligible by severity of injury, served as unfed controls and were studied prospectively to determine the risk of infection. Patients were evaluated for septic complications, antibiotic usage, and hospital and intensive care unit stay. Significantly fewer major infectious complications (6%) developed in patients who received the supplemented diet than patients in the isonitrogenous group (41%, P 5 0.02) or the control group (58%, P 5 0.002). Hospital stay was also significantly lower in patients receiving the supplemented diet than the other two groups. Unfed patients had the highest complication rate in this study.28 Other prospective, randomized clinical trials in an intensive care setting have demonstrated similar results using arginine, linolenic acid, beta-carotene, and hydrolyzed protein for up to 10 d.29,30 The results of prospective, randomized clinical trials involving the use of supplemental arginine are summarized in Table I.

Innate host cellular cytotoxicity, mediated in part by natural killer (NK) and lymphokine-activated killer (LAK) cells, is thought to play an important role in the inhibition of tumor growth and the reduction of metastatic spread. Arginine supplementation has been shown to enhance NK and LAK cytotoxicity.31 The effect of arginine on tumor growth has been examined over the past 50 y. In vivo animal studies have demonstrated both stimulation and inhibition of tumor growth.32 Park et al.33 has described an increase in tumor proliferation markers in patients with breast cancer treated with dietary arginine supplements. Human pancreatic cancer cell growth is inhibited by the arginine antimetabolite L-canavanine.34 Arginine supplementation in the tumor-bearing host may inhibit the growth and dissemination of immunogenic tumors by upregulating NK and LAK function. Reynolds et al.35 demonstrated that in non–tumor-bearing A/J mice, 1% arginine supplementation significantly enhanced thymic weight and spleen cell mitogenesis. Supplemental 1% arginine, when compared with 1.7% glycine, also enhanced interferoninduced NK cell activity, LAK cell generation, and macrophage cytotoxicity. In A/J mice, bearing C1300 murine neuroblastoma, 1% arginine enhanced splenocyte mitogenesis and IL-2 production and significantly (P , 0.05) retarded tumor growth as shown in Figure 4, and prolonged median survival time compared with glycine or no supplementation.35 A subsequent study assessed supplemental dietary arginine in mice as a means to potentiate the host antitumor response to interleukin-2 (IL-2) in the same murine neuroblastoma model. A/J mice received 1% arginine or isonitrogenous glycine (1.7%) in addition to a regular diet 14 d before subcutaneous inoculation with C1300 neuroblastoma cells. Twenty-four hours later, animals were administered IL-2 three times a day or saline intraperitoneally for 4 d. On days 4 and 10 post-C1300 neuroblastoma inoculation, mice were killed for assessment of NK cell and tumor specific cytotoxicity. Remaining animals were followed for tumor incidence, tumor growth, and duration of host survival. IL-2

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IMMUNONUTRITION: THE ROLE OF ARGININE

therapy in mice receiving dietary arginine compared with those receiving glycine resulted in significantly augmented NK cell cytotoxicity. C1300 neuroblastoma engraftment and growth were retarded, and prolonged host survival (P , 0.05) were also demonstrated in the IL-2, arginine-treated animals as shown in Figure 5. This study demonstrated that supplemental dietary L-arginine enhances lymphocyte cytotoxic mechanisms and may potentiate IL-2 immunotherapy.36 A separate study by Yeatman et al.37 examined the effects of dietary arginine on the growth of a murine colon tumor, metastatic to the liver in a model of advanced neoplastic disease. An arginine-depleted diet inhibited tumor growth. This effect was attributed to the dependence of this particular tumor model on arginine for growth in vitro and in vivo. Arginine has multiple, potent secretagogue activities on several endocrine glands.22 The relationship of the metabolic effects to the secretagogue properties of arginine is unclear. Arginine infusion induces the secretion of growth hormone, insulin, glucagon, prolactin, and somatostatin.38 – 41 An intact hypothalamo-pituitary axis is essential in order for arginine to mediate its effects.1 However, no significant differences between arginine enterally supplemented and nonsupplemented patient groups in mean insu-

lin, glucagon, cortisol, and growth hormone levels were found in a separate study.3 In this study, mean somatomedin C levels were significantly higher in the arginine group on day 7 suggesting a stimulatory effect of arginine on the pituitary. SUMMARY

Clearly, arginine has great potential as an immunomodulator and may prove useful in catabolic conditions such as severe sepsis and postoperative stress. There is a body of evidence suggesting that supplemental arginine upregulates immune function and reduces the incidence of postoperative infection. More modest improvements in nitrogen balance have been observed. Tumor response to arginine appears to depend on the immunogenicity of the particular tumor and on the requirement of arginine by that tumor as a growth substrate. Of note, ornithine shares the thymotrophic, immunostimulatory and secretagogue effects of arginine.3 It is, therefore, likely that these compounds share the same cellular mechanism of action or that arginine acts via increasing the concentration of available ornithine. The role of arginine in the injured patient and in the tumor-bearing host demands additional study based on the promising experimental evidence regarding the supplemental use of arginine.

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20. Pegg AE, McCann PP. Polyamine metabolism and function. Am J Physiol 1982;243:C212 21. Cynober L. Can arginine and ornithine support gut functions? Gut 1994;35(suppl 1):S42 22. Barbul A. Arginine: biochemistry, physiology, and therapeutic implications. JPEN 1986;10:227 23. Barbul A, Fishel RS, Shimazu S, et al. Intravenous hyperalimentation with high arginine levels improves wound healing and immune function. J Surg Res 1985;38:328 24. Hurson M, Regan MC, Kirk SJ, Wasserkrug BL, Barbul A. Metabolic effects of arginine in a healthy elderly population. JPEN 1995;19:227 25. Daly JK, Weintraub FN, Shou J, Rosato EF, Lucia M. Enteral nutrition during multimodality therapy in upper gastrointestinal cancer patients. Ann Surg 1995;221:327 26. Bower RH, Cerra FB, Bershadsky B, et al. Early enteral administration of a formula (Impact) supplemented with arginine, nucleotides, and fish oil in intensive care unit patients: results of a multicenter, prospective, randomized, clinical trial. Crit Care Med 1995;23:436 27. Braga M, Vignali A, Gianotti L, et al. Immune and nutritional effects of early enteral nutrition after major abdominal operations. Eur J Surg 1996;162:105 28. Kudsk KA, Minard G, Croce MA, et al. A randomized trial of isonitrogenous enteral diets after severe trauma. An immune-enhancing diet reduces septic complications. Ann Surg 1996;224:531 29. Brown RO, Hunt H, Mowatt-Larssen CA, et al. Comparison of specialized and standard enteral formulas in trauma patients. Pharmacotherapy 1994;14:314 30. Cerra FB, Lehmann S, Konstantinides N, et al. Improvement in immune function in ICU patients by enteral nutrition supplemented with arginine, RNA, and menhaden oil is independent of nitrogen balance. Nutrition 1991;7:193 31. Brittenden J, Park KG, Heys SD, et al. L-arginine stimulates host defenses in patients with breast cancer. Surgery 1994;115:205 32. Hester JE, Fee WE. Effect of arginine on growth of squamous cell carcinoma in the C3H/KM mouse. Arch Otolaryngol Head Neck Surg 1995;121:193 33. Park KG, Heys SD, Blessing K, et al. Stimulation of human breast cancers by dietary L-arginine. Clin Sci 1992;82:413 34. Swaffar DS, Ang CY, Desai PB, Rosenthal GA. Inhibition of the growth of human pancreatic cancer cells by the arginine antimetabolite L-canavanine. Cancer Res 1994;54:6045 35. Reynolds JV, Daly JK, Shou J, et al. Immunologic effects of arginine supplementation in tumor-bearing and non-tumor-bearing hosts. Ann Surg 1990;211:202

IMMUNONUTRITION: THE ROLE OF ARGININE 36. Lieberman MD, Nishioka K, Redmond BP, Daly JM. Enhancement of interleukin-immunotherapy with L-arginine. Ann Surg 1992;215:157 37. Yeatman TJ, Risley GL, Brunson ME. Depletion of dietary arginine inhibits growth of metastatic tumor. Arch Surg 1991;126:1376 38. Merimee TJ, Lillicrap DA, Rabinowitz D. Effect of arginine on serum levels of human growth hormone. Lancet 1965;2:668 39. Palmer JP, Walter RM, Ensinck JW. Arginine-stimulated acute phase of insulin and glucagon. 1. In normal man. Diabetes 1975;24:735 40. Rakoff JS, Siver UA, Sinha YN. Prolactin and growth hormone release in response to sequential stimulation by arginine and TRF. J Clin Endocrinol Metab 1973;37:641

617 41. Utsumi M, Makimura H, Isihara K. Determination of the immunoreactive somatostatin in rat plasma and responses to arginine, glucose and glucagon infusion. Diabetologia 1979;17:319 42. Moore FA, Moore EE, Kudsk KA, et al. Clinical benefits of an immune-enhancing diet for early postinjury enteral feeding. J Trauma 1994;37:607 43. Gottschlich MM, Jenkins M, Warden GD, et al. Differential effects of three enteral dietary regimens on selected outcome variables in burn patients. JPEN 1990;14:225 44. Gardiner KR, Gardiner RE, Barbul A. Reduced intestinal absorption of arginine during sepsis. Crit Care Med 1995;23:1227