Effects of dietary arginine supplementation on antibody production
and antioxidant enzyme activity in burned mice
Huey-Fang Shang
a, Hui-Ju Tsai
b, Wan-Chun Chiu
b, Sung-Ling Yeh
b,∗ aDepartment of Microbiology and Immunology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan, ROCbInstitute of Nutrition and Health Science, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan, ROC
Accepted 20 August 2002
Abstract
This study investigated the effect of arginine (Arg) supplementation on specific antibody production and antioxidant enzyme activities in burned mice vaccinated with detoxified Pseudomonas exotoxin A linked with the outer membrane proteins I and F, named PEIF. Also, the survival rate of burned mice complicated with Pseudomonas aeruginosa was evaluated. Experiment 1: Thirty BALB/c mice were assigned to two groups. One group was fed a control diet with casein as the protein source, while the other group was supplemented with 2% Arg in addition to casein. The two groups were isonitrogenous. The mice were immunized twice with PEIF, and the production of specific antibodies against PEIF was measured every week. After 8 weeks, all mice received a 30% body surface area burn injury. Mice were sacrificed 24 h after the burn. The antioxidant enzyme activities and lipid peroxides in the tissues as well as the specific antibody production were analyzed. Experiment 2: Twenty-eight mice were divided into two groups and vaccinated as described in experiment 1. After the burn the mice were infected with P. aeruginosa, and the survival rate was observed for 8 days. The results demonstrated that antioxidant enzyme activities and lipid peroxides in tissues were significantly lower in the Arg group than in the control group after the burn. The production of specific antibodies against P. aeruginosa significantly increased in the Arg group at 4 and 7 weeks after immunization, and 24 h after the burn. The survival rates of vaccinated burned mice after bacterial infection did not significantly differ between the two groups. These results suggest that vaccinating mice with Arg supplementation may enhance humoral immunity and attenuate the oxidative stress induced by burn injury. However, Arg supplementation did not improve survival in vaccinated mice complicated with P. aeruginosa infection.
© 2003 Elsevier Science Ltd and ISBI. All rights reserved.
Keywords: Burns; Arginine; Antioxidant enzyme activity; Antibody; Vaccination; P. aeruginosa
1. Introduction
A burn injury can give rise to a post-traumatic inflam-matory disease. Major burns are often associated with sec-ondary damage to tissues distant from the injured skin[1–4]. Nishigaki et al. [5] reported that lipid peroxide levels in-crease in burned rat skin, and that lipid peroxide generated in the burn wound accumulates in the liver, lung, kidney, and gut of injured animals. In addition, a characteristic and critical feature of burn injury is a decrease in host resistance to infection. This complication has been related to a depres-sion of both humoral and cellular components of the host defense system[6–9].
∗Corresponding author. Tel.:+886-2-27361661x6550-115; fax:+886-2-27373112.
E-mail address: [email protected] (S.-L. Yeh).
Arginine (Arg) is a semi-essential amino acid. Previous reports have shown that Arg stimulates anabolic hormone release and improves nitrogen balance[10,11]. Studies have also revealed that Arg enhances T lymphocyte responses for surgical patients [10], accelerates wound healing, and improves survival when Arg is supplemented in the diet of humans and injured animals[12–15]. A report by Stinnett et al.[16]showed that after severe burn injury, plasma Arg declined 30–40%. Dietary Arg supplementation replenishes the Arg level in plasma [17]. A study by Cui et al. [18] showed that dietary Arg supplementation promotes protein anabolism and attenuates muscle protein catabolism after thermal injury. Previous work in our laboratory demon-strated that Arg supplementation attenuates oxidative stress at the hypercatabolic stage after burn injury. Also, a better in vitro macrophage response was observed[19]. Arg is con-sidered a conditionally essential amino acid in burn patients [17,20].
0305-4179/03/$30.00 © 2003 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 2 ) 0 0 2 4 3 - 7
Pseudomonas aeruginosa is an opportunistic pathogen
that often infects burn patients[21,22]. Therapy for P.
aerug-inosa infection is hindered by its well-known antibiotic
resistance[23]. Production of specific antibodies is impor-tant for resolving bacterial infections, because antibodies neutralize the bacterial toxins and attract phagocytic cells to ingest and kill the bacteria. Saito et al. [24] demon-strated that Arg supplementation improved survival rates in a non-infected burned animal model. To our knowledge, there is no study, so far, investigating the effect of Arg sup-plementation on the production of specific antibodies and the potential benefit of Arg on survival rates in burned ani-mals complicated with infection. We have designed a novel vaccine, PEIF, against P. aeruginosa, which can effectively
block P. aeruginosa challenge in burned mice [25]. The
chimeric protein is composed of the receptor binding and membrane translocation domains of Pseudomonas exotoxin A (PE) linked with the outer membrane proteins I and F, to-gether designated as PEIF[25]. In this study, we immunized mice with this novel vaccine against P. aeruginosa before burn injury to investigate whether Arg supplementation has beneficial effects on antioxidant enzyme activity, T lym-phocyte subpopulations, and specific antibody production against PEIF. In addition, the survival rate in vaccinated burned mice complicated with a lethal dose of P. aeruginosa was also evaluated.
2. Materials and methods
2.1. Animals
Male BALB/c mice weighing 10–15 g (4 weeks of age) were used in this study. All mice were housed in temperature- and humidity-controlled rooms, and allowed free access to standard chow for 1 week prior to the ex-periment. Animals included in this study were kept under standard experimental animal care protocols.
2.2. Study protocol 2.2.1. Experiment 1
Thirty mice were randomly assigned to two groups, 15 mice to a group. One group was fed a control diet (con-trol), in which all amino acids were provided by casein. The other group was fed arginine (Arg), by which 2% of total kcal was Arg in addition to casein. Both diets were isoni-trogenous (Table 1). Mice were anesthetized with ether, and blood was taken from the retrobulbar vessels before immu-nizing with the novel PEIF vaccine against P. aeruginosa. The production and purification of the recombinant PEIF protein followed procedures described previously[25]. The emulsified vaccine was prepared by mixing the purified re-combinant PEIF protein with an equal volume of complete Freund’s adjuvant, and then each mouse was vaccinated sub-cutaneously at a dose of 2g per mouse on day 1. A booster
Table 1
Composition of the experimental diets (g/kg)
Component Arg Control
Casein 200 248 Arginine 24 – Protein N 39.7 39.7 Soybean oil 50 50 Corn starch 470 446 Salt mixturea 35 35 Vitamin mixtureb 10 10 Methylcellulose 30 30 Choline chloride 1 1 dl-Methionine 3 3 Sucrose 200 200
aSalt mixture contains the following (mg/g): calcium phosphate
dia-basic, 500; sodium chloride, 74; potassium sulfate, 52; potassium citrate monohydrate, 220; magnesium oxide, 24; manganese carbonate, 3.5; fer-ric citrate, 6; zinc carbonate, 1.6; curpfer-ric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01; chromium potassium sulfate, 0.55.
bVitamin mixture contains the following (mg/g): thiamin
hydrochlo-ride, 0.6; riboflavin, 0.6; pyridoxine hydrochlohydrochlo-ride, 0.7; nicotinic acid, 3; calcium pantothenate, 1.6; d-biotin, 0.02; cyanocobalamin, 0.001; retinyl palmitate, 1.6;dl-␣-tocopherol acetate, 20; cholecalciferol, 0.25; menaquinone, 0.005.
injection was given at a dose of 4g per mouse of PEIF
emulsified with an equal volume of incomplete Freund’s ad-juvant on day 28. Before the burn, immunized mice were bled (50l) from the retrobulbar vessels on days 21, 28, 35, 42, 49 and 56. The respective sera were isolated and stored at−70◦C until assay. After 8 weeks, a modification of the burned mouse procedure was used[26,27]. Mice were anes-thetized with sodium pentobarbitol (0.71l/g body weight) and shaved dorsally prior to burning. A Teflon template with a precisely cut window (2.5 cm × 3 cm) was pressed firmly against the shaved back. Ethanol (95% (v/v), 0.5 ml) was evenly spread over the area of the back outlined by the win-dow, ignited, and allowed to burn for 15 s[25,26]. Animals were immediately resuscitated by an intraperito6neal injec-tion of sterile 0.9% saline (10 ml/100 g body weight)[28]. This procedure produced a full-thickness burn injury on ap-proximately 30% of the total body surface area. They were deprived of food for 24 h with only free access to water, in order to induce a hypermetabolic state in the burned mice [29]. These experimental conditions simulate metabolic dis-orders observed in burn patients [29,30]. Mice were anes-thetized and sacrificed by cardiac puncture 24 h after the burn. Blood samples for analysis of T lymphocyte subpop-ulations were collected in tubes containing heparin, and other blood samples were centrifuged to isolate the sera. Other tissues including liver, lungs, and kidney were rapidly
excised. All samples were stored at −70◦C until being
assayed.
2.2.2. Experiment 2
Twenty-eight mice were divided into two experimental groups, with each group containing 14 mice. All mice were immunized twice with the novel PEIF vaccine against P.
aeruginosa and were fed control or Arg diets for 8 weeks
as described in experiment 1. After 8 weeks, burn injury was induced and P. aeruginosa strain PAO1 (ATCC 15692; in 0.2 ml PBS with about 2× LD50 of 3.2 × 105CFU) was
immediately subcutaneously injected into the burned area. The mice were also deprived of food for 24 h with only free access to water as mentioned above. Survival of the burned mice was noted every 6 h in the first 3 days, and then every 12 h until the end of 8 days.
2.3. Measurements of antioxidant enzymes and TBARS
A 15% tissue homogenate was prepared at 4◦C in 0.01 M phosphate buffer (pH 7.4) with 1.15% KCl, using a
homog-enizer [31]. Homogenates were centrifuged at 12000× g
for 20 min to remove cell debris and mitochondria. The su-pernatant was used for analysis of superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities (en-zyme kits of Randox, Antrim, Ireland) as described pre-viously [32]. Protein concentrations of supernatants were measured using Lowry et al.’s method[33]. The production of thiobarbituric acid-reactive substances (TBARS, assumed to be mainly malondialdehyde and its precursors) in mouse liver, lung, and kidney homogenates was determined by the method of Uchiyama and Mihara[34]. The molar extinction coefficient of malondialdehyde was assumed to be 156,000 [31].
2.4. Analysis of specific antibody production against PEIF
The specific antibody production of vaccinated mice
was measured by ELISA as described previously [35].
Briefly, purified recombinant PEIF protein was coated on polyvinylchloride, flat-bottom, 96-well Falcon microtiter plates overnight at 4◦C with a protein concentration of
3g/ml in coating buffer (pH 9.6 carbonate buffer). The
coated plates were then blocked with 0.5% BSA–PBS. Mouse sera from each group were diluted 1000-fold with
0.5% BSA–PBS, and 50l of diluted sera was added to
the coated well and incubated for 1 h at 37◦C. Bound spe-cific antibodies were detected using peroxidase-conjugated
goat anti-mouse secondary antibody (Sigma). After
three washings, 100l of substrate solution (0.54 mg/ml 2,2-azinobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and 0.03% H2O2 in 0.1 M citric acid) was added to each
well, and the absorbance was read after 15 min using a mi-croplate reader at 405 nm. Normal mouse serum was used as the negative control.
2.5. Analysis of T lymphocyte subpopulations
Flow cytometry was used to determine the
propor-tions of CD4+ and CD8+ T lymphocytes in fresh blood.
One hundred microliters of blood was incubated for 15 min at 4◦C with 10l of fluorescein-conjugated (FITC)
mouse monoclonal anti-mouse CD4+ (0.1 mg/ml) and
phycoerythrin-conjugated (PE) mouse anti-mouse CD8+
(1 mg/ml) (Serotec, Oxford, UK). After this, red blood cells were lysed with lysing buffer (Serotec). Fluorescence data were collected on 5× 104viable cells and analyzed by flow cytometry (Coulter, Miami, FL, USA).
2.6. Statistics
Data are expressed as the mean ± S.D. Differences
among groups were analyzed by analysis of variance using Duncan’s test. Survival rate was measured by Kaplan–Meier survival analysis. A P-value<0.05 was considered statisti-cally significant.
3. Results
There were no differences in initial body weights and weights after experimental diets for 8 weeks between the two experimental groups in either experiment 1 or 2 (data not shown). There were no differences in the percentages of CD4, CD8, and CD3 T cells or the CD4/CD8 T cell ratio between the Arg and control groups after the burn (Table 2). Antibody production increased logarithmically af-ter the second boosaf-ter and reached a plateau afaf-ter 7 weeks (data not shown). The specific antibody production in the Arg group was significantly higher than in the control group at various times (Fig. 1). The SOD and GSHPx activi-ties in liver, lung, and kidney homogenates were signifi-cantly lower in the Arg group than in the control group after the burn (Figs. 2 and 3). Also, lipid peroxidation products in liver and kidney homogenates were significantly lower in the Arg group after the burn than in the control group (Fig. 4).
In experiment 2, there were 13 survivors among the 14 mice in the Arg group, and 10 survivors in the control group after challenge with 2× LD50of P. aeruginosa to vaccinated
burned mice and observing them for 8 days. The survival rate of vaccinated burned mice in the Arg group tended to be higher than that of the control group after bacterial infection, however, no statistically significant difference was observed between the two groups (Fig. 5).
Table 2
Blood CD4, CD8, CD3 cells and the CD4/CD8 ratio between the two groups after the burn
CD4 (%) CD8 (%) CD4/CD8 CD3 (%) Arg 29.4± 6.1 12.1± 1.5 2.4± 0.3 46.9± 10.5 Control 29.3± 4.1 11.2± 2.0 2.6± 0.5 49.3± 6.6 There were no significant differences in the CD4, CD8, or CD3 popula-tions or the CD4/CD8 ratio between the two groups.
Fig. 1. Production of PEIF-specific antibodies in the Arg and control groups. Mice were immunized twice with recombinant PEIF protein on days 1 and 28, and sera antibody titers were measured by ELISA at weeks 0, 3, 4, 5, 6, 7 and 8. The dilution of mice antiserum was 1:1000. Significant difference between the two groups (∗P < 0.05).
Fig. 2. Glutathione peroxidase (GSHPx) activities in tissue homogenates between the two groups after the burn. Significant difference between the two groups (∗P < 0.05).
Fig. 5. Survival curves of vaccinated burned mice complicated with P. aeruginosa infection. There was no significant difference in the survival rate between the two groups.
Fig. 3. Superoxide dismutase (SOD) activities in tissue homogenates between the two groups after the burn. Significant difference between the two groups (∗P < 0.05).
Fig. 4. Malondialdehyde (MDA) concentrations in liver and kidney be-tween the two groups after the burn. Significant difference bebe-tween the two groups (∗P < 0.05).
4. Discussion
Supplemental Arg has been demonstrated to improve im-munologic response in both in vivo and in vitro studies. Augmentation of cell-mediated immunity was seen by Bar-bul et al. [36]with Arg supplementation. Saito et al. [24] also confirmed this observation by demonstrating that di-etary supplementation of Arg had a dose-response effect on a delayed hypersensitivity test. Since P. aeruginosa is a ma-jor cause of nosocomial infections in burned patients, an effective protective mechanism of burn patients against P.
aeruginosa infection is based on the production of specific
antibodies against bacterial virulent factors. Therefore, in this study, we investigated the effect of Arg supplementa-tion on humoral and cellular immunity, in order to determine whether Arg together with PEIF vaccination might have a synergistic protective effect in burned mice with P.
aerugi-nosa infection. In this study, 2% of total energy was supplied
by Arg; this amount of Arg was found to reduce mortality in burned guinea pigs [24]. Also, a shortened hospital stay and reduced wound infection were observed in burn patients consuming this level of Arg when compared to those using other enteral formulations[37].
A previous study reported by Daly et al. [10]
demon-strated that supplemental Arg increased the mean CD4+ T lymphocytes in surgical patients. Reynolds et al.[38]also showed that Arg supplementation significantly enhanced cy-totoxic T lymphocyte development and natural killer cell activity. In this study, CD4+helper, CD8+ suppressor-type cells, and the CD4+/CD8+ ratio did not differ between the two groups. Also, there were no differences in CD3 popu-lations between the two groups. Our finding was inconsis-tent with the reports mentioned above. It is possible that the metabolic stress in different disease conditions varies, which may lead to different immune response. However, we found that the production of specific antibodies against P.
aeruginosa was significantly higher in the Arg group than
in the control group at various times. This result suggests that burned mice supplemented with Arg had obviously
en-hanced humoral immunity, but the proliferation of CD4+
T cells might not be responsible for the production of spe-cific antibodies. Whether the stimulating effect of Arg on humoral immunity is due to the regulation of cytokines is currently being investigated.
After burn injury, generalized tissue inflammation is present in uninjured organs within hours[39]. Organ injury remote from the region of thermal injury has been shown to be due to intravascular action of complements, resulting in stimulation of intravascular neutrophils, leading to the formation of toxic oxygen products[40]. Lipid peroxide is thought to be one of the most harmful substance produced after burns [41]. Studies have shown that lipid peroxide in lung, liver, kidney and other tissues is seen early post-burn [1–5]. SOD and GSHPx are enzymes which protect tis-sues from the effects of free radicals and lipid peroxides, and the activities of both SOD and GSHPx increase after
free-radical-mediated injury and lipid peroxidation [42]. Saitoh et al. [41] demonstrated that Mn-SOD activities in lung and kidney were significantly higher than in the control group after a burn. The results of this study reveal that SOD and GSHPx activities in liver, kidney, and lung were sig-nificantly lower in the Arg group when compared with the control group. Also, lipid peroxide concentrations in liver and kidney were lower in the Arg group than in the control group after the burn of the vaccinated mice. These results were similar to our previous report[19]. This finding may indicate that Arg supplementation attenuates the oxidative stress induced by burn injury. This was true whether the mice were vaccinated or not, and increasing humoral immunity may play a role in reducing oxygen radicals after the burn. Arg is known to stimulate the local wound immune sys-tem, mainly lymphocyte activation, thereby modulating in-fection and healing. Saito et al.[24]demonstrated that Arg supplementation improved survival rates in a non-infected animal model. Our previous study showed that the survival rate of burned mice after 1× LD50 of P. aeruginosa
infec-tion was 26.7% for both the Arg and control groups after observation for 8 days (unpublished data). In this study, we
infected immunized burned mice with 2× LD50 P.
aerug-inosa to increase the mortality. The survival rates of the
Arg and control groups were 92 and 71%, respectively, at 8 days after the burn. This result indicates that PEIF vaccina-tion effectively reduced the mortality of burned mice after
P. aeruginosa infection. Although there was a tendency for
mice with Arg supplementation to have higher survival rates than mice in the control group, no significant difference was observed between the two groups. It is possible that the vac-cination effect of PEIF against P. aeruginosa infection is too strong to observe the beneficial effect of Arg supplementa-tion on the survival rates in burned mice. Whether a higher challenge dose of P. aeruginosa to burned vaccinated mice is needed to observe the difference of survival rates between these two groups requires further investigation.
In conclusion, the findings of this study suggest that Arg supplementation may enhance humoral immunity in vacci-nated mice. Also, oxidative stress induced by burn injury was attenuated. However, Arg demonstrated no appreciable benefit on enhancing cellular immunity, and survival rates were not improved when vaccinated burned mice were com-plicated with P. aeruginosa infection.
Acknowledgements
This study was supported by research grant NSC 90-2320-B-038-037 from the National Science Council, ROC.
References
[1] Till GO, Beauchamp C, Menapace D, et al. Oxygen radical dependent lung damage following thermal injury of rat skin. J Trauma 1983;23:269–77.
[2] Till GO, Hatherill JR, Tourtellotte WW, et al. Lipid peroxidation and acute lung injury after thermal trauma to skin. Am J Pathol 1985;119:376–84.
[3] Cetinkale O, Konukoglu D, Senel O, et al. Modulating the functions of neutrophils and lipid peroxidation by FK506 in a rat model of thermal injury. Burns 1999;25:105–12.
[4] Gurbur V, Corak A, Yegen BC, et al. Oxidative organ damage in a rat model of thermal injury: the effect of cyclosporin A. Burns 1997;23:37–42.
[5] Nishigaki I, Hagihara M, Hiramatsu M, et al. Effect of thermal injury on lipid peroxide levels of rat. Biochem Med 1980;24:185–9. [6] Farrell MF, Day NK, Tsakraklides V, et al. Study of lymphocyte
depletion and serum complement perturbations following acute burn trauma. Surgery 1973;73:697–705.
[7] Goldman AS, Rudloff HB, McNamee R, et al. Deficiency of plasma humoral recognition factor activity following burn injury. J Reticuloendothel Soc 1974;15:193–8.
[8] Miller CL, Baker CC. Changes in lymphocyte activity after thermal injury: the role of suppressor cells. J Clin Invest 1979;63:202–10. [9] Ninnemann JL. Immunosuppression following thermal injury through
B cell activation of suppressor T cells. J Trauma 1980;20:206–13. [10] Daly JM, Reynolds J, Thom A, et al. Immune and metabolic effects
of arginine in the surgical patients. Ann Surg 1988;208:512–23. [11] Evoy D, Lieberman MD, Fahey III TJ, et al. Immunonutrition: the
role of arginine. Nutrition 1998;14:611–7.
[12] Barbul A, Rettura G, Levenson SM, et al. Wound healing and thymotrophic effects of arginine: a pituitary mechanism of action. Am J Clin Nutr 1983;37:786–94.
[13] Barbul A, Lazarou SA, Efron DT, et al. Arginine enhances wound healing and lymphocyte immune response in humans. Surgery 1990;108:331–7.
[14] Cui XL, Iwasa M, Iwasa Y, et al. Arginine-supplemented diet decreases expression of inflammatory cytokines and improves survival in burn rats. J Parenter Enteral Nutr 2000;24:89–96. [15] Gianotti L, Alexander JW, Pyles T, et al. Arginine-supplemented
diets improve survival in gut-derived sepsis and bacterial clearance. Ann Surg 1993;217:644–53.
[16] Stinnett JD, Alexander JW, Watanabe C, et al. Plasma and skeletal muscle amino acids following severe burn in patients and experimental animals. Ann Surg 1982;195:75–89.
[17] Yu YM, Young VR, Castillo L, et al. Plasma arginine and leucine kinetics and urea production rates in burn patients. Metabolism 1995;44:659–66.
[18] Cui XL, Iwasa M, Iwasa Y, et al. Effects of dietary arginine supplementation on protein turnover and tissue protein synthesis in scald-burn rats. Nutrition 1999;15:563–9.
[19] Tsai HJ, Shang HF, Yeh CL, Yeh SL. Effects of arginine supplementation on antioxidant enzyme activity and macrophage response in burned mice. Burns 2002;28:258–63.
[20] Demling RH, Seigne P. Metabolic management of patients with severe burn. World J Surg 2000;24:673–80.
[21] O’Sullivan ST, O’Connor TPF. Immunosuppression following thermal injury: the pathogenesis of immunodysfunction. Br J Plast Surg 1997;50:615–23.
[22] Bodey GP, Bolivar R, Fainstein V, et al. Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 1983;5:279–313.
[23] Hancock REW. Intrinsic antibiotic resistance of Pseudomonas aeruginosa. J Antimicrob Chemother 1986;18:653–6.
[24] Saito H, Trocki O, Wang SL, et al. Metabolic and immune effects of dietary arginine supplementation after burn. Arch Surg 1987;122:784–9.
[25] Chen TY, Shang HF, Chen TL, et al. Recombinant protein composed of Pseudomonas exotoxin A, outer membrane proteins I and F as vaccine against P. aeruginosa infection. Appl Microbiol Biotechnol 1999;52:524–33.
[26] Holder IA. The pathogenesis of infection owing to Pseudomonas aeruginosa using the burned mouse model: experimental studies from the Shriners Burns Institute, Cincinnati. Can J Microbiol 1985;31:393–402.
[27] Holder IA, Jogan M. Enhanced survival in burned mice treated with antiserum prepared against normal and burned skin. J Trauma 1971;11:1041–6.
[28] Boucher JL, Obled C, Farges MC, et al. Ornithine␣-ketoglutarate modulates tissue protein metabolism in burn-injured rats. Am J Physiol 1997;273:E557–63.
[29] Vaubourdolle M, Coudray-Lucas C, Jardel A, et al. Action of enterally administered ornithine ␣-ketoglutarate on protein breakdown in skeletal muscle and liver of the burned rats. J Parenter Enteral Nutr 1991;15:517–20.
[30] Cynober L. Amino acid metabolism in the thermal burns. J Parenter Enteral Nutr 1989;13:196–205.
[31] Hu ML, Frankel EN, Leibovitz BE, et al. Effect of dietary lipids and Vitamin E on in vitro lipid peroxidation in rat liver and kidney homogenates. J Nutr 1989;119:1574–82.
[32] Yeh SL, Chang KY, Huang PC, et al. Effects of n-3 and n-6 fatty acids on plasma eicosanoids and liver antioxidant enzymes in rats receiving total parenteral nutrition. Nutrition 1997;13:32–6. [33] Lowry OH, Rosebrough NJ, Farr AC, et al. Protein measurement
with the folin–phenol reagent. J Biol Chem 1951;193:265–75. [34] Uchiyama M, Mihara M. Determination of malonaldehyde precursor
in tissues by thiobarbituric acid test. Anal Chem 1978;86: 271–8.
[35] Voller A, Bidwell DE, Bartlett A. The enzyme-linked immunosorbent assay ELISA. Alexandria, VA: Dynatech Laboratories; 1979. p. 1–32. [36] Barbul A, Saito D, Wasserkrug HL, et al. Arginine stimulates lymphocyte immune response in healthy human beings. Surgery 1981;90:244–51.
[37] Alexander JW, Gottschlich MM. Nutritional immunomodulation in burn patients. Crit Care Med 1990;18:S149–53.
[38] Renolds JV, Daly JM, Zhang S, et al. Immunomodulatory mechanisms of arginine. Surgery 1988;104:142–51.
[39] Youn YK, LaLonde C, Demling R. The role of mediators in the response to thermal injury. World J Surg 1992;16:30–6.
[40] Till GO, Johnson KJ, Kunkel R, et al. Intravascular activation of complement and acute lung injury. Dependency on neutrophils and toxic oxygen metabolites. J Clin Invest 1982;69:1126–35. [41] Saitoh D, Okada Y, Ookawara T, et al. Prevention of ongoing lipid
peroxidation by wound excision and superoxide dismutase treatment in the burned rat. Am J Emerg Med 1994;12:142–6.
[42] Gonzales R, Auclair C, Voisin E, et al. Superoxide dismutase, catalase, and glutathione peroxidase in red blood cells from patients with malignant disease. Cancer Res 1984;44:4137–9.
