Effect of dietary glutamate on chemotherapy-induced immunosuppression

BASIC NUTRITIONAL INVESTIGATION

Nutrition Vol. 15, No. 9, 1999

Effect of Dietary Glutamate on Chemotherapy-Induced Immunosuppression CHENG-MAO LIN, PHD, STEVE F. ABCOUWER, PHD, AND WILEY W. SOUBA, MD, SCD From the Surgical Oncology Research Laboratories, Department of Surgery, Massachusetts General Hospital; and the Harvard Medical School, Boston, Massachusetts, USA ABSTRACT

Chemotherapy causes severe host immune depression and consequently increases susceptibility to infection. Dietary glutamate (GLU) serves as a stable substrate for the formation of glutamine (GLN), which is an important fuel and metabolic precursor for the immune cells. The effect of addition of GLU to a GLN/GLU-free amino acid diet upon immune response was studied in rats recovering from chemotherapy. Animals were fed a 0, 4, or 8% GLU diet and received a single intraperitoneal injection of methotrexate (MTX, 20 mg/kg BW). Two in vivo immune tests, delayed-type hypersensitivity (DTH) and popliteal lymphoproliferation (PLP), were performed 3 and 7 d after MTX treatment. Food intake and body weight decreased significantly immediately after MTX treatment and gradually recovered after 8 d with no significant difference among treatment groups. In a 23-d feeding study, no significant difference was found in the DTH response, but the PLP response increased in a GLU dose related fashion (83 and 133% increases for the 4 and 8% GLU diets, respectively). In a 44-d feeding study, the DTH response increased 61 and 83%, while the PLP response increased 191 and 382% for the 4 and 8% GLU diets, respectively. Plasma GLN, GLU, or glutathione (GSH) levels were increased by dietary GLU, but only in the immediate postprandial state. In summary, dietary GLU improves immune status of rats recovering from MTX treatment. The immune-enhancing effect of dietary GLU was dose-dependent and more pronounced after a longer duration of dietary GLU intake. Nutrition 1999;15:687– 696. ©Elsevier Science Inc. 1999 Key words: glutamate, glutamine, glutathione, amino acid diet, immunity, chemotherapy, rats

INTRODUCTION

It is well known that chemotherapy can cause severe depression of the immune system and consequently increase the host’s susceptibility to infection. Countless efforts have been directed toward identifying the optimal nutritional regimen to enhance host immune function and improve survival of cancer patients. Specific nutrients, such as glutamine (GLN), arginine, nucleotides, and ␻-3 fatty acids, have been shown to affect immune response under certain circumstances.1 As an essential substrate for nucleotide synthesis in most dividing cells, GLN is an important fuel and metabolic precursor for rapidly growing cells such as enterocytes, lymphocytes, and fibroblasts. It is thought that under physiologic stresses, GLN synthesis is insufficient to meet the body’s needs. GLN supplementation has been found to affect various cell functions and is beneficial to patients in several disease states.2– 6 Although higher plasma levels of glutamate (GLU) were suggested as indications of unfavorable immune and disease status,7,8 there is no direct evidence concerning the effect of dietary GLU

on immune function. GLU has been studied mainly for its role as the excitatory neurotransmitter in brain function and neurologic disease.9 In addition, GLU serves as a precursor of glutathione (GSH), which has been associated with immunocompetence in both animal and human studies.10,11 With the knowledge that there is a continuous metabolic conversion between GLN and GLU12 and that GLU is a necessary constituent for GSH synthesis,13 we tested the hypothesis that dietary GLU reduces the severity of the immunosuppressive effect induced by a chemotherapeutic agent. Studies were designed to evaluate the in vivo immune response at different stages of recovery from chemotherapy. Since there are likely to be many factors (such as GLU levels, feeding duration, and time after chemotherapy) which potentially affect immune response, experiments were designed to investigate multiple dietary treatment groups with different feeding durations, and immune responses were tested at different time points using two in vivo immune assays. Plasma GLN, GLU, and GSH levels were analyzed at the end of each experiment to evaluate the impact of dietary GLU on blood

Supported by NIH Grant R01-CA57690 (Dr. Souba). Correspondence to: Steve F. Abcouwer, PhD, Massachusetts General Hospital, Surgical Oncology Research Laboratories, 55 Fruit Street, Jackson 918, Boston, MA 02114-2696, USA. E-mail: [email protected] or [email protected]

Nutrition 15:687– 696, 1999 ©Elsevier Science Inc. 1999 Printed in the USA. All rights reserved.

0899-9007/99/$20.00 PII S0899-9007(99)00153-7

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chemistry and to seek the possible metabolic mechanism(s) responsible for the immune modulation effect.

TABLE I. COMPOSITION OF GLU DIETS

MATERIALS AND METHODS

Animal Model

Ingredient

8% GLU (g/kg)

4% GLU (g/kg)

0% GLU (g/kg)

Adult female Fischer 344 rats (weighing 150 –175 g) were obtained from Charles River Laboratories (Wilmington, MA, USA). Animals were housed in stock cages (4 rats per cage) and given free access to water and the stock laboratory rodent diet (Lab Diet 5001, PMI Feeds, Inc., Indianapolis, IN, USA). Animals were maintained in a temperature-controlled (approximately 24°C) animal facility with constant 12-h light and dark cycles. Animals were allowed to acclimate to the facility for at least 1 wk before the experiment. They were then randomly assigned to treatment groups and given free access to water and one of the amino acid diets containing different levels of GLU purchased from Harlan Teklad (Madison, WI, USA). GLU comprised 0, 22.3, and 36.4% of the total amino acids, and total nitrogen contents were 20.6, 24.4, and 28.2 g nitrogen/kg in the 0, 4, and 8% GLU diets, respectively. To maintain the caloric density of the diets, differences in GLU content were adjusted with the same amount of sucrose. Complete compositions of these diets (Table I) have been published previously.14 Food intake and body weights of animals were measured throughout the experiments as indices of basic nutritional parameters. Methotrexate (MTX, Immunex Corp., Seattle, WA, USA) was given to animals according to their body weight (BW). A single intraperitoneal injection of MTX (20 mg/kg BW) was administered 9 or 30 d after initiating dietary treatment in the short-term or long-term feeding study, respectively. In initial studies, two in vivo immune tests, delayed-type hypersensitivity (DTH) and popliteal lymphoproliferation (PLP) were performed 3 and 7 d after MTX treatment, respectively. In a later study, the DTH assay was performed 7 d after MTX treatment. At the end of each immune function study, blood samples were collected, and plasma GLU, GLN, and GSH levels were analyzed. The animal experiment procedures were performed in accordance with the National Institutes of Health guidelines on the use of experimental animals and were approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital.

Amino acid L-Alanine L-Arginine HCl L-Asparagine L-Aspartic acid L-Cystine L-Glutamic acid L-Glutamine Glycine L-Histidine HCl H2O L-Isoleucine L-Leucine L-Lysine HCl L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine Total Sucrose Corn starch Corn oil Cellulose (fiber) Mineral mix, AIN-76 Calcium phosphate, dibasic Vitamin mix (Harlan Tekland) Ethoxyquin (antioxidant) Nitrogen (g/kg) Calorie (kcal/kg)

3.5 12.1 6.0 3.5 3.5 80.0 0.0 23.3 4.5 8.2 11.1 18.0 8.2 7.5 3.5 3.5 8.2 1.8 5.0 8.2 219.6 450.88 150.0 100.0 30.0 35.0 4.5 10.0 0.02 28.2 4181.9

3.5 12.1 6.0 3.5 3.5 40.0 0.0 23.3 4.5 8.2 11.1 18.0 8.2 7.5 3.5 3.5 8.2 1.8 5.0 8.2 179.6 490.88 150.0 100.0 30.0 35.0 4.5 10.0 0.02 24.4 4181.9

3.5 12.1 6.0 3.5 3.5 0.0 0.0 23.3 4.5 8.2 11.1 18.0 8.2 7.5 3.5 3.5 8.2 1.8 5.0 8.2 139.6 530.88 150.0 100.0 30.0 35.0 4.5 10.0 0.02 20.6 4181.9

Immune Function Assay 15

In the DTH assay, rats were sensitized by topical application of 100 ␮L of 1% 2,4-dinitrofluorobenzene (DNFB) (Sigma Chemical, St. Louis, MO, USA) in a 4:1 mixture of acetone and olive oil to the shaved abdomen for two consecutive days. Four days after the second DNFB sensitization, one ear of the rat was challenged with 50 ␮L of 0.5% DNFB in the same vehicle. The other ear was treated with the vehicle and served as a non-stimulated control. The degree of immunocompetence was assessed 24 h later by measuring the increment of ear thickness (swelling) with a micrometer (Fisher Scientific, Pittsburgh, PA, USA). The degree of immune response was quantified by the difference in ear thickness (10⫺2 mm) between the stimulated and the contralateral (nonstimulated) ear in the same animal. In the PLP assay,16 the status of immune function was evaluated by quantitating the spontaneous proliferation of lymphocytes in the popliteal lymph node (PLN) induced by local subcutaneous injection of sheep red blood cells (SRBC, Sigma Chemical) into the hind-leg footpad. One footpad of the rat was injected subcutaneously with 100 ␮L of sterile saline containing 1 ⫻ 108 SRBC. The contralateral footpad was injected with the same volume of sterile saline and served as the non-stimulated control. Because there is no crossover effect, the injected antigen stimulates only

the PLN in the leg which drains that foot and does not affect the other PLN. Seven days after antigen injection, the PLNs were excised, cleaned of adherent fat, and weighed on a digital scale (A-200DS, Denver Instrument Co., Arvada, CO, USA). The degree of immune response was defined as the difference in PLN weight (mg) between the stimulated and the contralateral (nonstimulated) PLN in the same animal. Plasma GLU and GLN Analysis At the end of each immune function study, whole blood samples were obtained at approximately 1 PM from the inferior vena cava via venapuncture from animals anesthetized with intraperitoneal ketamine hydrochloride (75 mg/kg BW, Fort Dodge Laboratories, Inc., Fort Dodge, IA, USA) and acepromazine maleate (1 mg/kg BW, Fort Dodge Laboratories). For the study with blood sampling at different times, blood was obtained by phlebotomy of anesthetized animals at approximately 9 AM and 5 PM on the same day. Blood samples were heparinized (heparin sodium, ElkinsSinn, Inc., Cherry Hill, NJ, USA) and centrifuged at 14 000⫻ g for 10 min at 4°C. Plasma samples were obtained and stored at ⫺80°C until analysis. Plasma GLU and GLN levels were measured by a modified spectrophotometric assay17 adapted to a 96-well plate format using an Anthos HT-2 microplate spectro-

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FIG. 1. Effect of a glutamate (GLU)-free diet on food intake and body weight in rats. Animals were fed a laboratory rodent chow or the 0% GLU diet throughout the experiment. Two in vivo immune tests (the delayed type hypersensitivity [DTH] response 2,4-dinitrofluorobenzene [DNFB] and the popliteal lymphoproliferation [PLP] response to sheep red blood cells [SRBC]) were performed at various time points as indicated. Data were the mean for a group of three rats in each treatment.

photometer (Anthos Labtec, Inc., Frederick, MD, USA), as reported previously.18 Plasma GSH Analysis Plasma GSH levels were analyzed using a modification of the Tietze method19 adapted to a 96-well plate format. Plasma samples were deproteinized by adding 100% tricholoroacetic acid (TCA) in a ratio of 9:1 (v/v) to obtain a final concentration of 10% TCA. After precipitation on ice for 15 min, the mixtures were then centrifuged at 14 000⫻ g for 15 min to separate the protein. The supernatants (in 10% TCA) were removed and assessed for GSH levels. Briefly, in each well of a MicroTest III 96-well assay plate (Becton Dickinson, Bedford, MA, USA), 10 ␮L of deproteinized samples were mixed with 190 ␮L of the Tietze reaction solution, a mixture of 24.4 mL Tietze buffer (200 mM NaH2PO4 and 0.01 M EDTA, pH 7.3; Sigma Chemical), 6.72 mg of 5⬘-5⬘-dithio-bis2-nitrobenzoic acid (DTNB; Sigma Chemical), 10 mg of ␤nicotinamide adenine dinucleotide (NADH; Sigma Chemical) and 165 U of GSH reductase (Sigma Chemical). Kinetic readings of absorbance at 405 nm (with a 492 nm reference filter) of the triplicate samples were obtained every 15 s for six cycles using an Anthos HT2 microplate spectrophotometer (Anthos Labtec). The maximum slope was obtained and compared to a standard curve generated by adding known amounts of reduced GSH diluted with deproteinized dialized fetal bovine serum (dFBS) in 10% TCA.

Statistical Analysis Data are expressed as the mean plus the standard error of the mean (SEM) for each group. Analysis of variance (ANOVA) and Fisher’s Least Significant Difference (LSD) were used to identify significant differences among the treatment groups. Differences between the means of plasma profile at different sampling times were compared by using the paired t test. A p value of ⱕ0.05 was defined as the level of significance. All statistics were performed using the StatView program (SAS Institute Inc., Cary, NC, USA) on a personal computer. RESULTS

Effect of Amino Acid Diet on Immune Response The effect of the amino acid diet on growth and immune response was tested in a preliminary study. Adult female Fischer 344 rats were fed either a laboratory rodent diet (chow) or an amino acid diet containing 0% GLU (GLN/GLU-free). Food intake was slightly lower (average 2.9 g/d less) in the 0% GLU group throughout the experiment (Fig. 1A) which was in keeping with the higher caloric content of the amino acid diet. Animals fed the 0% GLU diet showed a decrease in body weight right after switching to the amino acid diet, but they gradually regained weight and returned to level similar to that of the animals fed chow diet (Fig. 1B). The two in vivo immune function assays (DTH and PLP) were also tested in these animals. DTH response

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GLUTAMATE ENHANCES IMMUNE RESPONSE AFTER CHEMOTHERAPY without GLU (Fig. 4). Rats fed the 4% GLU diet had an 83% increase, and rats fed the 8% GLU diet had a 133% increase in the PLP response, compared to that of rats fed the 0% GLU diet.

FIG. 2. Effect of a glutamate (GLU)-free diet on immune response in rats. Animals were fed a laboratory rodent chow or the 0% GLU diet throughout the experiment. Animals were challenged with 2,4-dinitrofluorobenzene (DNFB) 26 d after initiating dietary treatment, and their delayed type hypersensitivity (DTH) response (defined as the difference in ear thickness, 10⫺2 mm) was assessed 24 h later. Animals were injected with sheep red blood cells (SRBC) 27 d after dietary treatment, and their popliteal lymphoproliferation (PLP) response (defined as the difference in popliteal lymph node [PLN] weight, mg) was assessed 7 d later. Data were the mean plus SEM for a group of three rats in each treatment.

was assessed at 27 d after switching half of the animals to the amino acid diet (challenged at day 26). The PLP response was assessed at 34 d after switching half of the animals to the amino acid diet (challenged at day 27). There was no significant difference between the immune responses of rats in the two dietary treatment groups (Fig. 2). Effect of Short-Term GLU Feeding on Immune Response After Chemotherapy To examine the hypothesis that dietary GLU enhances immune response after chemotherapy, rats were fed amino acid diets containing different levels of GLU (0, 4, and 8%) throughout the experiment. Nine days after initiating dietary treatment, MTX (20 mg/kg BW) was administrated by a single intraperitoneal injection. Food intake decreased significantly immediately after MTX treatment in all three dietary treatment groups (Fig. 3A). Three days after MTX treatment, diarrhea was observed in all animals. Two days later, diarrhea subsided, and food intake gradually increased and returned to the normal levels. Body weight change followed the trend of food intake (Fig. 3B). However, by the end of the study, the mean body weight of each treatment group had not reached the original value. There were no significant differences in food intake and final body weight among the dietary treatment groups. Animals were challenged with DNFB 3 d after MTX treatment, and their DTH responses were assessed 24 h later. Seven days after MTX treatment, animals were injected with SRBC, and their PLP responses were evaluated 7 d later. In this short-term feeding study (23 d total), there was no significant difference in the mean DTH response among the treatment groups (Fig. 4). However, there was a GLU dose-related effect in the PLP response, as rats fed diets containing higher amounts of GLU had significantly increased PLP responses compared to that of rats fed a diet

Effect of Long-Term GLU Feeding on Immune Response After Chemotherapy A long-term feeding study (44 d total) was initiated to determine whether a longer duration of GLU feeding has a greater effect on immune function after chemotherapy and whether this effect would be detectable by the DTH assay. The basic protocol used in the previous short-term feeding study was followed, except that animals were fed GLU diets for 30 d (3 wk more than the short-term feeding study) before MTX treatment was introduced. As seen in the short-term feeding study (Fig. 3A), food intake of rats in all three dietary treatment groups decreased significantly immediately after MTX treatment (Fig. 5A). Three days after MTX treatment, diarrhea was observed in all animals. Two days later, diarrhea subsided, and food intake gradually increased and returned to the normal levels. Similar to the results of the shortterm feeding study (Fig. 3B), body weight change followed the trend of food intake (Fig. 5B). Again, by the end of the study, the mean body weight of each treatment group had not reached the original value. There was no significant difference in food intake and final body weight among the treatment groups. The results indicated that even with a longer period of adjustment to the amino acid diet, animals were still unable to overcome the effect of a chemotherapeutic agent upon food intake. When animals were on the dietary treatment for a longer period of time (44 d total), results of immune function assessed by both DTH and PLP assays showed significant differences among the dietary treatment groups. The DTH response increased 61% in rats fed the 4% GLU diet and increased 83% in rats fed the 8% GLU diet, compared to that of rats fed the 0% GLU diet (Fig. 6). Thus, dietary GLU did affect immune function during the early, neutropenic stage after MTX treatment. As the significant difference in the DTH response was not seen in the short-term feeding study, the results indicated that an adaptation in metabolic change accounted (at least partially) for the immune-enhancing effect of dietary GLU. Results of the PLP assay were more dramatic than that of the DTH assay. Rats fed the 4% GLU diet had a 191% increase, and rats fed the 8% GLU diet had a 382% increase in the PLP response, compared to that of rats fed the 0% GLU diet (Fig. 6). In this experiment, rats fed the 0% GLU diet had a similar PLP response as that of rats with the same dietary treatment in the short-term feeding study. Thus, the increased relative response in rats fed either the 4 or 8% GLU diet was not due to exaggerated detrimental effects of the 0% GLU diet but, rather, to an even higher PLP response brought about by the long-term GLU feeding. Confirmation of the Effect of GLU on the Immune Response After Chemotherapy To further confirm the immune-enhancing effect of dietary GLU in the late (recovery) stage after MTX treatment, the DTH assay was performed at the same time point as the PLP assay in the previous (short-term and long-term feeding) studies. Consistent with the previous studies, MTX was administered 9 or 30 d after initiating dietary treatment for the short-term or long-term study, respectively. Animals were challenged with DNFB 7 d after MTX treatment, and their DTH responses were assessed 24 h later. In the short-term study, there were 24 and 26% increases in the DTH response in rats fed the 4 and 8% GLU diets, respectively. In the long-term study, the increases in the DTH response were 15 and 32% for rats fed the 4 and 8% GLU diets, respectively (Fig. 7). Although the difference was not as marked as seen in the PLP response, the results confirmed that dietary GLU improved im-

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FIG. 3. Effect of short-term glutamate (GLU) feeding on food intake and body weight in methotrexate (MTX) treated rats. Animals were fed different GLU diets throughout the experiment. A single intraperitoneal injection of MTX (20 mg/kg BW) and two in vivo immune tests (the delayed type hypersensitivity [DTH] response 2,4-dinitrofluorobenzene [DNFB] and the popliteal lymphoproliferation [PLP] response to sheep red blood cells [SRBC]) were performed at various time points as indicated. Data were the mean for a group of four rats in each treatment.

mune status of rats at the late (recovery) stage after MTX treatment. Further analyses were, therefore, performed to seek the metabolic mechanism(s) responsible for the beneficial effect of dietary GLU upon immune status after chemotherapy. Effect of Dietary GLU on Plasma Profile It was hypothesized that dietary GLU may stimulate proliferation and/or function of the immune cells by directly raising plasma GLU concentration or indirectly elevating plasma GLN concentration. To examine the impact of dietary GLU on plasma GLU and GLN levels, blood samples were collected from animals at the end of the short-term (23 d) and long-term (44 d) feeding studies. Plasma samples were obtained from animals at approximately 1 PM (the middle of the light cycle) and analyzed for GLU and GLN levels. There was no significant difference in plasma GLU (Fig. 8A) and GLN (Fig. 8B) levels among dietary treatment groups in both studies, except for plasma GLU levels in rats fed the 8% GLU diet in the long-term feeding study, which were significantly decreased. Although in the long-term feeding study, plasma GLN levels in rats fed either the 4 or 8% GLU diet were slightly higher compared to that of rats fed the 0% GLU diet, the difference was not statistically significant. Thus, it was concluded that dietary GLU did not exert its immune-enhancing effects through sustained elevation of either plasma GLN or GLU levels. GSH depletion has been shown to impair T cell and macrophage immune function in rats11 and affect the levels of CD4⫹ cells in humans.10 Given the demonstrated link between enteral

GLU and GSH synthesis,13 it was hypothesized that dietary GLU may enhance immune response by providing precursors for de novo GSH synthesis, which may exert its immune-enhancing effects via regulating the production of prostaglandin E2 (PGE2)20 and interleukin-2 (IL-2).21 However, in the present study, there was no significant difference in plasma GSH from both the shortterm and long-term feeding studies, although rats fed the 8% GLU diet had a nominally higher mean plasma GSH level in the long-term feeding study (Fig. 8C). It, therefore, seemed that dietary GLU at the levels and duration studied had no significant impact on plasma GSH levels. In this study, ingestion of considerable amounts of GLU did not cause a significant sustained elevation in plasma levels of GLU, GLN, or GSH. However, it was suspected that the lack of impact by dietary GLU on plasma GLU profile in this study might be due to the time of sample collection. Therefore, plasma samples were obtained from another set of animal groups fed the 0, 4, or 8% GLU diets for 2 wk and, as the rats consume the majority of their food at night, two time points were selected to examine the plasma profile at different postprandial stages. Blood samples were first collected from the animals at 2 h after the beginning of the light cycle (9 AM). Animals were then given free access to water only and blood samples were again obtained 8 h later (5 PM). In samples obtained at 9 AM, the levels of plasma GLU were well correlated to the GLU levels in the diet (Fig. 9A). In contrast, when blood samples were collected at 5 PM, the plasma GLU levels were lower than 0.05 mM in all three dietary treatment groups. Interestingly, plasma GLN levels were also correlated to the dietary GLU levels

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FIG. 4. Effect of short-term glutamate (GLU) feeding on immune response in methotrexate (MTX) treated rats. Animals were fed different GLU diets throughout the experiment. MTX (20 mg/kg BW) was administered by intraperitoneal injection 9 d after initiating dietary treatment. Animals were challenged with 2,4-dinitrofluorobenzene (DNFB) 3 d after MTX treatment, and their delayed type hypersensitivity (DTH) response (defined as the difference in ear thickness, 10⫺2 mm) was assessed 24 h later. Animals were injected with sheep red blood cells (SRBC) 7 d after MTX treatment, and their popliteal lymphoproliferation (PLP) response (defined as the difference in popliteal nymph node [PLN] weight, mg) was assessed 7 d later. Data were the mean plus SEM for a group of four rats in each treatment. *P ⱕ 0.05 versus the 0% GLU.

in the samples collected in the morning and they become inversely correlated to the dietary GLU levels in the afternoon (Fig. 9B). Although not as apparent, the plasma GSH levels also followed the same trend as the GLU and GLN (Fig. 9C). DISCUSSION

The effect of dietary GLU upon immune function following treatment with the chemotherapeutic agent MTX was tested using a GLN/GLU-free amino acid diet. To our knowledge, this diet formulation represents the only published GLN/GLU-free animal diet. These 0, 4, and 8% GLU-containing diets were previously used by Horvath et al. to study the effects of GLU upon the function of the rat small intestine.14 To determine immune status of the same animals at different time points after MTX treatment, a combination of two in vivo immune tests was selected. The DTH assay is a faster immune function test that evaluates the degree of skin (i.e., ear) contact sensitivity elicited by DNFB. The maximal response is usually obtained 24 – 48 h after DNFB challenge in the sensitized rats. The PLP assay is an accumulated immune function test that measures proliferation of lymphocytes in the PLN induced by subcutaneous injection of SRBC into the hind footpad. The antigens (i.e., SRBC) that migrate into the lymphatic system are sequestered by the PLN in the leg, which drains that foot. The immune cells present in the lymph node proliferate in response to the antigen, which leads to an increase in the size of the lymph node over a period of time. To assess the immune response of animals receiving chemotherapy, the two in vivo immune function tests were performed at different time points. The time points were selected based on the results of a preliminary study in which differential blood cell

counts were performed to assess the temporal change in immune status of animals after MTX treatment (data not shown). Results showed that the neutrophil population was reduced to nearly 0 in samples collected 5 d after MTX treatment and gradually recovered after 14 d. The DTH assay was used to assess immune status at the early (neutropenic) stage, while the PLP assay was employed to assess the late (recovery) stage after MTX treatment. The immune assays were selected based on their characteristics. Although both assays evaluate the cell-mediated immune response, the DTH assay is a non-invasive survival assay, while the PLP assay requires tissue removal and is non-survival. As the objective of the experiment was to assess immune response in the same animal at different stages of recovery from chemotherapy, the DTH assay was performed at the early stage and the PLP assay was utilized at the later stage. In the short-term feeding study, results of the two immune tests showed the effect of dietary GLU on immune function at different stages after chemotherapy (Fig. 4). The DTH data were obtained 4 d after MTX treatment, when animals were neutropenic, while the PLP data were obtained 14 d after MTX treatment, when animals had recovered from the immunosuppressive effect of MTX. Results of this study indicated that dietary GLU enhances immune response at the late stage after MTX treatment but has no effect at the early stage. However, it is possible that the lack of immune-enhancing effect from dietary GLU at the early stage after MTX treatment was because the duration of GLU feeding was too short to have a significant effect on the immune system. Furthermore, since different immune assays were used, it is also possible that the effect of dietary GLU on the immune system is not detectable by the DTH assay. To determine whether the lack of effect from dietary GLU at the early stage of immunosuppression induced by MTX treatment was due to the duration of GLU feeding or due to the assay (DTH) used to evaluate immune response, a long-term feeding study was performed. Results of the long-term feeding study indicated that dietary GLU does affect immune responses at both the early and late stages after MTX treatment (Fig. 6). The data demonstrated that considerable time is needed to promote the immune-enhancing effect of dietary GLU and that the DTH assay can be used to detect the effect of dietary GLU upon the immune system in rats. In the long-term experiment, the effects of GLU were greater than those observed after short-term feeding. However, in this experiment, rats fed the 0% GLU diet exhibited a PLP response similar to those rats fed this diet in the short-term feeding study. Thus, the increased responses in long-term GLU fed rats was not due to exaggerated detrimental effects of the 0% GLU diet but, rather, to an even higher PLP response brought about by the long-term GLU feeding. This observation supports the hypothesis that the body requires more time to adjust to the dietary change and experience the beneficial immune-enhancing effect. It also suggests that this immune-enhancing effect is not simply a reversal of the adverse effects of the GLN/GLU-free diet. In the confirming study, the DTH assay was applied 7 d after MTX treatment (the same time point as the PLP assay in the previous short-term and long-term feeding studies), rather than 3 d following chemotherapy. Although the differences between dietary groups was not as marked as seen in the PLP response (Fig. 7), this test confirmed that dietary GLU improved immune status of rats at the late (recovery) stage after MTX treatment regardless of the assay used. In this study, the amino acid diet was used to ablate the dietary intake of GLN and GLU. The diet itself had no apparent detrimental effect upon body weight or immune function in rats fed for 4 –5 wk (Figs. 1 and 2). However, it has been shown that elemental diets have adverse effects when combined with chemotherapy.

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FIG. 5. Effect of long-term glutamate (GLU) feeding on food intake and body weight in methotrexate (MTX) treated rats. Animals were fed different GLU diets throughout the experiment. A single intraperitoneal injection of MTX (20 mg/kg BW) and two in vivo immune tests (the delayed type hypersensitivity [DTH] response 2,4-dinitrofluorobenzene [DNFB] and the popliteal lymphoproliferation [PLP] response to sheep red blood cells [SRBC]) were performed at various time points as indicated. Data were the mean for a group of four rats in each treatment.

FIG. 6. Effect of long-term glutamate (GLU) feeding on immune response in methotrexate (MTX) treated rats. Animals were fed different GLU diets throughout the experiment. MTX (20 mg/kg BW) was administered by intraperitoneal injection 30 d after initiating dietary treatment. Animals were challenged with 2,4-dinitrofluorobenzene (DNFB) 3 d after MTX treatment, and their delayed type hypersensitivity (DTH) response (defined as the difference in ear thickness, 10⫺2 mm) was assessed 24 h later. Animals were injected with sheep red blood cells (SRBC) 7 d after MTX treatment, and their popliteal lymphoproliferation (PLP) response (defined as the difference in popliteal nymph node [PLN] weight, mg) was assessed 7 d later. Data were the mean plus SEM for a group of four rats in each treatment. *P ⱕ 0.05 versus the 0% GLU.

FIG. 7. Effect of dietary glutamate (GLU) on immune response in methotrexate (MTX) treated rats. Animals were fed different GLU diets throughout the experiment. MTX (20 mg/kg BW) was administered by intraperitoneal injection 9 or 30 d after initiating dietary treatment for the short-term or long-term feeding study, respectively. Animals were challenged with 2,4-dinitrofluorobenzene (DNFB) 7 d after MTX treatment, and their delayed type sensitivity (DTH) response (defined as the difference in ear thickness, 10⫺2 mm) was assessed 24 h later in both studies. Data were the mean plus SEM for a group of four rats in each treatment. *P ⱕ 0.05 versus the 0% GLU.

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FIG. 8. Effect of dietary glutamate (GLU) on plasma profile in methotrexate (MTX) treated rats. Animals were fed different GLU diets throughout the experiment. MTX (20 mg/kg BW) was administered by intraperitoneal injection 9 or 30 d after initiating dietary treatment for the short-term or long-term feeding study, respectively. Blood samples were collected 2 wk after MTX treatment. Data were the mean plus SEM for a group of four rats in each treatment. *P ⱕ 0.05 versus the 0% GLU.

FIG. 9. Effect of dietary glutamate (GLU) on plasma profile at different sampling time in rats. Animals were fed different GLU diets throughout the experiment. Blood samples were collected 2 wk after initiating dietary treatment at different time points as indicated. Data were the mean plus SEM for a group of five rats in each treatment. *P ⱕ 0.05 versus the 0% GLU. #P ⱕ 0.05 versus the 9 AM sample of the same dietary treatment.

GLUTAMATE ENHANCES IMMUNE RESPONSE AFTER CHEMOTHERAPY McAnena et al.22 found that administration of MTX (20 mg/kg BW) to rats fed an elemental diet in which all of the protein was provided as amino acids was extremely toxic to the gastrointestinal tract. Results from other investigators in similar studies suggested that elemental diets containing free amino acids lead to chemotherapeutic intolerance.23 Although the amino acid diets used in these studies are designed for studying specific amino acids and are more nutrient complete than elemental diets, the adverse effects of MTX observed in the present study may, nonethe-less, be attributable to the nature of the diets used. Indeed, when rats were fed a standard chow diet and treated intraperitoneally with MTX up to 60 mg/kg BW, much less effect upon food intake and body weight was observed (data not shown). Results of this study indicate a detrimental combination of an amino acid diet and chemotherapy and suggest that dietary GLU does not diminish this interaction. It could be that this chemotherapy-induced starvation contributed significantly to the immune depression of MTX-treated rats and it is possible that dietary GLU exerts a positive effect only in such malnourished rats. The results demonstrated that the dietary GLU does have a positive impact on plasma GLU, GLN, and GSH levels at the immediate postprandial stage (Fig. 9). However, the inverted change in the plasma profile 8 h later indicated that the animals provided dietary GLU exhibited increased utilization and/or removal of this amino acid from the bloodstream during this fasting period. The parallel effect upon plasma GLN and GSH levels may be a direct result of these fluctuations in plasma GLU concentrations. At this time, it is not known whether the transient increase in plasma GLU, GLN, or GSH levels or increased utilization of these metabolites plays a causal role in enhancement of immune recovery following chemotherapy. It should be noted that the nitrogen content of these isocaloric diets varied by 37% (from 20.6 g/kg to 28.2 g/kg). It is possible that this increased nitrogen delivery played a role in the enhancement of immune recovery, irrespective of the form in which it was delivered.

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In conclusion, dietary GLU increased immune response observed in rats following MTX treatment. The effect of GLU was dose-dependent and more pronounced after a longer duration of dietary GLU intake. These results suggest that dietary GLU improves the immune status of rats recovering from chemotherapeutic treatment by reducing the severity of the immunosuppressive effect induced by the chemotherapeutic agent and enhancing recovery after chemotherapy. However, the mechanism underlying the immune-enhancing effect of GLU is not apparent. It is possible that the observed effects of GLU were the result of alterations in MTX toxicity or pharmacokinetics. GLU could alter MTX retention within cells by serving as a substrate for MTX polyglutamation or could alter MTX toxicity or clearance by altering folate metabolism. However, we are not aware of any publication showing a direct in vivo interaction between GLU and MTX. Charland et al. observed that feeding rats a 3% GLN-containing diet reduced the rate of serum MTX clearance by 25% and reduced the rate of renal MTX elimination by 65%.24 It stands to reason, however, that any such effect of dietary GLU upon MTX clearance and elimination would only serve to increase the detrimental effects upon the immune system, not decrease immune depression or promote immune recovery. Further studies are needed to identify the factor(s) and mechanism(s) responsible for the beneficial effect of dietary GLU in MTX chemotherapy.

SUMMARY

Dietary GLU reduces the severity of the immunosuppressive effect induced by methotrexate and improves immune status of rats recovering from chemotherapy. This immune-enhancing effect of dietary GLU was dose-dependent, more pronounced after a longer duration of dietary intake, and coincided with transient elevations of plasma GLN, GLU, or GSH levels.

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