Supplemental glutamine augments phagocytosis and reactive oxygen intermediate production by neutrophils and monocytes from postoperative patients in vitro

APPLIED NUTRITIONAL INVESTIGATION

Supplemental Glutamine Augments Phagocytosis and Reactive Oxygen Intermediate Production by Neutrophils and Monocytes From Postoperative Patients In Vitro Satoshi Furukawa, MD, Hideaki Saito, MD, Tomomi Inoue, MD, Takeaki Matsuda, MD, Kazuhiko Fukatsu, MD, Ilsoo Han, MD, Shigeo Ikeda, MD, and Akio Hidemura, MD From the Department of Surgery, Faculty of Medicine and the Surgical Center, University of Tokyo, Tokyo, Japan The energy substrate for neutrophils has been believed to be glucose. However, a recent investigation has demonstrated that neutrophils use glutamine (Gln) as well as glucose. Nevertheless, little is known about the effects of Gln on neutrophil function. Thus, this study was designed to investigate the effects of Gln on phagocytosis and reactive oxygen intermediate (ROI) production by neutrophils from postoperative patients in vitro. Eleven patients who had undergone major gastrointestinal surgery were randomly selected. Peripheral blood was drawn before surgery and on postoperative days (PODs) 1, 3, and 7. The blood was washed with medium to remove plasma. Washed whole blood was incubated in RPMI 1640 medium containing neither Gln nor glucose for 24 h at 37°C. The medium was supplemented with Gln at a concentration of 0, 500, 1000, or 2000 ␮M. Whole blood was then assessed for phagocytosis by flow cytometry using fluorescent beads. ROI production by phagocytes was measured by flow cytometry using dihydrorhodamine 123. In each assay, the neutrophil population was gated and analyzed. Serum amino acids were also measured. Postoperative serum Gln level decreased significantly until POD 7. Phagocytosis by neutrophils on PODs 3 and 7 was significantly greater at 2000 ␮M Gln than at other Gln concentrations. Neutrophil ROI production was significantly greater at 2000 ␮M Gln than at 0 ␮M Gln at each time point. In conclusion, supplemental Gln enhances both phagocytosis and ROI production by neutrophils from postoperative patients in vitro. Nutrition 2000;16:323–329. ©Elsevier Science Inc. 2000 Key words: glutamine, neutrophil, phagocytosis, reactive oxygen intermediate, postoperative patient, monocyte

INTRODUCTION In randomized, double-blind, controlled trials, parenterally or enterally administered glutamine (Gln) lowered the incidence of infection in patients with bone-marrow transplantation1 and multiple trauma.2 However, the precise mechanisms are not well understood. Gln is an important fuel for lymphocytes and macrophages; in fact, Gln enhances the functions of these immune cells.3 However, supplemental Gln augments the in vitro bacterial killing activity of neutrophils in burned or postoperative patients.4,5 Nevertheless, the mechanisms underlying the enhancing effect of Gln on neutrophil bactericidal capacity have not been fully studied. To eliminate bacteria, neutrophils phagocytose and then kill the target. Bacterial killing by neutrophils can be ascribed to two mechanisms, oxidative and non-oxidative. As to the oxidative mechanisms, activated neutrophils produce several antimicrobial reactive oxygen intermediate (ROI) products, including superoxide, hydrogen peroxide, hydroxyl radicals, hypochlorous acid, and singlet oxygen.6 These ROIs are generated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase, located on the plasma membrane, that reduces molecular oxygen to superoxide anion. The energy substrate for neutrophils has long been thought to

be glucose.7 However, it was suggested that neutrophils can use Gln in severe infection where glucose use is restricted.8 In addition, Gln has recently been shown to be used by rat peritoneal neutrophils.9 Therefore, neutrophils apparently use Gln as well as glucose. Nevertheless, the effects of Gln on neutrophil function remain to be clarified. Therefore, the present study focused on the effects of Gln in vitro on phagocytosis and ROI production by neutrophils from postoperative patients. Macrophages also ingest and kill bacteria within phagocytic vacuoles by using ROI.10 Macrophages use Gln at a high rate,11 and the phagocytosis of unopsonized zymosan in vitro by murine peritoneal macrophages has been shown to depend on Gln concentration in vitro.3 In addition, an in vivo study showed that a Gln-enriched enteral diet significantly increased superoxide production and Candida albicans killing by rat peritoneal macrophages.12 Therefore, the effects of Gln on phagocytosis and ROI production by monocytes from postoperative patients were also examined in vitro with the whole-blood method.

PATIENTS AND METHODS Patients

Correspondence to: Satoshi Furukawa, MD, Department of Surgery, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Date accepted: Jan. 13, 2000. Nutrition 16:323–329, 2000 ©Elsevier Science Inc., 2000. Printed in the United States. All rights reserved.

Informed consent was obtained from each patient. Eleven patients scheduled to undergo various kinds of major gastrointestinal operations were randomly selected to determine which patient group could benefit from Gln supplementation. 0899-9007/00/$20.00 PII S0899-9007(00)00228-8

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Blood Sampling and Incubation of Washed Whole Blood With Glutamine Peripheral blood was drawn at 7 AM before surgery and on postoperative days (PODs) 1, 3, and 7. At the time of blood drawing, at least 8 h had passed since the last administration of intravenous prophylactic antibiotics. The whole-blood method was adopted.13 First, 4 mL of venous blood was collected from the patients into a syringe containing 0.2 mL of heparin sodium (1000 U/mL; Novo Nordisk A/S, Copenhagen, Denmark). Then 46 mL of Iscove’s Modified Dulbecco’s Medium (IMDM; Nikken Biomedical Laboratory, Kyoto, Japan) was added, and the mixture was centrifuged at 400 g for 5 min to remove plasma. IMDM was then added to achieve a volume of 5 mL. Washed whole blood was incubated in RPMI 1640 medium containing neither glucose nor Gln (Nikken Biomedical Laboratory) for 24 h at 37°C in 5% CO2. The medium was supplemented with Gln at concentrations of 0, 500, 1000, and 2000 ␮mol/L. After drawing each blood sample, the serum was separated and stored at ⫺80°C until analysis of C-reactive protein (CRP) and amino acids, as described below. A part of each serum sample was used to opsonize the fluorescent latex beads on each occasion. Phagocytosis Fluorescent latex beads (1.0 ␮m in diameter; Fluoresbrite, Polysciences Inc., Warrington, PA, USA) were opsonized with the subjects’ serum for 30 min at 37°C. The beads were then washed twice with phosphate buffered saline (PBS; Nikken Biomedical Laboratory) and adjusted to 2.5 ⫻ 107/50 ␮L in PBS. Gln-treated washed whole blood (100 ␮L) was combined with 50 ␮L of fluorescent latex beads. After a 30-min incubation at 37°C, these reactions were stopped by placing the samples on ice. The samples were then washed with PBS. Each sample was subjected to erythrocyte lysis by addition of 2 mL of FACS-lysing solution (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) for 10 min at room temperature, washed with PBS, and stored in 500 ␮L of 0.5% paraformaldehyde (PFA; Sigma Chemical Co., St. Louis, MO, USA) at 4°C until flow cytometric analysis. Flow cytometric analyses were performed using FACScan (Becton Dickinson Immunocytometry Systems). In each sample, at least 5000 cells were collected. Samples were analyzed with CELLQuest software (Becton Dickinson Immunocytometry Systems). Neutrophil and monocyte populations were gated according to a combination of forward light scatter and perpendicular light scatter, and mean fluorescence intensity was analyzed. All measurements were performed under the same instrument settings. Production of Reactive Oxygen Intermediates Whole blood was assessed for ROI production using dihydrorhodamine 123 (DHR, Molecular Probes, Eugene, OR, USA).14 Briefly, after preincubation of the washed whole blood with different concentrations of Gln, 1 mL of each sample was incubated with or without 100 ng/mL of phorbol myristate acetate (PMA; Sigma Chemical Co.) stimulation for 30 min at 37°C. The reaction was stopped by placing the samples on ice. The samples were then incubated with 7.5 ␮mol/L of DHR for 10 min at 37°C. DHR becomes fluorescent with oxidation by ROI. DHR is sensitive to the generation of not only hydrogen peroxide but also other ROIs.15 After the reaction had been stopped by placing the samples on ice, cells were subjected to erythrocyte lysis by addition of 2 mL of FACS-lysing solution for 10 min at room temperature. Samples were washed with PBS and fixed with 500 ␮L of 0.5% ice-cold PFA for 10 min at 4°C. The samples were then assessed for ROI production by flow cytometry, i.e., FACScan. The stimulation index of ROI production was calculated by dividing the

mean fluorescence intensity of PMA-stimulated phagocytes (neutrophils and monocytes) by that of unstimulated phagocytes.15 Analyses of Serum C-Reactive Protein and Amino Acids Serum CRP and amino acids were measured by turbidimetric immunoassay and high-performance liquid chromatography, respectively. Statistical Analysis Data are presented as mean ⫾ standard error of the mean (SEM). Statistical analyses were performed by using one-way repeatedmeasures analysis of variance followed by contrast testing using SuperANOVA software (Abacus Concepts, Inc., Berkeley, CA, USA). Linear regression analysis was also used. Statistical significance was set at P ⬍ 0.05.

RESULTS Clinical and Laboratory Data of Patients Clinical and laboratory data of the patients are shown in Table I. Patients’ ages ranged from 50 to 89 y, with mean of 65 y. Diagnoses included esophageal cancer, gastric cancer, ascending colon cancer, descending colon cancer, and rectal cancer. All patients could eat normally before the operation. Preoperative serum albumin level and percentage of ideal body weight were 3.4 ⫾ 0.2 g/dL and 94% ⫾ 3%, respectively. Associated diseases were left hemiplegia due to cerebral infarction, liver cirrhosis, diabetes mellitus, and hypertension in one patient. No patient underwent preoperative chemotherapy or radiotherapy. The operations included subtotal esophagectomy, total gastrectomy, distal gastrectomy, right hemicolectomy, left hemicolectomy, low anterior resection, and low anterior resection followed by resection of metastases in the liver. Operative time ranged from 100 to 765 min, with a mean of 305 min. Intraoperative blood loss ranged from 30 to 1910 mL, with a mean of 417 mL. The patients received standard parenteral nutrition containing no Gln. Mean energy intake on POD 1 was 473 kcal. Mean nitrogen determined on POD 1 was 0.62 g. Administered energy and nitrogen were increased gradually, reaching a plateau on POD 5 and continuing throughout the study period. Mean energy intake and nitrogen determined on POD 5 were 1087 kcal and 4.82 g, respectively. Peripheral white blood cell count before surgery and on PODs 1, 3, and 7 was 5800, 10 700, 9000, and 8400/mm3, respectively. Serum CRP level before surgery and on PODs 1, 3, and 7 was 0.4, 8.2, 10.7 and 8.3 mg/dL, respectively. Serum Glutamine Level Postoperative serum Gln level decreased significantly until POD 7, with the lowest level on POD 3 (before surgery, 590 ⫾ 30 ␮mol/L; POD 1, 474 ⫾ 22 ␮mol/L; POD 3, 391 ⫾ 21 ␮mol/L; POD 7, 483 ⫾ 36 ␮mol/L; P ⬍ 0.01 versus preoperative level). Serum Gln concentration was negatively correlated with serum CRP level on PODs 3 and 7 (POD 3, r ⫽ ⫺0.67; POD 7, r ⫽ ⫺0.63; and for both, P ⬍ 0.05) (Fig. 1). On POD 1, there was also a negative correlation between serum Gln concentration and serum CRP level, but it did not reach statistical significance (r ⫽ ⫺0.51, P ⬍ 0.11). Phagocytosis Before surgery and on POD 1, there was no difference in the phagocytic activity of neutrophils at the Gln concentrations used. However, phagocytosis by neutrophils on PODs 3, and 7 was

GLN ENHANCES POSTOPERATIVE PATIENTS’ LEUKOCYTE PHAGOCYTOSIS AND ROI PRODUCTION

325

TABLE I. CLINICAL AND LABORATORY DATA

Patient age (y)/sex

Diagnosis

66/M

Esophageal cancer

61/M

Esophageal cancer

69/M 64/M 53/M 76/F

Gastric cancer Gastric cancer Gastric cancer Ascending colon cancer

82/M 89/M 50/M

Ascending colon cancer Ascending colon cancer Descending colon cancer Rectal cancer Rectal cancer with liver metastasis

50/M 50/F

Associated disease

LC

Operation Subtotal esophagectomy Subtotal esophagectomy Total gastrectomy Total gastrectomy Distal gastrectomy Right hemicolectomy

Cerebral infarction DM Right hemicolectomy HT Right hemicolectomy Left hemicolectomy LAR LAR with partial liver resection

Serum albumin (g/dL)

%IBW

Operative time (min)

Intraoperative blood loss (mL)

3.9

100.6

765

970

3.9

93.9

520

190

Right pleural abscess, wound infection Wound infection

3.4 3.1 3.8 2.4

104.1 98.3 85.9 82.8

325 210 195 135

190 155 130 120

Anastomotic leakage (⫺) (⫺) (⫺)

2.7 3.4 4.0

69.3 89.6 102.5

100 100 245

30 75 520

(⫺) (⫺) (⫺)

4.1 3.2

107.4 94.5

220 535

295 1910

Wound infection Anastomotic leakage

Postoperative complications

DM, diabetes mellitus; HT, hypertension; IBW, ideal body weight; LAR, low anterior resection; LC, liver cirrhosis.

significantly higher at 2000 ␮mol/L of Gln than at other Gln concentrations studied (Table II). The phagocytic activity of monocytes did not differ among Gln groups before surgery. However, on PODs 1, 3, and 7, phagocytosis by monocytes was significantly greater at 2000 ␮mol/L of Gln than at 0 ␮mol/L of Gln (Table II). Moreover, the monocyte phagocytic activity was significantly higher at 1000 ␮mol/L of Gln than at 0 ␮mol/L of Gln on POD 1. In addition, its activity was significantly greater at 500 and 1000 ␮mol/L of Gln than at 0 ␮mol/L of Gln on POD 7.

We defined the improvement index of phagocytosis as the mean fluorescence intensity of phagocytes after incubation for 24 h at 2000 ␮mol/L of Gln divided by the mean fluorescence intensity of phagocytes after incubation for 24 h at 0 ␮mol/L of Gln. On POD 3, the serum Gln level of the patients was negatively correlated with the improvement index of the phagocytic activity of both neutrophils and monocytes (neutrophils, r ⫽ ⫺0.62; monocytes, r ⫽ ⫺0.61; and for both, P ⬍ 0.05) (Fig. 2). There were no significant correlations between the preoperative nutritional parameters (preoperative serum albumin level and per-

FIG. 1. Correlation between patients’ serum CRP level and serum glutamine concentration on postoperative days 3 and 7 (POD 3, r ⫽ ⫺0.67; POD 7, r ⫽ ⫺0.63; and for both, P ⬍ 0.05). POD, postoperative day; CRP, C-reactive protein; Gln, glutamine.

326

GLN ENHANCES POSTOPERATIVE PATIENTS’ LEUKOCYTE PHAGOCYTOSIS AND ROI PRODUCTION TABLE II. EFFECT OF GLUTAMINE ON PHAGOCYTOSIS BY PHAGOCYTES* Neutrophils

Glutamine (␮M) Before surgery POD 1 POD 3 POD 7

Monocytes

0

500

1000

2000

0

500

1000

2000

66 ⫾ 11 108 ⫾ 37 51 ⫾ 8 120 ⫾ 15

100 ⫾ 20 102 ⫾ 23 60 ⫾ 10 135 ⫾ 18

84 ⫾ 21 99 ⫾ 26 67 ⫾ 10 137 ⫾ 17

88 ⫾ 20 120 ⫾ 30 95 ⫾ 20†‡§ 177 ⫾ 27†‡§

56 ⫾ 8 43 ⫾ 8 41 ⫾ 9 55 ⫾ 9

66 ⫾ 11 47 ⫾ 7 44 ⫾ 10 67 ⫾ 12†

65 ⫾ 11 53 ⫾ 8† 45 ⫾ 10 67 ⫾ 13†

66 ⫾ 9 60 ⫾ 10†‡ 54 ⫾ 11†‡§ 74 ⫾ 12†

* Data are presented as mean ⫾ SEM (mean fluorescence intensity). † P ⬍ 0.05 versus 0 ␮mol/L of glutamine. ‡ P ⬍ 0.05 versus 500 ␮mol/L of glutamine. § P ⬍ 0.05 versus 1000 ␮mol/L of glutamine. POD, postoperative day.

centage of ideal body weight) and in vitro phagocytosis by phagocytes (neutrophils and monocytes) from postoperative patients. Reactive Oxygen Intermediate Production Neutrophil ROI production was significantly greater at 2000 ␮mol/L of Gln than at 0 ␮mol/L of Gln at each time point (Table III). In addition, ROI production by neutrophils was significantly higher at 1000 ␮mol/L of Gln than at 0 ␮mol/L of Gln on PODs 3 and 7. In contrast, Gln supplementation caused no appreciable change in monocyte ROI production before surgery. However, ROI production by monocytes was significantly greater at 2000 ␮mol/L of Gln than at 0 and 500 ␮mol/L of Gln postoperatively (Table III). Furthermore, monocyte ROI production was significantly higher at 500 and 1000 ␮mol/L of Gln than at 0 ␮mol/L of Gln on POD 3 and at 1000 ␮mol/L of Gln than at 0 ␮mol/L of Gln on POD 7.

We defined the improvement index of ROI production as the phagocyte stimulation index of ROI production after incubation for 24 h at 2000 ␮mol/L of Gln divided by the phagocyte stimulation index of ROI production after incubation for 24 h at 0 ␮mol/L of Gln. On POD 1, the serum CRP level of the patients was positively correlated with the improvement index of ROI production by neutrophils (r ⫽ 0.61, P ⬍ 0.05) (Fig. 3). No significant correlations were found between the preoperative nutritional parameters and in vitro ROI production by phagocytes from postoperative patients.

DISCUSSION In the present study, phagocytosis by neutrophils on PODs 3 and 7 was significantly greater at 2000 ␮mol/L of Gln than at other Gln

FIG. 2. Correlation between patients’ serum glutamine level and improvement index of phagocytic activity of both neutrophils and monocytes on postoperative day 3 (neutrophils, r ⫽ ⫺0.62; monocytes, r ⫽ ⫺0.61; and for both, P ⬍ 0.05). Improvement index of phagocytic activity ⫽ MFI of phagocytes after incubation for 24 h at 2000 ␮mol/L of Gln divided by the MFI of phagocytes after incubation for 24 h at 0 ␮mol/L of Gln. Gln, glutamine; MFI, mean fluorescence intensity.

GLN ENHANCES POSTOPERATIVE PATIENTS’ LEUKOCYTE PHAGOCYTOSIS AND ROI PRODUCTION

327

TABLE III. EFFECT OF GLUTAMINE ON ROI PRODUCTION BY PHAGOCYTES* Neutrophils Glutamine (␮M) Before surgery POD 1 POD 3 POD 7

Monocytes

0

500

1000

2000

0

500

1000

2000

21.0 ⫾ 3.8 13.7 ⫾ 2.7 18.9 ⫾ 6.9 18.5 ⫾ 4.3

25.7 ⫾ 5.7 15.5 ⫾ 4.2 20.9 ⫾ 7.7 20.1 ⫾ 4.5

26.1 ⫾ 6.1 15.7 ⫾ 3.5 22.5 ⫾ 7.4† 21.8 ⫾ 4.9†

27.6 ⫾ 5.8† 17.7 ⫾ 4.1† 25.9 ⫾ 8.5†‡§ 28.0 ⫾ 6.1†‡§

10.4 ⫾ 2.7 6.3 ⫾ 0.9 7.4 ⫾ 2.5 8.5 ⫾ 1.5

10.0 ⫾ 2.4 6.4 ⫾ 1.3 8.9 ⫾ 2.5† 9.1 ⫾ 1.8

11.4 ⫾ 2.8 7.3 ⫾ 1.1 9.5 ⫾ 2.5† 10.4 ⫾ 2.1†

11.6 ⫾ 2.8 8.2 ⫾ 1.6†‡ 10.4 ⫾ 2.8†‡ 12.4 ⫾ 2.3†‡§

* Data are presented as mean ⫾ SEM of the stimulation index of ROI production. The stimulation index of ROI production was calculated by dividing the mean fluorescence intensity of phagocytes stimulated by phorbol myristate acetate (neutrophils and monocytes) by that of unstimulated phagocytes. † P ⬍ 0.05 versus 0 ␮mol/L of glutamine. ‡ P ⬍ 0.05 versus 500 ␮mol/L of glutamine. § P ⬍ 0.05 versus 1000 ␮mol/L of glutamine. POD, postoperative day; ROI, reactive oxygen intermediates.

concentrations. Moreover, neutrophil ROI production was significantly greater at 2000 ␮mol/L of Gln than at 0 ␮mol/L of Gln before surgery and on PODs 1, 3, and 7. In addition, both phagocytosis and ROI production by monocytes on PODs 1, 3, and 7 were significantly greater at 2000 ␮mol/L of Gln than at 0 ␮mol/L of Gln. A recent investigation has shown that neutrophils use Gln as well as glucose.9 However, the effects of Gln on neutrophil functions remain to be established. Therefore, the primary purpose of this study was to investigate the effects of Gln on phagocytosis and ROI production by neutrophils from postoperative patients. In this experiment, depletion of energy substrates for neutrophils, i.e., glucose and Gln, was simulated by using RPMI 1640 medium containing neither glucose nor Gln. However, 0 mg/dL of glucose and 0 ␮mol/L of Gln would most likely not occur in the in

FIG. 3. Correlation between patients’ serum CRP level and improvement index of ROI production by neutrophils on postoperative day 1 (r ⫽ 0.61, P ⬍ 0.05). Improvement index of ROI production ⫽ stimulation index of ROI production by neutrophils after incubation for 24 h at 2000 ␮mol/L of Gln divided by the stimulation index of ROI production by neutrophils after incubation for 24 h at 0 ␮mol/L of Gln. CRP, C-reactive protein; Gln, glutamine; ROI, reactive oxygen intermediates.

vivo setting. Moreover, 2000 ␮mol/L of Gln is not easily obtained in vivo. Nevertheless, glucose metabolism is impaired in septic patients.16 Severe hypoglycemia reportedly occurs in sepsis; the lowest serum glucose level in patients with sepsis was 5 mg/dL, with a mean of 22 mg/dL.17 Moreover, neutrophils show a 2- to 10-fold increase in glucose oxidation during particle ingestion.18 In addition, uptake of glucose by injured tissue in vivo is increased, and this increased uptake is localized to inflammatory cells at the injury site.19 Therefore, glucose depletion may occur in critically ill patients, especially at inflammatory sites. Plasma Gln concentration in patients with sepsis20 or severe head injuries21 decreases to approximately 250 ␮mol/L. Furthermore, during enteral administration of approximately 860 ␮mol 䡠 kg⫺1 䡠 h⫺1 of Gln, the mean plasma Gln level is nearly 1300 ␮mol/L.22 Thus, the concentrations of glucose and Gln used in the present study seemed to be appropriate for the study objective. The whole-blood method was adopted in this study because its conditions are close to those of an in vivo setting, where monocytes, lymphocytes, erythrocytes, and platelets exist together with neutrophils. The whole-blood method also has an advantage in that an adequate number of neutrophils are obtained from only a small amount (4 mL) of blood. Therefore, the method allowed frequent blood drawing even from postoperative patients. In addition, we modified this method by washing the whole blood to avoid the influence of serum, which contains Gln.23 Thus, the whole-blood method was considered appropriate for our study. We have shown that supplemental Gln augmented ROI production by neutrophils from postoperative patients in vitro. It is speculated that Gln may be used by neutrophils as an energy source by way of the citric acid cycle. Gln is metabolized to ␣-ketoglutarate by way of glutamate and then enters the citric acid cycle.24 In addition, citric-acid-cycle enzymes are present in leukocytes.7 Therefore, Gln may enhance NADPH oxidase activity, thereby augmenting superoxide production by neutrophils. Consequently, neutrophil ROI production may be increased. Ogle et al. showed that Gln had no significant effect on phagocytosis by neutrophils from burn patients.4 Conversely, the present study showed that supplemental Gln augmented phagocytosis by neutrophils from postoperative patients. The discrepancies in the effects of Gln on neutrophil phagocytosis may be due to several differences between the two experiments. First, the patient population was different. Second, isolated neutrophils were used in the study by Ogle et al., whereas washed whole blood was employed in ours. Third and most important of all, the incubation time with Gln was very different. Ogle et al. cultured neutrophils for only 30 min with Gln4; in contrast, we incubated neutrophils with Gln for

328

GLN ENHANCES POSTOPERATIVE PATIENTS’ LEUKOCYTE PHAGOCYTOSIS AND ROI PRODUCTION

up to 24 h. Gln may promote neutrophil phagocytosis by serving as an energy source and increasing cellular adenosine triphosphate (ATP). Our secondary purpose was to examine the effects of Gln on phagocytosis and ROI production by monocytes from postoperative patients in vitro. Spittler et al. reported the effect of Gln on functions of monocytes harvested from healthy human volunteers.25 Low Gln levels resulted in reduced expression of HLA-DR on monocyte-derived macrophages and decreased tetanus-toxoidinduced antigen presentation. In addition, low Gln level downregulated the expression of intercellular adhesion molecule-1 (CD54), Fc receptor for immunoglobulin G (Fc␥ R1/CD64), and complement receptor types 3 (CD11b/CD18) and 4 (CD11c/CD18). Furthermore, depletion of Gln was associated with a significant reduction in cellular ATP, which may have influenced cell-surface marker expression and phagocytosis. We demonstrated that Gln enhanced phagocytosis and ROI production by monocytes from postoperative patients in vitro. Gln probably enhanced monocyte phagocytosis and ROI production by serving as an energy substrate and increasing cellular ATP. A negative correlation was demonstrated between serum Gln concentration and serum CRP level on PODs 3 and 7. In addition, there was a positive correlation between the patients’ serum CRP level and the improvement index of ROI production by neutrophils on POD 1. These findings indicate that, as the surgical stress increased, there was a decrease in serum Gln level. Moreover, at decreased serum Gln levels, an increase in the degree of improvement in neutrophil ROI production by Gln was demonstrated in in vitro tests. Therefore, supplemental Gln may be useful, especially for patients who have suffered severe surgical stress. The patients’ serum Gln level negatively correlated with the improvement index of phagocytic activity of both neutrophils and monocytes on POD 3. This finding indicates that, as the serum Gln concentration decreased, there was enhancement of phagocytic activity of phagocytes by supplemental Gln in in vitro tests. This observation suggests that Gln is especially beneficial for patients whose serum Gln levels are severely depressed. There are no reports, to our knowledge, that describe the postoperative change in intracellular Gln levels of neutrophils and monocytes. In contrast, postoperative intracellular Gln level in muscle decreased until POD 30, reaching the lowest level on POD 3.26 Therefore, it is speculated that intracellular Gln levels of neutrophils and monocytes may decrease postoperatively and reach the lowest level within a few days after surgery. Thus, starting supplemental Gln immediately after surgical insult may be most effective for promotion of host defenses. No significant relation was demonstrated between the patients’ serum Gln level and the improvement index of ROI production by phagocytes. The reason for this is unclear. However, it is noteworthy that supplemental Gln augmented ROI production by neutrophils and monocytes in vitro. The total amino acid concentration was increased by 11%, 23%, and 45% when 0.5, 1, and 2 mM of Gln was added, respectively, to the culture medium used in this study. In addition, the total nitrogen content was increased by the same percentage as the total amino acid concentration. However, we did not investigate the effects of other glucogenic amino acids such as alanine on phagocyte ROI production and phagocytosis. Therefore, the enhancement of ROI production and phagocytosis by phagocytes in the present study may not have been Gln specific but due to increased total amino-acid concentration, increased nitrogen content, or increased concentration of glucogenic amino acids in the culture medium. Clinically, the serum Gln level decreases after burns,3 trauma,27 and surgery.28 Moreover, neutrophils isolated from patients after burns,29 trauma,29 and surgery30,31 demonstrate reduced bactericidal function. Therefore, Gln supplementation in critical illness may enhance neutrophil function. The subjects of the present study were all cancer patients. Other types of postoperative or catabolic

patients may respond differently. However, Gln has been shown to augment the in vitro bacterial killing activity of neutrophils from burn or postoperative patients.4,5 Furthermore, parenterally or enterally administered Gln lowered patients’ infection rate in randomized, double-blind, controlled trials.1,2 Although we did not actually show that there was improved bacterial killing when Gln was added to neutrophils in this particular study, our results support these beneficial effects of Gln on host defense against infection. In conclusion, supplemental Gln enhances both phagocytosis and ROI production by neutrophils and monocytes from postoperative patients in vitro. The enhancing effect of Gln on neutrophil bactericidal capacity may be partly explained by increased phagocytosis or ROI production.

REFERENCES 1. Ziegler TR, Young LS, Benfell K, et al. Clinical and metabolic efficacy of glutamine-supplemented parenteral nutrition after bone marrow transplantation. A randomized, double-blind, controlled study. Ann Intern Med 1992; 116:821 2. Houdijk AP, Rijnsburger ER, Jansen J, et al. Randomised trial of glutamineenriched enteral nutrition on infectious morbidity in patients with multiple trauma. Lancet 1998;352:772 3. Parry-Billings M, Evans J, Calder PC, et al. Does glutamine contribute to immunosuppression after major burns? Lancet 1990;336:523 4. Ogle CK, Ogle JD, Mao JX, et al. Effect of glutamine on phagocytosis and bacterial killing by normal and pediatric burn patient neutrophils. JPEN 1994; 18:128 5. Furukawa S, Saito H, Fukatsu K, et al. Glutamine-enhanced bacterial killing by neutrophils from postoperative patients. Nutrition 1997;13:863 6. Smolen JE, Boxer LA. Functions of neutrophils. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Williams hematology, 5th ed. New York: McGraw-Hill, 1995:779 7. Beutler E. Metabolism of neutrophils. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Williams hematology, 5th ed. New York: McGraw-Hill, 1995:767 8. Vlessis AA, Goldman RK, Trunkey DD. New concepts in the pathophysiology of oxygen metabolism during sepsis. Br J Surg 1995;82:870 9. Curi TC, De Melo MP, De Azevedo RB, et al. Glutamine utilization by rat neutrophils: presence of phosphate-dependent glutaminase. Am J Physiol 1997; 273:C1124 10. Lincoln JA, Lefkowitz DL, Cain T, et al. Exogenous myeloperoxidase enhances bacterial phagocytosis and intracellular killing by macrophages. Infect Immun 1995;63:3042 11. Newsholme P, Gordon S, Newsholme EA. Rates of utilization and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages. Biochem J 1987;242:631 12. Shou J, Daly JM. Enteral glutamine augments host immune function. Surg Forum 1994;45:33 13. Elbim C, Bailly S, Chollet-Martin S, et al. Differential priming effects of proinflammatory cytokines on human neutrophil oxidative burst in response to bacterial N-formyl peptides. Infect Immun 1994;62:2195 14. Watson RW, Redmond HP, Wang JH, et al. Neutrophils undergo apoptosis following ingestion of Escherichia coli. J Immunol 1996;156:3986 15. Vowells SJ, Sekhsaria S, Malech HL, et al. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J Immunol Methods 1995;178:89 16. Scheeren T, Susanto F, Reinauer H, et al. Prostacyclin improves glucose utilization in patients with sepsis. J Crit Care 1994;9:175 17. Miller SI, Wallace R Jr, Musher DM, et al. Hypoglycemia as a manifestation of sepsis. Am J Med 1980;68:649 18. Paraskevas F. Phagocytosis. In: Lee GR, Foerster J, Lukens J, et al., eds. Wintrobe’s clinical hematology, 10th ed. Baltimore: Williams & Wilkins, 1999:415 19. Forster J, Morris AS, Shearer JD, et al. Glucose uptake and flux through phosphofructokinase in wounded rat skeletal muscle. Am J Physiol 1989;256: E788 20. Vente JP, von Meyenfeldt MF, van Eijk HM, et al. Plasma-amino acid profiles in sepsis and stress. Ann Surg 1989;209:57 21. Petersen SR, Jeevanandam M, Holaday NJ, et al. Arterial-jugular vein free amino acid levels in patients with head injuries: important role of glutamine in cerebral nitrogen metabolism. J Trauma 1996;41:687

GLN ENHANCES POSTOPERATIVE PATIENTS’ LEUKOCYTE PHAGOCYTOSIS AND ROI PRODUCTION 22. Hankard RG, Darmaun D, Sager BK, et al. Response of glutamine metabolism to exogenous glutamine in humans. Am J Physiol 1995;269:E663 23. Inoue T, Saito H, Matsuda T, et al. Growth hormone and insulin-like growth factor I augment bactericidal capacity of human polymorphonuclear neutrophils. Shock 1998;10:278 24. Mayes PA. The citric acid cycle: the catabolism of acetyl-coA. In: Murray RK, Granner DK, Mayes PA, Rodwell VW, eds. Harper’s biochemistry, 23rd ed. London: Prentice-Hall International Inc., 1993:164 25. Spittler A, Winkler S, Gotzinger P, et al. Influence of glutamine on the phenotype and function of human monocytes. Blood 1995;86:1564 26. Petersson B, Vinnars E, Waller SO, et al. Long-term changes in muscle free amino acid levels after elective abdominal surgery. Br J Surg 1992;79:212

27. Askanazi J, Carpentier YA, Michelsen CB, et al. Muscle and plasma amino acids following injury. Influence of intercurrent infection. Ann Surg 1980; 192:78 28. Parry-Billings M, Baigrie RJ, Lamont PM, et al. Effects of major and minor surgery on plasma glutamine and cytokine levels. Arch Surg 1992;127:1237 29. Alexander JW, Hegg M, Altemeier WA. Neutrophil function in selected surgical disorders. Ann Surg 1968;168:447 30. El-Maallem H, Fletcher J. Effects of surgery on neutrophil granulocyte function. Infect Immun 1981;32:38 31. Shigemitsu Y, Saito T, Kinoshita T, et al. Influence of surgical stress on bactericidal activity of neutrophils and complications of infection in patients with esophageal cancer. J Surg Oncol 1992;50:90

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