APPLIED NUTRITIONAL INVESTIGATION Nutrition Vol. 13, No.

10, 1997

Glutamine-Enhanced Bacterial Killing by

Neutrophils From Postoperative Patients

SATOSHI FURUKAWA, MD, HIDEAKI SAITO, MD, KAZUHIKO FUKATSU, MD,

YOJIRO HASHIGUCHI, MD, TSUYOSHI INABA, MD, MING-TSAN LIN, MD, TOMOMI INOUE, MD,
ILSOO HAN, MD, TAKEAKI MATSUDA, MD, AND TETSUICHIRO MUTO, MD

From the Department of Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan

Date accepted: 7 February 1997

ABSTRACT
Neutrophils play an important role in host defense by phagocytosing and destroying invading bacteria. A recent investigation
revealed that glutamine (Gln) augmented the in vitro bactericidal activity of neutrophils from bum patients. However, it is unclear
whether Gln enhances the function of neutrophils in postoperative patients. This study was designed to investigate the effect of
Gln on the in vitro Escherichia c&i-killing activity of neutrophils from postoperative patients. Nine randomly selected patients
were included in this study. On the morning of the first postoperative day, blood was drawn and neutrophils were isolated. Eight
healthy volunteers served as controls. E. coli was opsonized with pooled normal serum. Neutrophils (5 X 106), together with
opsonized E. coli (5 X 105), were incubated for 2 h at 37°C in Hanks’ balanced salt solution supplemented with 0, 100,500, or
1000 nmol/mL of Gln. The bactericidal function of neutrophils was determined by counting the number of viable bacteria. Tumor
necrosis factor (TNF)-(Y, interleukin (IL)-lp, IL-8, and granulocyte elastase levels in the cell culture supematant were measured.
Plasma C-reactive protein (CRP), cortisol, and amino acids were also analyzed. The plasma concentration of Gln was
significantly lower in the postoperative patients than in the controls. Following culture with patient neutrophils, the number of
viable E. coli decreased by 26% as the in vitro Gln concentration was increased from 500 to 1000 nmol/mL (P < 0.01). We
defined the Gln lOOO/Gln 500 ratio of the number of viable bacteria as the number of viable E. coli at an in vitro Gln
concentration of 1000 nmol/mL divided by the number of viable E. coli at an in vitro Gln concentration of 500 nmol/mL. A
positive correlation was thus demonstrated between the plasma Gln level and the Gln lOOO/Gln 500 ratio of the number of viable
bacteria in the patients (r = 0.69, P = 0.04). This finding indicated that as plasma Gln fell, there was an enhancement of
neutrophil E. coli-killing activity by neutrophils in in vitro tests when the Gln concentration was increased from 500 to 1000
nmol/mL. Gln supplementation caused no appreciable changes in TNF-a, &l/3, R-8, or granulocyte elastase levels in cell
culture supematants. A negative correlation was recognized between the patient plasma Gln level and the Gln lOOO/Gln 500 ratio
of the cell culture supematant IL-8 level (r = -0.73, P = 0.025). In conclusion, Gln supplementation enhanced the in vitro
bactericidal function of neutrophils from postoperative patients. Nutrition 1997;13:863-869. OElsevier Science Inc. 1997

Key words: glutamine, neutrophils, bacterial killing, Escherichia coli

INTRODUCTION Host defense mechanisms are impaired following surgery.4

Despite major advances in surgical techniques and periopera-
tive care, infectious complications remain the major cause of
postoperative morbidity and mortality.’ Currently, antibiotic ther-
apy, adequate nutrition, and debridement of infected tissues are
the major means of reducing infectious complications following
surgery.* However, postoperative infectious complications still
occur at a high incidence. Thus, new approaches are needed.
Recently developed specific nutritional substrates have been
shown to augment host immune function and improve survival.3
Glutamine (Gln) is one of these new substrates.3
Neutrophils play a pivotal role in host defense by phagocytosing
and destroying invading bacteria.5 Neutrophils isolated from pa-
tients after surgery,6s7 bums,s and trauma* show depressed bacte-
ricidal function. However, little information is available concem-
ing means of augmenting the bactericidal activity of neutrophils.
Gln has been shown to enhance the functions of lymphocytes
and macrophages.9 Moreover, a recent study revealed that Gln
augmented the in vitro bactericidal activity of neutrophils from
bum patientslO However, it remains unclear whether Gln en-
hances the function of neutrophils in postoperative patients. This

Correspondence to: Satoshi Fumkawa, MD, Department of Surgery, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyoku, Tokyo, Japan.

Nutrition 13:863-869, 1997

OElsevier Science Inc. 1997 0899-9007/97/$17.00
Printed in the USA. All rights reserved. ELSEVIER PI1 SO899-9007(97)00271-2
864 GLUTAMINE-ENHANCED BACTERIAL KlLLING BY NEUTROPHILS

TABLE I.

CLINICAL AND LABORATORY DATA

Preoperative Operative Intraoperative Intraoperative WBC on

Patient Associated serum Alb time blood loss transfusion POD Postoperative
age/sex Diagnosis disease Operation (g/dL) % IBW (min) (mL) (mL) 1 (/mm3) complications

1. 46 Arteriosclerosis DM Aortobifemoral 4.0 102.8 325 400 0 15 800 (-)

M obliterans bypass
2. 64 Esophageal DM Subtotal 3.2 85.1 350 250 600 14 100 (-)
M cancer esophagectomy
3. 66 Pharyngeal (y) Total 3.7 76.2 720 2000 840 22 900 (-)
M cancer laryngectomy
4. 59 Ulcerative DM Total colectomy 3.5 91.2 330 1040 400 14 600 anastomotic
M colitis leakage
5. 71 Pharyngeal (-) Middle 3.9 113.1 590 760 0 13 700 (-)
M cancer pharyngectomy
6. 41 F Breast cancer (-) Modified radical 4.2 90.8 275 350 0 9900 (-)
mastectomy
7. 66 Arteriosclerosis (-) Femoropopliteal 3.8 104.4 210 250 0 9200 (-)
M obliterans bypass
8. 75 Rectal cancer (-) Low anterior 3.5 93.9 190 260 0 8400 (-)
M resection
9. 67 Sigmoid colon DM, LC Sigmoidectomy 3.5 93.6 220 500 0 10900 (-)
M cancer

Alb, albumin; DM, diabetes mellitus; IBW, ideal body weight; LC, liver cirrhosis; POD, postoperative day; WBC, white blood cell count.

study was therefore designed to investigate the effect of Gln on the
in vitro Escherichiu c&killing activity of neutrophils from pa-
tients undergoing various kinds of major surgery.
Production of interleukin-2 (IL-2) by concanavalin A-stimu-
lated rat lymphocytes depends upon the Gln concentration.lt
Moreover, the ability of murine peritoneal macrophages to secrete
IL-l also depends on the Gln level.11 However, the effect of Gln
on neutrophil production of cytokines has not been studied exten-
sively. On the other hand, granulocyte elastase has been shown to
be a component of neutrophils, and this enzyme contributes to the
nonoxidative bacterial killing capacity of neutrophils.5 Therefore,
we measured the levels of tumor necrosis factor (TNF)-cq IL-l&
B-8, and granulocyte elastase in cell culture supernatants to
elucidate the mechanism by which Gln enhances the bactericidal
function of neutrophils.
PATIENTS AND METHODS
Patients
We randomly selected nine patients who had undergone vari-
ous kinds of operations. Informed consent was obtained from each
patient. Clinical and laboratory data on the patients are shown in
Table I. Patient ages ranged from 41 to 75 y. Diagnoses included
arteriosclerosis obliterans, esophageal cancer, pharyngeal cancer,
ulcerative colitis, breast cancer, rectal cancer, and sigmoid colon
cancer. All but patient 2 could eat normally before the operation.
Patient 2 was given a 1500 kcal/d elemental diet. The average
preoperative serum albumin level and percent ideal body weight
were 3.8 2 0.1 g/dL and 95 ? 4%, respectively. Associated
diseases were diabetes mellitus in four patients and liver cirrhosis
in one.
The operations included aortobifemoral bypass, subtotal
esophagectomy, total laryngectomy, total colectomy, middle phar-
yngectomy, modified radical mastectomy, femoropopliteal by-
pass, low anterior resection, and sigmoidectomy. Operative time
ranged from 190-720 min with an average of 357 min. Intraop-
erative blood loss ranged from 250-2000 mL, averaging 646 mL.
One patient (patient 4) undergoing total colectomy developed
anastomotic leakage. There were no postoperative mortalities. We
conducted the same measurements in eight healthy, male, fasting
volunteers with an average age of 33 y.
Blood Sampling and Isolation of Neutrophils
Blood was drawn at 0700 h on the first postoperative day
(POD). No amino acid solution had been included in the postop-
erative parenteral nutrition regimens prior to the blood draw. At
the time of blood drawing, at least 8 h had passed since the last
administration of intravenous prophylactic antibiotics. The mean
peripheral white blood cell count on POD 1 was 13 300/mm3,
ranging from 8400-22 900/mm3. The percentage of neutrophils
was counted using an automatic counter (Blood Cell Analyzer
8200, HITACHI, Tokyo, Japan) and varied from 52-81%.
Neutrophils were obtained by a modification of the methods of
Ogle et al.iO At 0700 h on POD 1, we drew 20 mL peripheral
blood into a syringe containing 4 mL 6% dextran (Midori Juji,
Osaka, Japan) and 0.6 mL heparin sodium (1000 U/mL) (Novo
Nordisk A/S, Copenhagen, Denmark). Similarly, at 0700 h, we
collected from the controls 40 mL peripheral blood in a syringe
containing 8 mL 6% dextran and 1.2 mL heparin sodium (1000
U/mL). The controls had fasted for 8 h prior to the blood drawing.
The syringe was placed upright for 45-60 min to allow for
separation of red cells and plasma. The plasma layer was then
removed through a bent needle and collected in a 50-mL centri-
fuge tube. The plasma layer was underlaid with 10 mL Ficoll
(Pharmacia Biotech A/B, Uppsala, Sweden) and centrifuged at
500 X g for 30 min at 27°C. After centrifugation, the mononuclear
layer was removed and the pellet containing the neutrophils and a
GLUTAMINE-ENHANCED BACTERIAL KILLING BY NEUTROPHILS
few contaminating erythrocytes was resuspended. The erythro-
cytes were lysed with distilled water. The neutrophils were
washed once with Hanks’ balanced salt solution (HBSS) (without
calcium and magnesium; Nikken Biomedical Laboratory, Kyoto,
Japan) supplemented with 0.1% gelatin (Iwaki Glass, Tokyo,
Japan) and 10 mM HEPES (Cosmo Bio Co., Tokyo, Japan). The
cells were then suspended with HBSS containing 0.1% gelatin and
10 mM HEPES. The neutrophils were >95% pure and viable as
determined by microscopic observation. Live neutrophils were
counted by trypan blue exclusion and adjusted to 1 X 107/mL.
Preparation of Bacteria
E. coli (American Type Culture Collection 25922) were cul-
tured in a brain-heart infusion broth (Nissui Pharmaceutical Co.,
Tokyo, Japan) for 18 h at 37°C. The culture was centrifuged at
1700 X g for 10 min at 4°C to pellet the E. coli, then washed twice
and resuspended in sterile normal saline. A lOO-PL suspension
was serially diluted with sterile saline, plated on sheep blood agar
plates (Nissui Pharmaceutical), and incubated for 18 h for deter-
mination of the bacterial concentration. The remainder was stored
at 4°C until use. Just before use, the bacterial suspension was
adjusted to 1 X 10’ colony-forming units (CFU) /mL.
Opsonization of E. coli
E. coli (1 X lo*), together with 1 mL pooled normal serum and
2 mL HBSS containing 0.1% gelatin and 10 mM HEPES, were
cultured for 15 min at 37°C. The culture was centrifuged at
2500 X g for 10 min at 4°C to pellet the E. coli, then washed once
and resuspended in HBSS supplemented with 0.1% gelatin and 10
mM HEPES. The final concentration of the opsonized E. coli was
adjusted to 5 X lo6 CFUlmL. As our intent was to examine the
bactericidal function of the neutrophils themselves, we opsonized
the E. coli with pooled normal serum, rather than with subject
serum.
Measurement of Bactericidal Activity of Neutrophils
Our preliminary examination showed the most appropriate
neutrophil to E. coli ratio for assessing neutrophil bactericidal
function to be 1O:l. Neutrophils (5 X 106), together with 5 X 10’
opsonized E. coli, were incubated in HBSS containing 0.1%
gelatin and 10 mM HEPES supplemented with 0, 100, 500, or
1000 nmol/mL Gln in a final volume of 1 mL. After a 2-h
incubation at 37”C, the reactive mixtures were vigorously vor-
texed and a 0. I-mL aliquot from each sample was added to 9.9 mL
sterile saline. This dilution and one more 1:lOO serial dilution
were plated on sheep blood agar plates and cultured for 18 h at
37°C. Viable E. coli counts were then determined. In handling a
large number of samples at one measurement, isolated neutrophils
may lose viability, thereby diminishing the validity of the exper-
iment. Therefore, we determined viable bacterial counts in cell
culture supematants of the samples on four separate days. Cell
culture supematants were separated and stored at -20°C until the
measurement of TNF-(Y, IL-l& IL-8, and granulocyte elastase
levels, as described below.
Assay of TNF-a, IL-Ifi IL-& and Granulocyte Elastase
TNF-(r, IL-l& and IL-8 levels in the cell culture supematant
were measured using commercially available enzyme-linked im-
munosorbent assay methods (TNF-c~ and IL-lp from Genzyme
Immunobiologicals, Cambridge, MA, USA; IL-8 from TORAY,
Tokyo, Japan). For each assay, a standard curve using recombi-
nant cytokine was constructed. We measured granulocyte elastase
levels in the cell culture supematant by chromogenic substrate
assay using S-2484 (Chromogenix A/B, Molndal, Sweden).
865
Analyses of Plasma C-reactive Protein, Cortisol, and Amino
Acids
Blood samples were centrifuged at 1700 X g for 10 min at
4°C. The plasma was separated and stored at -20°C until analy-
sis. Plasma C-reactive protein (CRP), cortisol, and amino acids
were measured by turbidimetric immunoassay, radioimmunoas-
say, and high-performance liquid chromatography, respectively.
Statistical Analysis
Results are expressed as the mean ?SEM. To achieve a normal
distribution, viable bacterial counts were transformed, if neces-
sary, to a logarithmic scale before statistical analysis. An unpaired
Student’s t test was used for the plasma amino acid concentrations
of both patients and controls. Concerning the number of viable E.
coli in the culture medium, a test with a one-way, repeated-
measures analysis of variance model showed that significant dif-
ference(s) existed among the four in vitro Gln levels in the patient
group. Therefore, a contrast test was carried out within the patient
group to determine the Gln concentration at which viable bacterial
counts differed from those at other Gln concentration(s). Linear
regression analysis was also used. A P value of CO.05 was
considered significant.
RESULTS
Plasma Amino Acid Concentrations
The plasma concentrations of the following amino acids on
POD 1 were significantly lower in patients than in controls:
threonine (P < O.OOOl), serine (P < 0.05), asparagine (P < O.OS),
Gln (P < O.OOl), glycine (P < 0.05), alanine (P < 0.05), citrulline
(P < O.OOl), valine (P < O.OOl), isoleucine (P < O.OOOl), leucine
(P < O.Ol), omithine (P < O.OOl), lysine (P < O.OOOl), and
histidine (P < 0.001) (Table II).
Bactericidal Function of Neutrophils at Various in vitro Gin
Concentrations
Following culture with neutrophils from patients, the number
of viable E. coli decreased by 26% as the in vitro Gln concentra-
tion was increased from 500 to 1000 nmol/mL, and this decrease
was statistically significant (P < 0.01) (Table III). Gln supple-
mentation caused no appreciable changes in the number of viable
E. coli cultured with neutrophils from healthy volunteers. There
was no difference in the number of viable E. coli between the
patients and the volunteers at any of the Gln concentrations used.
Correlation Between Plasma Concentrations of Gln and
Bactericidal Function of Neutrophils
There were no statistically significant correlations between
plasma Gln levels and the number of viable E. coli at any of the
supplementary Gln concentrations used, in either patients or con-
trols. We defined the Gln lOOO/Gln 500 ratio of the number of
viable bacteria as the number of viable E. coli at an in vitro Gln
concentration of 1000 nmoYmL divided by the number of viable
E. coli at an in vitro Gln concentration of 500 nmol/mL. Thus, a
positive correlation was demonstrated between the plasma Gln
level and the Gln lOOO/Gln 500 ratio of the number of viable
bacteria in the patients (r = 0.69; P = 0.04) (Fig.1).
White Blood Cell Count, Plasma CRP Values, and Plasma
Corns01 Levels
T ere were positive correlations between the peripheral white
bloo $ cell count on POD 1 and the number of viable bacteria at all
concentrations of Gln studied (Gln 0 nmol/mL, r = 0.71; 100
nmoYmL, r = 0.74; 500 nmol/mL, r = 0.72; 1000 nmol/mL,
r = 0.67; and for all concentrations, P 5 0.05) (Fig.2). The mean
866 GLUTAMINE-ENHANCED BACTERIAL KILLING BY NEUTROPHILS

TABLE II.

PLASMA AMINO ACID CONCENTRATIONS IN PATIENTS

AND HEALTHY VOLUNTEERS

Amino acid concentration (nmol/mL)

Patients Controls
Amino acid (n = 9) (n = 8) P value

Phosphoserine 11.8 t 2.0 10.8 +- 0.8 NS

Tatnine 57.1 t 8.4 54.2 k 2.9 NS
Aspartate 12.1 ? 5.5 8.9 2 3.0 NS
Threonine 79.0 2 5.8 125.4 k 3.3 <O.OOOl
Serine 91.3 -c 7.4 113.4 2 2.9 co.05
Asparagine 36.5 k 3.7 47.2 ? 3.0 co.05
Glutamate 61.8 2 7.7 70.1 ? 10.8 NS
Glutamine 506.9 2 20.6 616.9 2 12.2 <O.OOl I I I
Proline 119.8 2 17.8 169.5 ? 17.1 NS 400 500 600
Glycine 191.8 2 12.4 241.1 _’ 13.9 co.05
Plasma Glutamine (nmol/ml)
Alanine 312.9 2 33.5 416.7 2 23.4 co.05
Citrulline 21.4 2 3.1 37.5 k 2.0 <O.OOl
Valine 181.1 2 12.2 261.0 k 13.0 <O.OOl FIG. 1. Correlation between plasma glutamine (Gin) level and the Gln
Cysteine 32.9 -c 6.8 17.4 2 3.7 NS lOOO/Gln 500 ratio of the number of viable bacteria in the patients (n = 9;
Methionine 19.6 2 2.8 26.0 2 0.9 NS r = 0.69; P = 0.04). Gin lOOO/Gln 500 ratio of the number of viable
Isoleucine 47.3 2 5.1 83.7 2 4.3 <0.0001 bacteria equals the number of viable E. coli at an in vitro Gln concentration
111.9 2 8.3 152.2 k 7.3 co.01 of 1000 nmol/mL divided by the number of viable E. coli at an in vitro Gln
Leucine
concentration of 500 nmol/mL.
Tyrosine 59.5 5 6.6 70.5 _’ 7.0 NS
Phenylalanine 77.5 2 9.7 64.6 k 2.7 NS
Ornithine 45.4 2 3.2 80.0 k 7.2 <O.OOl
Lysine 129.6 5 8.7 188.3 ?z 6.6 <O.OOOl at any of the Gln concentrations tested. The number of viable
Hi&dine 53.3 2 4.5 77.3 2 3.1 <O.OOl bacteria showed no significant correlation with either operative
Arginine 57.2 k 13.1 88.3 ? 7.5 NS time or intraoperative blood loss.
TNF-a, IL-I/3 IL-8, and Granulocyte Elastase Levels in Cell
Values are mean + SEM. NS, not significant.
Culture Supenatants
TNF-a, IL-l& IL-8, and granulocyte elastase levels in the cell
culture supematants did not differ significantly, in patients or
plasma CRP values in the patient (POD 1) and control groups
were 12.2 and 0.1 mg/dL, respectively (P < 0.0001). In the controls, as the in vitro concentration was changed (Table IV). In
patients, a positive correlation was recognized between the plasma the patients, a negative correlation was demonstrated between cell
CRP level and the number of viable E. coli at all Gin concentra- culture supematant granulocyte elastase levels and the number of
tions studied (Gln 0 nmol/mL, r = 0.74; 100 nmol/mL, r = 0.72; viable bacteria (Gln 0 nmol/mL, r = -0.67; 100 nmollml,
r = -0.85; 500 nmoYmL, r = -0.73; 1000 nmol/mL,
500 nmol/mL, r = 0.72; 1000 nmol/mL, r = 0.68; and for all
concentrations, P < 0.05) (Fig. 3). The mean plasma cortisol
values in the patients and controls were 16.5 and 13.2 pg/dL,
respectively. No correlations were recognized in either group
between the plasma cortisol level and the number of viable E. cob

TABLE III.

BACTERICIDAL FUNCTION OF NEUTROPHILS AT DIFFERENT

GLUTAMINE CONCENTRATIONS*

In vitro glutamine concentration (nmol/mL)

0 100 500 1000

I I
Patients (n = 9) 3.6 2 1.4 3.4 r 1.2 3.7 k 1.3 2.8 ” 0.9t
Controls (n = 8) 3.0 2 1.3 3.0 5 1.3 2.7 5 1.1
10000 15000 20000
3.0 2 1.1
Peripheral White Blood Cell Count (/mn?)
Values are mean t SEM.
* X 16 colony-forming units of E. coli per milliliter of cell culture FIG. 2. Correlation between peripheral white blood cell count on postop-
supematant. erative day one and the number of viable bacteria at an in vitro glutamine
7 P < 0.05versus in vitro glutamine 500 nmoVmL in the patients. concentration of 0 nmol/mL (n = 9; r = 0.71; P = 0.03).
GLUTAMINE-ENHANCED BACTERIAL KILLING BY NEUTROPHILS 867

0
6 Patients (n=9) 0
HI.74 0
1 p.o.02 /

4 0
15’00 2600 2ioo
Cell Culture Supernatant Granulocyte Elastase (t-g/ml)

I I I I I
FIG. 4. Correlation between cell culture supematant granulocyte elastase
5 15 20 levels and the number of viable bacteria in the patients at an in vitro
PIas: CRP( mg/dl ) glutamine concentration of 1000 nmol/mL (n = 9; I = -0.78; P = 0.01).

FIG. 3. Correlation between plasma C-reactive protein (CRP) level and the
number of viable bacteria in the patients at an in vitro glutamine concen-
tration of 0 nmoVmL (n = 9; r = 0.74; P = 0.02). CFU, colony-forming
units.
r = -0.78; and for all concentrations, P 5 0.05) (Fig. 4). In the
patients, there was no relationship between the plasma Gln con-
centration and the cell culture supematant level of TNF-(Y, IL-lp,
IL-8 or granulocyte elastase. We defined the Gln lOOO/Gln 500
ratio of the cell culture supernatant IL-8 level as the IL-8 level in
the supernatant at an in vitro Gln concentration of 1000 nmol/mL
TABLE IV.
CELL CULTURE SUPERNATANT TNF-a, IL-lp, IL-8, AND
GRANULOCYTE ELASTASE LEVELS
divided by the IL-8 level at an in vitro Gln concentration of 500
nmol/mL. A negative correlation was demonstrated between the
plasma Gln level and the Gln lOOO/Gln500 ratio of the cell culture
supematant IL-8 level (r = -0.73, P = 0.025) (Fig. 5).
DISCUSSION
In the present study, the in vitro bactericidal function of neu-
trophils from postoperative patients was significantly greater at
1000 nmol/mL than at 500 nmoYmL of Gln.
The bactericidal function of neutrophils after surgery has been
shown to be depressed as compared with the preoperative level
and to be lowest on POD 1.6.7Therefore, we examined the effect
of Gln on the in vitro E. co&killing activity of neutrophils on POD
1. The serum Gln level decreases after surgery,iz trauma,i3 and
bums.9 Our results confirmed this decreased serum Gln concen-
tration in surgical illness. Depressed serum Gln in critically ill

In vitro Gln Healthy

concentration Patients volunteers
Pawsi (n=9)
(nmol/mL) (n = 9) (n = 8)
p=o:o25
TNF-(Y (pg/mL) 0 158? 53 186 2 45
100 132 f 33 225 5 49
500 169 2 57 223 t 45
1000 141 + 41 253 ? 55
IL-l p (pg/mL) 0 405 2 107 292 2 65
100 405 % 99 297 + 74
500 397 ? 119 273 2 59
1000 366 5 108 246 t 56
IL-8 (pg/mL) 0 940 2 274 1163 f 251
100 979 + 234 1041 2 228
500 937 -t 233 1089 2 241 t I I

1000 792 2 202 1048 t 230 400 500 600

Granulocyte elastase 0 1743 I 192 1827 -c 124 Plasma Glutamine (nmol/ml)
(ng/mL)
100 1749 2 135 1638 f 105
500 1762 2 132 1721 ? 124 FIG. 5. Correlation between plasma glutamine (Gln) level and the Gln
lOOO/Gln 500 ratio of the cell culture supematant interleukin-8 (IL-8) level
1000 1867 + 142 1625 ? 121
in the patients (n = 9; r = -0.73; P = 0.025). Gln lOOO/Gln 500 ratio of
IL-8 level equals IL-8 level at an in vitro Gln concentration of 1000
Values are mean ? SEM. nmoYmL divided by IL-8 level at an in vitro Gln concentration of 500
Gln, glutamine; IL, interleukin; TNF, tumor necrosis factor. nmol/mL.
868 GLUTAMINE-ENHANCED
patients might be partly explained by the increased consumption
of Gln by lymphocytes,14 macrophages,i5 and intestinal epithelial
cellsi Gln depletion results in immunosuppression.il Thus, it
appears that Gln supplementation in critical illness enhances the
function of lymphocytes and macrophages. Gln has, in fact, been
shown to augment the in vitro activity of lymphocytes and mac-
rophages.9
A recent study revealed that Gln augmented the in vitro bac-
tericidal activity of neutrophils from bum patients. lo We demon-
strated herein that the in vitro bactericidal function of neutrophils
from postoperative patients was significantly greater, i.e., by 26%,
at 1000 nmol/mL than at 500 nmoVmL of Gin. Thus, Gln clearly
enhances the bactericidal function of neutrophils.
The large SEM of the bacterial number shown in Table III may
be attributable to the sample measurements having been done on
separate days. To reduce the variability due to differences among
individuals (between-subject), we applied a test with a one-way,
repeated-measures analysis of variance model followed by a con-
trast test. This statistical method allowed intra-individual variation
(within-subject) to be determined. We demonstrated that the num-
ber of viable E. coli decreased significantly as the in vitro Gln
concentration was increased from 500 to 1000 nmol/mL.
Following culture with neutrophils from patients, the number
of viable E. coli decreased significantly as the in vitro Gln con-
centration was increased from 500 to 1000 nmol/mL, but not from
0 to 1000 nmol/mL. Therefore, we chose the Gln lOOO/Gln 500
ratio to interpret our results. The plasma Gln level of the postop-
erative patients was approximately 500 nmol/mL. On the other
hand, the mean plasma Gln concentration of the patients receiving
parenteral Gln supplementation (163 mmol * kg-’ - h-t) was
approximately 950 nmol/mL.i7 Thus, we simulated in vivo Gln
supplementation by using the Gln lOOO/Gln 500 ratio of the
number of viable bacteria in this in vitro study. We demonstrated
a positive correlation between the plasma Gln level and the Gln
lOOO/Gln 500 ratio of the number of viable bacteria in postoper-
ative patients. This finding indicated that as plasma Gln fell,
neutrophil E. co&killing activity was enhanced in in vitro tests
when the Gin concentration was increased from 500 to 1000
nmol/mL.
In contrast, supplemental Gln had no significant effect on the
bactericidal function of neutrophils from the controls. The mean
age of our controls was lower than that of the patients. As age
affects neutrophil function,” it would be worthwhile to study the
effect of Gln on the bactericidal function of neutrophils from
elderly, healthy volunteers.
Opsonized E. coli are ingested and killed by neutrophils within
phagocytic vacuoles, where they are exposed to an array of reac-
tive oxygen metabolites and toxic lysosomal components.ig Thus,
the bactericidal-enhancing effect of Gln in this study may have
occurred at the level of phagocytosis, the formation of reactive
oxygen metabolites, or degranulation. Ogle et al.i” showed that
Gln had no effect on phagocytosis by neutrophils from bum
patients. Therefore, Gln may enhance neutrophil bactericidal func-
tion by other mechanism(s) such as increasing the formation of
reactive oxygen metabolites and/or promoting the degranulation
of neutrophils.
Neutrophils produce TNF-a, IL-l, and IL-8.2o Neutrophils
release approximately 1 ng/mL IL-8 in response to stimulation
with 10 nM formyl-methionyl-leucyl-phenylalanine for 2 h.21
Moreover, neutrophils stimulated with 5 mg/mL lipopolysaccha-
ride for 2 h have been shown to secrete 36 pg/mL TNF.22 There-
fore, neutrophils can produce measurable amounts of cytokines
with a 2-h incubation. However, in our study, Gln supplementaion
produced no appreciable changes in TNF-(Y, IL-l/3, or IL-8 levels
in cell culture supematants after a 2-h incubation. Further inves-
BACTERIAL KILLING BY NEUTROPHILS
tigation is needed to determine cytokine productions resulting
from an incubation period of more than 2 h.
Neutrophil activation by TNF-(Y, IL-l, or IL-8 in vitro in-
creases the production of reactive oxygen metabolites.23 We did
not determine reactive oxygen metabolite levels in cell culture
supematants. Thus, the possibility remains that Gln supplementa-
tion increased the formation of reactive oxygen metabolites by
neutrophils. However, the enhancement of oxidative burst produc-
tion, if any, may not be directly mediated by TNF-cr, IL-lp, or
IL-8 based on our results.
We demonstrated a negative correlation, in the postoperative
patients, between cell culture supematant granulocyte elastase
levels and the number of viable bacteria. This finding indicated
that as the cell culture supematant granulocyte elastase level rose,
there was an enhancement of E. co&killing activity by neutrophils
in in vitro tests. However, these granulocyte elastase levels did not
differ significantly, in the patients, as the in vitro Gln concentra-
tion was changed. This observation suggests that the nonoxidative
bacteria-killing capacity of neutrophils may not mediate the bac-
tericidal-enhancing effect of Gln. Thus, the mechanisms underly-
ing the enhancing effect of Gln on neutrophil bactericidal capacity
are apparently not based on the increased production of the cyto-
kines studied or granulocyte elastase.
The energy source neutrophils use to produce reactive oxygen
metabolites is believed to be glucose.24 There have been no studies
investigating whether Gln is utilized by neutrophils. However, in
a recent review, Vlessis and associatesz5 described new concepts
in the pathophysiology of oxygen metabolism during sepsis. The
progression of sepsis limits the aerobic oxidation of glucose. In
contrast, aerobic oxidation of glutamate (and also Gln following
transamination) is not inhibited in sepsis. Therefore, it appears that
Gln is being utilized for aerobic metabolism in preference to
glycolytic substrates. It was shown that glutaminase, the key
enzyme in the utilization of Gln as an energy source, is located
within the mitochondria of lymphocytes and macrophages.26
There are no reports, to the best of our knowledge, that neutrophils
possess glutaminase. However, in light of the present results, it is
tempting to speculate that Gln enhances the in vitro bactericidal
function of neutrophils by serving as a neutrophil substrate.
We showed that as the plasma Gln level fell, there was an
elevation of the cell culture supematant IL-8 level in in vitro tests
when the Gln concentration was increased from 500 to 1000
nmol/mL. Since IL-8 is a neutrophil chemoattractant, the in-
creased IL-8 level at an infected site results in more neutrophils
migrating to the lesion in vivo. This migration of neutrophils is
advantageous for host defense.
In the patients who had undergone various operations, there
was a positive correlation between the peripheral white blood cell
count on POD 1, and the number of viable bacteria in neutrophil-
killing assays at all Gln concentrations. In addition, we observed
in the patients positive correlations between the plasma CRP level
and the number of viable bacteria at all Gln concentrations stud-
ied. These findings indicate that as the surgical stress increased,
there was a decrease in the E. coli-killing capacity of neutrophils
in in vitro tests. Thus, it is important to prevent this reduction in
bactericidal activity under severe surgical stress. Ziegler et ali7
reported that bone marrow transplantation patients receiving par-
enteral Gln had a lower incidence of infection and had a shorter
hospital stay than patients receiving Gln-free parenteral nutrition.
Our results support the beneficial effects of Gln on host defense
against infection. Further trials are needed to clarify the immune-
enhancing effect of Gln supplementation in postoperative patients.
In conclusion, Gln supplementation enhanced the in vitro bac-
tericidal function of neutrophils from postoperative patients.
GLUTAMINE-ENHANCED BACTERIAL KILLING BY NEUTROPHILS 869

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(For an additional perspective, see Editorial Comment on page 914.)