Department of Physiology and Biophysics and Department of Histology and Embryology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The capacity of rat neutrophils to utilize glutamine was
investigated by 1) determination of
oxygen consumption in the presence of glucose or glutamine,
2) measurement of maximal activity
of phosphate-dependent glutaminase,
3) Northern blot, Western blot, and
immunocytochemical detection of glutaminase, and
4) measurement of glutamine
utilization and also production of ammonia, glutamate, aspartate,
alanine, and lactate and decarboxylation of
[U-14C]glutamine in
cells incubated for 1 h. The rate of respiration by isolated
neutrophils in the absence of added substrate was 5.0 nmol · min1 · 107
cells
1. Maximal activity of
phosphate-dependent glutaminase was 56 nmol · min
1 · mg
protein
1 in freshly
obtained neutrophils; the Michaelis-Menten constant was 3.5 mM for
glutamine. This enzyme activity was inhibited by 2 mM glutamate, 2 mM
oxoglutarate, and 2 mM NH4Cl. The
presence of glutaminase protein (65 kDa) was confirmed by Western blot and immunocytochemical detection and the presence of the mRNA (6.0 kb)
by Northern blot analysis. Glutamine was utilized by neutrophils
incubated for 1 h at a rate of 12.8 nmol · min
1 · mg
protein
1 when the amino
acid was added to the medium at 2 mM, which is three to four times
higher than the physiological concentration. In the presence of 0.5 mM
glutamine, the amino acid was utilized at a rate of 2.9 nmol · min
1 · mg
protein
1. The addition of
0.5 mM glutamate to the incubation medium caused a marked reduction (by
70%) in glutamine utilization by neutrophils. Glucose was utilized at
7.7 nmol · min
1 · mg
protein
1 when cells were
incubated in 5 mM glucose. The conversion of [U-14C]glutamine to
14CO2
was very low: <1% was totally oxidized. The formation of ammonia was
~27% of glutamine utilization, and the conversion of glutamine to
glutamate, aspartate, alanine, and lactate accounted for ~84.6% of
the total amino acid utilized by neutrophils. In this study, evidence
is presented that, in addition to lymphocytes and macrophages, neutrophils also utilize glutamine.
oxygen consumption; glucose; oxidative metabolism
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
SEVERAL STUDIES HAVE SHOWN that the rate of glutamine utilization by lymphocytes and macrophages is similar to or greater than the rate of glucose utilization. Neither glutamine nor glucose is fully oxidized by these cells. Glucose is mainly converted to lactate and most of the glutamine to glutamate, lactate, and aspartate (8, 9). A high rate of glutamine utilization, but only partial oxidation, is also characteristic of other cells (e.g., enterocytes, thymocytes, fibroblasts, and tumor cells) (22).
Glutamine is an important precursor of nucleotides for RNA and DNA synthesis, and it has been recognized that this amino acid plays a key role in cell proliferation. Macrophages are terminally differentiated cells, but they also utilize glutamine at high rates (24). In these cells, glutamine has been assumed to take part in cytokine production (26). However, the significance of glutamine metabolism in macrophages remains to be established.
Neutrophils act as first-line-of-defense cells in the plasma and undergo phagocytosis alone or in cooperation with antigen-specific defenses. Neutrophils contain a characteristic lobulated chromatin-dense nucleus, which has given rise to the term polymorphonuclear leukocyte, and are 9-12 µm in diameter. At any time, >90% of the neutrophil population is located as newly differentiated cells within the bone marrow. The remaining neutrophils are distributed between the circulation and the vascular endothelium, where they are attached to marginated pools or located within specific tissues.
Along with the onset of phagocytosis of bacteria or tissue fragments by neutrophils, a number of different cellular processes, including motility, respiratory burst, and secretion of cytoplasmic (proteolytic) enzymes and immunomodulatory compounds, are initiated. The combination of these processes assists in the killing and digestion of the engulfed bacteria and, if prolonged, the development of a local inflammation. Increase of respiratory burst involves a sudden stimulus-induced increase in nonmitochondrial oxidative metabolism, which results in the production of the superoxide anion and associated reactive oxygen species (27). The processes of endocytosis, secretion of active compounds, and generation of reactive oxygen species have been assumed to be mostly dependent on glucose metabolism in neutrophils (28). The capacity of rat neutrophils to utilize glutamine was investigated by determination of 1) oxygen consumption in the presence of glucose or glutamine, 2) maximal activity and the Michaelis-Menten constant (Km) of phosphate-dependent glutaminase, 3) Northern blot, immunocytochemical detection, and Western blot of this enzyme, 4) inhibition of this enzyme activity by glutamate, oxoglutarate, and NH4Cl, and 5) glutamine utilization and production of ammonia, glutamate, aspartate, alanine, and lactate and decarboxylation of [U-14C]glutamine in cells incubated for 1 h.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Male Wistar rats weighing 180 g (~2 mo of age) were obtained from the Biomedical Institute (University of São Paulo, São Paulo, Brazil). The rats were maintained at 23°C under a 12:12-h light-dark cycle.
Chemicals and enzymes. All chemicals and enzymes were obtained from Sigma Chemical (St. Louis, MO). [U-14C]glucose (295 mCi/mmol) and [U-14C]glutamine (229 mCi/mmol) were purchased from Amersham International (Buckinghamshire, UK).
Peritoneal neutrophil preparation. The cells were obtained from 130- to 150-g Wistar rats killed by decapitation without anesthesia. Neutrophils were obtained by intraperitoneal lavage with 40 ml of sterile phosphate-buffered saline (PBS) 4 h after the intraperitoneal injection of 20 ml of 1% sterile oyster glycogen solution (type II, Sigma Chemical) in PBS. The cells were centrifuged (850 g for 8 min) three times in PBS. The number of viable cells, >95% neutrophils, was always counted in a Neubauer chamber using optical microscopy and 1% trypan blue solution.
Oxygen consumption. Oxygen consumption was monitored in an oxygen monitor (model 5300, Yellow Springs Instruments) for 10 min. In these experiments, 5 × 107 cells/ml in PBS at 37°C in a final volume of 3.0 ml were always used. Oxygen consumption was measured in freshly obtained cells and neutrophils incubated for 18 h in PBS containing 2% defatted bovine serum albumin (BSA). The viability of the neutrophils incubated under these conditions was always >95%, as indicated by exclusion of trypan blue.
Incubation procedure. Neutrophils were incubated (1.0 × 107 cells/flask) at 37°C in 1 ml of Krebs-Ringer medium with 2% (wt/vol) defatted BSA in the presence of glucose (5 mM) or glutamine (2 mM). After 1 h of incubation, the cells were disrupted by addition of 0.2 ml of 25% (wt/vol) perchloric acid. Protein was removed by centrifugation, and the supernatant fluid was neutralized with 40% KOH solution and a tris(hydroxymethyl)aminomethane (Tris) · KOH (0.5-2.0 M) solution for the measurement of metabolites.
[U-14C]glucose and [U-14C]glutamine decarboxylation. The 14CO2 produced from [U-14C]glucose or [U-14C]glutamine was collected as previously described (9). Neutrophils were incubated for 1 h in the presence of [U-14C]glucose or [U-14C]glutamine in a sheltered Erlenmeyer flask (25 ml) with one compartment for cell incubation and another for CO2 collection. Then the cells were disrupted using 200 µl of 25% perchloric acid solution. The labeled CO2 was collected over 1 h in 1:1 (vol/vol) phenylethylamine-methanol, and the radioactivity was counted in a liquid scintillation counter (Beckman-LS 5000 TD, Beckman Instruments, Fullerton, CA).
Metabolite measurements. Neutralized samples of the incubation medium were used for measurements of glucose (3), glutamine (33), ammonia (33), glutamate (5), aspartate (4), alanine (32), and lactate (14). The content of glutamine and glutamate in the cells freshly obtained from the rats was also determined using the same methods. The production of NADH or NADPH was monitored at 340 nm using a recording spectrophotometer (Gilford Response).
Glutaminase assay. For the measurement of phosphate-dependent glutaminase activity in neutrophils, the cells were obtained by centrifugation at 4°C and then homogenized in an extraction medium containing 150 mM potassium phosphate, 1 mM EDTA, and 50 mM Tris · HCl at pH 8.6. Phosphate-dependent glutaminase (EC 3.5.1.2) was assayed as described by Curthoys and Lowry (11). The assay medium consisted of 50 mM phosphate buffer, 0.2 mM EDTA, 50 mM Tris · HCl, 20 mM glutamine, and 0.05% (vol/vol) Triton X-100, to which 100 µl of homogenate were added. The total volume was 1.0 ml at pH 8.6. Assay media, in duplicate, were incubated at 37°C. The reaction was initiated by the addition of freshly prepared glutamine, promoting a 10-min linear reaction time course. The reaction was stopped by addition of 0.2 ml of 25% (wt/vol) perchloric acid solution and then neutralized. The amount of glutamate was determined as described by Bernt and Bergmeyer (5) at 340 nm in a Gilford Response spectrophotometer. The Km of glutaminase was determined using 0.1, 0.5, 1.0, and 2.0 mM glutamine. The inhibiting effect of 2.0 mM oxoglutarate, 2.0 mM NH4Cl, and 2.0 mM succinate on neutrophil glutaminase activity was also tested.
Assays of hexokinase, citrate synthase, glucose-6-phosphate
dehydrogenase, and lactate dehydrogenase.
The activities of hexokinase (EC 2.7.1.1), citrate synthase (EC
4.1.3.7), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were
determined as previously described (10, 24). The extraction medium for
hexokinase contained 50 mM Tris · HCl, 1 mM EDTA, 30 mM MgCl2, and 20 mM
-mercaptoethanol at pH 7.4. The extraction medium for citrate
synthase and glucose-6-phosphate dehydrogenase contained 50 mM
Tris · HCl and 1 mM EDTA; the final pH values were
7.4 and 8.0, respectively. The extraction medium for lactate dehydrogenase consisted of 10 mM phosphate buffer at pH 7.4. The activity of lactate dehydrogenase was determined as previously described (10). For all enzyme assays, 0.05% (vol/vol) Triton X-100
was added to the assay system to complete the extraction of the enzyme.
Western blot analysis. Immunoblot analysis was carried out essentially as described by Towbin et al. (31). The samples containing 50 µg of neutrophil protein and 10 µg of brain protein were subjected to electrophoresis on a preparative 10% (wt/vol) polyacrylamide slab gel in the presence of sodium dodecyl sulfate (18) and then transferred to nitrocellulose. After blocking with 20 mM Tris · HCl-500 mM NaCl, pH 7.4, containing 5% defatted milk (wt/vol), the nitrocellulose was incubated with affinity-purified antiglutaminase antibodies (gift from Dr. Norman Curthoys, Dept. of Biochemistry and Molecular Biology, Colorado State University, Ft. Collins, CO) (30). It was then incubated with a goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugate and revealed by using the enhanced chemiluminescence detection system (Amersham Life Science).
Immunocytochemical analysis. Neutrophils were spread on clean, grease-free slide fixative solution (60% methanol-30% chloroform-10% acetic acid) at 4°C for 5 min. Slides were washed twice for 2 min in PBS and treated for 15 min with 0.3% H2O2 in PBS to block endogenous peroxidases. The slides were washed with PBS and incubated with goat serum in BSA-PBS for 30 min. Then the preparation was incubated overnight with affinity-purified antiglutaminase antibodies (1:100). The primary antibody was omitted in the control. Slides were washed three times for 10 min in PBS and incubated with goat anti-rabbit IgG-peroxidase conjugate (1:2,000) for 2 h at room temperature. After they were washed in PBS, the slides were incubated with diaminobenzidine-H2O2 (Sigma Chemical) for 5 min at room temperature. Slides were washed in PBS for 10 min before they were stained with Mayer's hematoxylin for a few seconds.
Northern analysis.
Total RNA was isolated from rat neutrophils using Trizol solution
(GIBCO, Gaithersburg, MD). Aliquots containing 30 µg of the isolated
RNAs were fractionated by electrophoresis on a 1% agarose gel
containing 3% formaldehyde with the DNA probes that were labeled (2 × 106
counts · min1 · ml
1)
by random priming (15). The hybridized filters were exposed to
Hyperfilm MP (Amersham Life Science). The results of glutaminase mRNA
from neutrophils were compared with results from kidney, which are well
established (17).
cDNA glutaminase probe. cDNA of phosphate-dependent glutaminase (2) was kindly provided by Dr. Norman P. Curthoys.
Protein determination. Protein content of neutrophils was measured by the method of Lowry et al. (19) using BSA as standard. The total protein content of neutrophils obtained under the conditions of this study was 0.64 mg/107 cells.
Expression of results and statistical analysis. The enzyme activities are expressed as nanomoles per minute per milligram of protein. The consumption of glucose and glutamine, the production of ammonia, glutamate, aspartate, alanine, and lactate from glutamine and lactate from glucose, and the decarboxylation of [U-14C]glucose and [U-14C]glutamine of neutrophils incubated for 1 h are expressed as nanomoles per hour per milligram of protein. Analysis of the significance of differences using a standard Student's t-test for P < 0.05 is given in Table 1.
|
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rate of respiration by isolated neutrophils in the absence of added
substrate was 5.0 nmol · min1 · 107
cells
1, which was linear
with respect to time for 1 h (results not shown). This rate is higher
than that reported for lymphocytes and macrophages (21, 22). Incubation
of neutrophils for 18 h in PBS did not modify the rate of oxygen
consumption from endogenous substrate (Table 1). This finding may
indicate that the intracellular concentration of metabolites in rat
neutrophils is very high. Indeed, the intracellular concentrations of
glutamine and glutamate in freshly obtained neutrophils were 12.9 and
17.5 mM, respectively (data not shown). The addition of 5 mM glucose to
the incubation medium slightly raised oxygen consumption in freshly
obtained cells but provoked a marked increase in neutrophils cultured
for 18 h (64%). Similarly, the addition of glutamine also raised
oxygen consumption in neutrophils cultured for 18 h (61%).
Maximal activity of phosphate-dependent glutaminase in neutrophils was
56 nmol · min1 · mg
protein
1 in freshly
obtained cells (Table 2). This glutaminase activity was
higher than that reported for lymphocytes and macrophages (6, 22). The
activity of glutaminase in neutrophils was comparable to that of
hexokinase, citrate synthase, and glucose-6-phosphate dehydrogenase and
much lower than that of lactate dehydrogenase. The differences may be
partially a consequence of the temperature of the enzyme assays:
37°C for glutaminase and 25°C for the others.
|
The presence of glutaminase protein in neutrophils was confirmed by Western blot (Fig. 1) and immunocytochemical detection (Fig. 2). Rat neutrophils exhibited a positive immune reaction to a 65-kDa protein similar to that found in rat brain (16). The presence of the glutaminase mRNA in neutrophils was also evaluated by Northern blot (Fig. 3). Similar to results reported for kidney (29), neutrophils presented a significant amount of mRNA (6.0 kb) for glutaminase. The Km of neutrophil glutaminase for glutamine was determined to be 3.5 mM (data not shown). Addition of glutamate to the glutaminase assay inhibited this enzyme activity: 28% at 0.2 mM, 34% at 0.5 mM, and 70% at 2.0 mM. In addition to glutamate, other metabolites (at 2 mM) also inhibited this enzyme activity: 50% for 2-oxoglutarate, 32% for NH4Cl, and 13% for succinate, as reported by Ardawi and Newsholme (1) for lymphocytes. These findings provide further evidence that the phosphate-dependent glutaminase found in rat neutrophils is a kidney-type glutaminase (12).
|
|
|
|
Glutamine was utilized by 1-h-incubated neutrophils at a rate of 12.8 nmol · min1 · mg
protein
1 when added to the
medium at 2 mM, which is three to four times higher than the
physiological concentration (Table 3). In the presence
of 0.5 mM glutamine, the amino acid was utilized at a rate of 2.9 ± 0.1 (SE)
nmol · min
1 · mg
protein
1
(n = 5 incubations). The addition of
0.5 mM glutamate to the incubation medium reduced glutamine utilization
by neutrophils by ~70%: 0.9 ± 0.4 (SE)
nmol · min
1 · mg
protein
1
(n = 5 incubations). Therefore, the
discrepancy between the capacity to hydrolyze glutamine and glutamine
utilized was possibly due to the suppression caused by the
intracellular concentration of glutamate. Also, these findings led us
to speculate that the inhibition of glutamine utilization by
extracellular glutamate may occur in neutrophils under certain
pathophysiological conditions, such as acquired immune deficiency
syndrome (13).
Glucose was utilized at 7.7 nmol · min1 · mg
protein
1 when cells were
incubated in 5 mM glucose. The conversion of glucose to lactate was
high (60% of the amount utilized), whereas
[U-14C]glucose
decarboxylation was very low. Similar results have been reported for
lymphocytes and macrophages (22). However, the conversion of
[U-14C]glutamine to
14CO2
was very low: <1% was totally oxidized. The formation of ammonia was
~27% of glutamine utilization in neutrophils. The conversion of
glutamine to glutamate, aspartate, alanine, and lactate accounted for
~84.6% of the total amino acid utilized by these cells.
Glutamine supplementation improves survival rates in septic mice and prevents sepsis-induced multiple system organ failure (20). A complex interrelationship exists among infection, endotoxin, cytokines, and interorgan glutamine metabolism. The utilization of this amino acid by immune cells has been assumed to play an important role in the function of the immune system (21, 23). In fact, glutamine is actively utilized by lymphocytes and macrophages (22). In this study, evidence is presented that neutrophils also utilize glutamine at significant rates. Ogle et al. (25) showed that glutamine enhances the bactericidal function of neutrophils from normal subjects and burn patients. Further studies are necessary to fully address the significance of glutamine metabolism for neutrophil function.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful for the technical assistance of G. de Souza, J. R. Mendonça, M. Carnelós Filho, C. M. R. Pellegrini, and D. M. H. Tenório and to Prof. E. A. Newsholme for constant interest and encouragement.
![]() |
FOOTNOTES |
---|
This research has been supported by Fundação de Amparo a Pesquisa do Estado de São Paolo and Conselho Nacional de Desenvocuimente Cientifico e Tecnologico.
Address for reprint requests: R. Curi, Dept. de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900, Butantan, São Paulo, SP, Brasil.
Received 30 August 1996; accepted in final form 2 May 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ardawi, M. S. M.,
and
E. A. Newsholme.
Intracellular localization and properties of phosphate-dependent glutaminase in rat mesenteric lymph nodes.
Biochem. J.
217:
289-296,
1984[Medline].
2.
Banner, C.,
J.-J. Hwang,
R. A. Shapiro,
R. J. Wenthold,
Y. Nakatani,
K. A. Lampel,
J. W. Thomas,
D. Huie,
and
N. P. Curthoys.
Isolation of a cDNA for rat brain glutaminase.
Mol. Brain Res.
3:
247-254,
1988.
3.
Barham, D.,
and
P. Trinder.
An improved colour reagent for the determination of blood glucose by the oxidase system.
Analyst
97:
142-145,
1972[Medline].
4.
Bergemeyer, H. U.,
E. Bernt,
H. Mollering,
and
E. R. Pfleiderer.
L-Aspartate and L-asparagine.
In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. London: Academic, 1974, p. 1696-1700.
5.
Bernt, E.,
and
H. U. Bergmeyer.
L-Glutamate UV assay with glutamate dehydrogenase and NAD.
In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. London: Academic, 1974, p. 1704-1708.
6.
Costa Rosa, L. F. B. P.,
R. Curi,
C. Murphy,
and
P. Newsholme.
The effect of adrenaline and phorbol myristate or bacterial lipopolysaccharide on stimulation of pathways of macrophage glucose, glutamine and O2 metabolism. Evidence for cyclic AMP-dependent protein kinase-mediated inhibition of glucose-6-phosphate dehydrogenase and activation of NADP+-dependent "malic" enzyme.
Biochem. J.
310:
709-714,
1995[Medline].
7.
Crabtree, B.,
A. R. Leech,
and
E. A. Newsholme.
Measurement of enzyme activities in crude extracts of tissues.
In: Techniques in Metabolic Research, edited by C. Pogsons. Amsterdam: Elsevier/North Holland, 1979, p. 1-37.
8.
Curi, R.,
and
E. A. Newsholme.
The effect of adenine nucleotides on the rate of glutamine utilization by incubated mitochondria isolated from rat mesenteric lymph nodes.
Mol. Cell. Biochem.
86:
71-76,
1989[Medline].
9.
Curi, R.,
P. Newsholme,
and
E. A. Newsholme.
Metabolism of pyruvate by isolated rat mesenteric lymphocytes, lymphocyte mitochondria and isolated mouse macrophages.
Biochem. J.
250:
383-388,
1988[Medline].
10.
Curi, R.,
P. Newsholme,
and
E. A. Newsholme.
Intracellular distribution of some enzymes of the glutamine utilisation pathway in rat lymphocytes.
Biochem. Biophys. Res. Commun.
138:
318-322,
1986[Medline].
11.
Curthoys, N. P.,
and
O. H. Lowry.
The distribution of glutaminase isoenzymes in the various structures of the nephron in normal, acidotic, and alkalotic rat kidney.
J. Biol. Chem.
248:
162-168,
1973
12.
Curthoys, N. P.,
and
M. Watford.
Regulation of glutaminase activity and glutamine metabolism.
Annu. Rev. Nutr.
15:
133-159,
1995[Medline].
13.
Droge, W.,
K. K. Murthy,
H. C. Stahl,
S. Hartung,
R. Plesker,
S. Rouse,
E. Peterhans,
R. Kinscherf,
T. Fischbach,
and
H. P. Eck.
Plasma amino acid dysregulation after lentiviral infection.
AIDS Res. Hum. Retroviruses
9:
807-809,
1993[Medline].
14.
Engel, P. C.,
and
J. B. Jones.
Causes and elimination of erratic blanks in enzymatic metabolite assays involving the use of NAD+ in alkaline hydrazine buffers: improved conditions for the assay of L-glutamate, L-lactate, and other metabolites.
Anal. Biochem.
88:
475-484,
1978[Medline].
15.
Feinberg, A. P.,
and
B. Vogelstein.
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem.
137:
266-267,
1984[Medline].
16.
Haser, W. G.,
R. A. Shapiro,
and
N. P. Curthoys.
Comparison of the phosphate-dependent glutaminase obtained from rat brain and kidney.
Biochem. J.
229:
399-408,
1985[Medline].
17.
Hwang, J. J.,
and
N. P. Curthoys.
Effect of acute alterations in acid-base balance on rat renal glutaminase and phosphoenolpyruvate carboxykinase gene expression.
J. Biol. Chem.
266:
9392-9396,
1991
18.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
19.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
20.
Neu, J.,
V. Shenoy,
and
R. Chakrabarti.
Glutamine nutrition and metabolism: where do we go from here?
FASEB J.
10:
829-837,
1996
21.
Newsholme, E. A.,
P. Newsholme,
and
R. Curi.
The role of the citric acid cycle in cells of the immune system and its importance in sepsis, trauma and burns.
Biochem. Soc. Symp.
54:
145-161,
1987[Medline].
22.
Newshome, E. A.,
P. Newsholme,
R. Curi,
B. Crabtree,
and
M. S. M. Ardawi.
Glutamine metabolism in different tissues: its physiological importance.
In: Perspectives in Clinical Nutrition, edited by J. M. Kinney,
and P. G. Borum. Baltimore, MD: Urban & Schwarzenberg, 1989, p. 71-98.
23.
Newsholme, P.,
L. F. B. P. Costa Rosa,
E. A. Newsholme,
and
R. Curi.
The importance of fuel metabolism to macrophage function.
Cell Biochem. Funct.
14:
1-10,
1996[Medline].
24.
Newsholme, P.,
R. Curi,
S. Gordon,
and
E. A. Newsholme.
Metabolism of glucose, glutamine, long chain fatty acids and ketone bodies by murine macrophages.
Biochem. J.
239:
121-125,
1986[Medline].
25.
Ogle, C. K.,
J. D. Ogle,
J. X. Mao,
J. Simon,
J. G. Noel,
B. G. Li,
and
J. W. Alexander.
Effect of glutamine on phagocytosis and bacterial killing by normal and pediatric burn patient neutrophils.
J. Parent. Enteral Nutr.
18:
128-133,
1994[Abstract].
26.
Parry-Billings, M.,
R. J. Baigrie,
P. M. Lamont,
P. J. Morris,
and
E. A. Newsholme.
Effects of major and minor surgery on plasma glutamine and cytokine levels.
Arch. Surg.
127:
1237-1240,
1992[Abstract].
27.
Rosen, G. M.,
S. Pou,
C. L. Ramos,
M. S. Cohen,
and
B. E. Britigan.
Free radicals and phagocytic cells.
FASEB J.
9:
200-209,
1995
28.
Selvaraj, R. J.,
and
A. J. Sbarra.
Relationship of glycolytic and oxidative metabolism to particle entry and destruction in phagocytosing cells.
Nature
17:
1272-1276,
1966.
29.
Shapiro, R. A.,
L. Farrell,
M. Srinivasan,
and
N. P. Curthoys.
Isolation, characterization, and in vitro expression of a cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase.
J. Biol. Chem.
266:
18792-18796,
1991
30.
Shapiro, R. A.,
W. G. Haser,
and
N. P. Curthoys.
Immunoblot analysis of glutaminase peptides in intact and solubilized mitochondria isolated from various rat tissues.
Biochem. J.
242:
743-747,
1987[Medline].
31.
Towbin, H.,
T. Staehelin,
and
J. Gordon.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:
4350-4354,
1979[Abstract].
32.
Williamson, D. H.
L-Alanine determination with alanine dehydrogenase.
In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. London: Academic, 1974, p. 1679-1682.
33.
Windmueller, H. G.,
and
A. E. Spaeth.
Uptake and metabolism of plasma glutamine by the small intestine.
J. Biol. Chem.
249:
5070-5079,
1974