Comparison of a Fluorimetric and a Calorimetric Method for the Determination of Hydrogen Peroxide Production by Rat Pleural Macrophages
CLAUDIA VON REIN
A fluorimetric oxide
production
lower
comparison on H202
HzOz
by isolated
compared.
by rat pleural phenol
was detected
that the fluorimetric
for the determination
cells were
assay, using homovanillic
production
to the calorimetric
production
method
by isolated
of the fluorimetric
nificantly
assay with
acid (HVA)
sena sig-
was measured
A negative
in the calorimetric
HVA is unsuitable
a higher
as reagent,
macrophages
red method.
per-
of hydrogen
Despite
influence
assay. These
results
for determining
in
of HVA suggest
H202 formation
cells. Hydrogen
Key Words: try;
ROLF HIRSCHELMANN
and a calorimetric
(HzOz)
sitivity
AND
peroxide
production
in vitro;
Macrophages;
Fluorime-
Colorimetry
INTRODUCTION Hydrogen
peroxide
the pathogenesis Biemond, 1988;
belongs
to the
reactive
oxygen
species
that are involved
of inflammatory and other pathological processes Kedzioara and Bartosz, 1988; Parks, 1989). Several
(Arthur, methods
in
1988; have
been described for the determination of H202 production by cells (Ebermann and Couperus, 1987; Hasui et al., 1987; Hool and Nieman, 1988; Hyslop and Sklar, 1984; Malehorn et al., 1986; Pick and Keisari, 1980; Ruth et al., 1983; Wymann et al., 1987). We wanted
to compare
the calorimetric
assay of Pick and Keisari
(1980) and a
fluorimetric method (Ruth et al., 1983). Both methods are simple as they are based on horseradish peroxidase (HRP)-catalysed oxidation of a hydrogen donor. In the calorimetric assay phenol red is the H-donor and in the fluorimetric method HVA is oxidized method
to a highly
is the higher
fluorescent
sensitivity
dimer.
Obviously
in comparison
the advantage
to the calorimetric
of the
latter
assay. In the first
method (Ruth et al., 1983), a linear relationship between the H202 concentration and fluorescence intensity was found in the range of 0.05 to 4.5 nmol/mL. With the method of Pick and Keisari (1980) a linearity was found between 1 and 60 nmol H202/ mL. However, in comparing the two methods we measured a significantly lower H202 production in phorbol myristate acetate (PMA) stimulated rat pleural macrophages using the fluorimetric method. Therefore, we compared the two methods
From Sektion Pharmazie, Martin-Luther Universitat, Postfach, Halle 4010, Germany. Address reprint requests to: Dr. R. Hirschelmann, Sketion Pharmazie, Martin-Luther Universittit, Postfach, Halle 4010, Germany. Received October 1, 1990; revised and accepted January 1, 1991.
61 lournal of
Pharmacological
0 1991 Elsevier
Science
Methods Publishing
26, 61-65 Co..
Inc.,
0160.5402/911$3.50
(19911 655 Avenue
of the Americas,
New
York,
NY 10010
62
C. Von Rein and R. Hirschelmann systematically method. MATERIALS
in an attempt
AND
to explain
the inconsistent
results with the fluorimetric
METHODS
Reagents HVA was obtained from Merck, Darmstadt (FRG); phenol red was from Polskie Odczynniki Chemiczne Gliwice (Poland). HRP was purchased from VEB LCA Feinchemie Sebnitz (GDR) and PMA was supplied by Sigma Chemical Company, St. Louis (USA). PMA was dissolved in dimethylsulfoxide to a concentration of 10 t.r.M and stored at -20°C in aliquots small enough for one experiment. Powdered isotonic buffer for cell culture (IBC) was obtained from Staatliches lnstitut fur Immunpraparate und Nahrmedien, Berlin (GDR) and was used as the suspending medium for cells.
Buffers and Reagent Solution HVA-HRP-solution (for the fluorimetric method): 9.1 mg HVA was dissolved in 25 mL IBC yielding a HVA concentration of 200 PM (see Table 1). An aliquot of 10 mL of this solution and 1 mL of a solution of 5 mg HRP in 10 mL IBC were made up to 50 mL with
IBC. This solution
was prepared
fresh.
Buffered phenol red solution (for the calorimetric assay): Shortly before use amounts of 0.1 g phenol red and 0.2 g HRP were added to a volume of 1000 mL of a buffer containing 0.14 M of sodium chloride, 5 mM K2HP04, 5 mM KH2P04 and 5.5 mM glucose.
Animals and Preparation
of Cells
We used pleural macrophages from healthy female rats (outbred strain Barby: Wistar), body wt 200 to 250 g. The rats were injected in the pleural cavity with 2 mL of isotonic roform,
saline.
opening
cells were
Cells were the pleural
washed
collected
after 24 hr by killing
the animals
cavity and by lavage with IO mL of isotonic
in IBC and the total number
of cells was determined.
TABLE 1 Change in Hz02 Production by Rat Pleural Macrophages with Increasing HVA Concentration HVA
CONCENTRATION
[)LMOL
(20
MIN
*
L-‘]
%
Hz02
PREINCUBATION)
N
0 (controls)
[NMOL
*
(I@
CELLS
*
30
INHIBITION
MIN)-‘1
5
3.15
c 0.62
100
5
1.63
rt 0.45’:
48
200
5
1.12
2 0.39”
64
300
5
0.79
2 0.28**
75
** Significantly
different
from
controls;
p < 0.01 (Student’s
t-test)
in chlo-
saline.
The
Assays of Hz O2 Production by Rat leukocytes
Calorimetry The cells were
resuspended
in 2 mL buffered
phenol
red solution
and stimulated
by adding PMA to a final concentration of 25 nM. After 30 min incubation at 37”C, the cells were centrifuged and the reaction was stopped by adding 100 PL of a 1 M NaOH solution to the cell free system. The amount of oxidized phenol red was measured at 610 nm using a spectrophotometer Spekol (VEB Carl Zeiss Jena). For measuring the influence of HVA on H202 generation by the macrophages, the resuspended cells were incubated with HVA (for time of preincubation Tables 1 and 2). After washing the cells to remove HVA the macrophages resuspended
in buffered
phenol
red and stimulated
with PMA as described
see were above.
Fluorimetry H202
production
mixed solution concentration),
was assayed fluorimetrically
of IBC ad HVA-HRP 30 min incubation
after resuspension
of the cells in a
solution (I :I), stimulation with 25 nM PMA (final at 37”C, centrifugation of the cells, and stopping
the reaction by addition of 0.5 mL NaOH-EDTA-buffer, pH 12, containing 0.1 M NaOH and 30 mM EDTA. Emission was measured at 420 nm following excitation at 312 nm using a Perkin-Elmer MPF-44 spectrofluorimeter (Perkin-Elmer, Ueberlingen).
Cell Viability The viability
was tested
by using the method
of methylene
blue exclusion.
The
cells were incubated for 5 min at 37X in a buffer to which five drops of 1% methylene blue were added. Thereafter a microscopic determination of viability was performed. Dead cells cannot exclude the methylene blue.
Statistical Calculations All values are presented as mean + SD. Student’s t-test was used for significance calculation. Statistical significance was accepted at the 0.01 level of probability.
TABLE 2 Change in HzOz Production by Rat Pleural Macrophages with Increasing Time of Preincubation with HVA TIME [MINI N
[NMOL
*
(I@
CELLS
*
30
MIN)~‘]
INHIBITION
5
2.29 k 0.37
5
5
0.78 k 0.22**
66
10
5
0.63 k O.l**
72
20
5
0.78 " 0.16**
66
30
5
0.82 k 0.41**
64
0 (controls)
** Significantly
%
Hz02
(PREINCUBATIONWITH 200 JLMOL * L-' HVA)
different
from
controls;
p < 0.01
(Student’s
t-test)
63
64
C. Von Rein and R. Hirschelmann TABLE 3 HzOz Production by Rat Pleural Macrophages after Stimulation with 25 nM PMA (final concentration) HZOZ
RESULTS
AND
Despite
METHOD
N
calorimetric fluorimetric
10 6
* (I@’
CELLS
* 30
MIN)-‘I
3.99 k 1.01 0.77 k 0.28
DISCUSSION
the higher
nificantly lower assay (Table 3). Therefore
[NMOL
sensitivity
results
of the fluorimetric
for H,Oz
we determined
formation
the influence
HVA
than with
method
we obtained
the calorimetric
of HVA on the amount
The maximum
of inhibition
H202
production
tabolite
of the catecholamines.
is reached
sigred
of H202 generated
using the calorimetric method and we found a significant decrease mation. This impairment was dose-(Table 1) as well as time-dependent impairs
phenol
of H202 for(Table 2).
after 5 min (Table 2). Thus, HVA apparently
by rat pleural
macrophages.
Its concentration
HVA
is a physiological
me-
in rat plasma is in the range of 60-
70 nM (Seegal et al., 1986). In our experiments the HVA concentration of 200-300 FM (Tables 1 and 2) greatly exceeds physiological concentration. As there are no HVA receptors at the cell membrane (Watson and Abbot, 1990), a non-specific impairment of the cells by the high HVA concentrations is very likely. There may be an inhibition of the superoxide-generating membrane-bound NADPH oxidase, as H202 is formed mainly via dismutation of superoxide anion radicals lison, 1979), or even an impairment of a hitherto not characterized
(Segal and Alsystem for a
divalent oxygen reduction. Therefore, the fluorimetric assay described by Ruth et al. (1983) obviously has the disadvantage of HVA cytotoxicity against the cells, although we could not find a decreased viability of the cells after incubation with HVA. However, the time planation of cell damage
of incubation was rather short (maximal by HVA could be the rise in superoxide
centration after autoxidation sition metals would cause
of HVA in the buffer solution. generation of cell-damaging
Catalytic hydroxyl
30 min). An exand H202 contraces of tranradicals from
superoxide and H202 (Buettner, 1988; Halliwell, 1990). Thus the HVA method will produce inconsistent results unless transition metals are excluded, e.g., by chelation.
In any case, this method
should
be used with caution.
REFERENCES Arthur MJP (1988) Reactive oxygen intermediates and liver injury. / Hepato/6:125-131. Biemond P (1988) Superoxide dependent iron release from ferritin in inflammatory diseases. Free Radical
Biol Med
4:185-198.
Buettner GR (1988) In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals. / Biochem Biophys
Methods
16:27-40.
Ebermann R, Couperus A (1987) A nonenzymatic
Assays of Hz O2 Production by Rat leukocytes method for determination of hydrogen peroxide and organic peroxides. Anal Biochem 165:414419. Halliwell B (1990) How to characterize a biological antioxidant. Free Rad Res Commun 9:1-32. Hasui M, Hirabayashi Y, Kobayashi Y (1987) Simultaneous measurement by flow cytometry of phagocytosis and hydrogen peroxide production of neutrophils in whole blood. / lmmun Meth 117:53-58. Hool K, Nieman TA (1988) Immobilized luminol chemiluminescent reagent system for hydrogen peroxide determinations in flowing streams. Anal Chem 60:834-837. Hyslop PA, Sklar LA (1984) A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorometric assay of cellular activation processes. Anal Biochem 141: 280-286. Kedzioara j, Bartosz G (1988) Down’s syndrome: a pathology involving the lack of balance of reactive oxygen species. Free Radical Biol Med4:317330. Malehorn CL, Riehl TE, Hinze WL (1986) Improved determination of hydrogen peroxide or Iucigenin by measurement of lucigenin chemilumi-
nescence in 111:941-947.
organised
assemblies.
Analyst
Parks DA (1989) Oxygen radicals: mediators of gastrointestinal pathophysiology. CUT 30:293-298. Pick E, Keisari Y (1980) A simple calorimetric method for the measurement of hydrogen peroxide produced by cells in culture. / lmmunol Meth 38:161-170. Ruth W, Cooper PH, Baggiolini M (1983) Assay of H202 production by macrophages and neutrophils with homovanillic acid and horse radish peroxidase. / lmmunol Meth 63:347-357. Seegal RS, Brosch KO, Bush B (1986) High performance liquid chromatography of biogenic amines and metabolites in brain, cerebrospinal fluid, urine and plasma. / Chromat 377:131-144. Segal AW, Allison AC (1979) Oxygen consumption by stimulated human neutrophils. In: Oxygen free Radicals and Tissue Damage. Ciba Foundation Symposium 65 (new series); AmsterdamOxford-New York, Excerpta Medica, pp. 205219. Watson S, Abbot A (1990) Receptor Nomenclature Supplement. Tif?S 11. Wymann MP, von Tscharner V, Deranleau DA, Baggiolini M (1987) Chemiluminescence detection of H202 produced by human neutrophils during the respiratory burst. Anal Biochem 165:371-378.
65
CLAUDIA VON REIN
A fluorimetric oxide
production
lower
comparison on H202
HzOz
by isolated
compared.
by rat pleural phenol
was detected
that the fluorimetric
for the determination
cells were
assay, using homovanillic
production
to the calorimetric
production
method
by isolated
of the fluorimetric
nificantly
assay with
acid (HVA)
sena sig-
was measured
A negative
in the calorimetric
HVA is unsuitable
a higher
as reagent,
macrophages
red method.
per-
of hydrogen
Despite
influence
assay. These
results
for determining
in
of HVA suggest
H202 formation
cells. Hydrogen
Key Words: try;
ROLF HIRSCHELMANN
and a calorimetric
(HzOz)
sitivity
AND
peroxide
production
in vitro;
Macrophages;
Fluorime-
Colorimetry
INTRODUCTION Hydrogen
peroxide
the pathogenesis Biemond, 1988;
belongs
to the
reactive
oxygen
species
that are involved
of inflammatory and other pathological processes Kedzioara and Bartosz, 1988; Parks, 1989). Several
(Arthur, methods
in
1988; have
been described for the determination of H202 production by cells (Ebermann and Couperus, 1987; Hasui et al., 1987; Hool and Nieman, 1988; Hyslop and Sklar, 1984; Malehorn et al., 1986; Pick and Keisari, 1980; Ruth et al., 1983; Wymann et al., 1987). We wanted
to compare
the calorimetric
assay of Pick and Keisari
(1980) and a
fluorimetric method (Ruth et al., 1983). Both methods are simple as they are based on horseradish peroxidase (HRP)-catalysed oxidation of a hydrogen donor. In the calorimetric assay phenol red is the H-donor and in the fluorimetric method HVA is oxidized method
to a highly
is the higher
fluorescent
sensitivity
dimer.
Obviously
in comparison
the advantage
to the calorimetric
of the
latter
assay. In the first
method (Ruth et al., 1983), a linear relationship between the H202 concentration and fluorescence intensity was found in the range of 0.05 to 4.5 nmol/mL. With the method of Pick and Keisari (1980) a linearity was found between 1 and 60 nmol H202/ mL. However, in comparing the two methods we measured a significantly lower H202 production in phorbol myristate acetate (PMA) stimulated rat pleural macrophages using the fluorimetric method. Therefore, we compared the two methods
From Sektion Pharmazie, Martin-Luther Universitat, Postfach, Halle 4010, Germany. Address reprint requests to: Dr. R. Hirschelmann, Sketion Pharmazie, Martin-Luther Universittit, Postfach, Halle 4010, Germany. Received October 1, 1990; revised and accepted January 1, 1991.
61 lournal of
Pharmacological
0 1991 Elsevier
Science
Methods Publishing
26, 61-65 Co..
Inc.,
0160.5402/911$3.50
(19911 655 Avenue
of the Americas,
New
York,
NY 10010
62
C. Von Rein and R. Hirschelmann systematically method. MATERIALS
in an attempt
AND
to explain
the inconsistent
results with the fluorimetric
METHODS
Reagents HVA was obtained from Merck, Darmstadt (FRG); phenol red was from Polskie Odczynniki Chemiczne Gliwice (Poland). HRP was purchased from VEB LCA Feinchemie Sebnitz (GDR) and PMA was supplied by Sigma Chemical Company, St. Louis (USA). PMA was dissolved in dimethylsulfoxide to a concentration of 10 t.r.M and stored at -20°C in aliquots small enough for one experiment. Powdered isotonic buffer for cell culture (IBC) was obtained from Staatliches lnstitut fur Immunpraparate und Nahrmedien, Berlin (GDR) and was used as the suspending medium for cells.
Buffers and Reagent Solution HVA-HRP-solution (for the fluorimetric method): 9.1 mg HVA was dissolved in 25 mL IBC yielding a HVA concentration of 200 PM (see Table 1). An aliquot of 10 mL of this solution and 1 mL of a solution of 5 mg HRP in 10 mL IBC were made up to 50 mL with
IBC. This solution
was prepared
fresh.
Buffered phenol red solution (for the calorimetric assay): Shortly before use amounts of 0.1 g phenol red and 0.2 g HRP were added to a volume of 1000 mL of a buffer containing 0.14 M of sodium chloride, 5 mM K2HP04, 5 mM KH2P04 and 5.5 mM glucose.
Animals and Preparation
of Cells
We used pleural macrophages from healthy female rats (outbred strain Barby: Wistar), body wt 200 to 250 g. The rats were injected in the pleural cavity with 2 mL of isotonic roform,
saline.
opening
cells were
Cells were the pleural
washed
collected
after 24 hr by killing
the animals
cavity and by lavage with IO mL of isotonic
in IBC and the total number
of cells was determined.
TABLE 1 Change in Hz02 Production by Rat Pleural Macrophages with Increasing HVA Concentration HVA
CONCENTRATION
[)LMOL
(20
MIN
*
L-‘]
%
Hz02
PREINCUBATION)
N
0 (controls)
[NMOL
*
(I@
CELLS
*
30
INHIBITION
MIN)-‘1
5
3.15
c 0.62
100
5
1.63
rt 0.45’:
48
200
5
1.12
2 0.39”
64
300
5
0.79
2 0.28**
75
** Significantly
different
from
controls;
p < 0.01 (Student’s
t-test)
in chlo-
saline.
The
Assays of Hz O2 Production by Rat leukocytes
Calorimetry The cells were
resuspended
in 2 mL buffered
phenol
red solution
and stimulated
by adding PMA to a final concentration of 25 nM. After 30 min incubation at 37”C, the cells were centrifuged and the reaction was stopped by adding 100 PL of a 1 M NaOH solution to the cell free system. The amount of oxidized phenol red was measured at 610 nm using a spectrophotometer Spekol (VEB Carl Zeiss Jena). For measuring the influence of HVA on H202 generation by the macrophages, the resuspended cells were incubated with HVA (for time of preincubation Tables 1 and 2). After washing the cells to remove HVA the macrophages resuspended
in buffered
phenol
red and stimulated
with PMA as described
see were above.
Fluorimetry H202
production
mixed solution concentration),
was assayed fluorimetrically
of IBC ad HVA-HRP 30 min incubation
after resuspension
of the cells in a
solution (I :I), stimulation with 25 nM PMA (final at 37”C, centrifugation of the cells, and stopping
the reaction by addition of 0.5 mL NaOH-EDTA-buffer, pH 12, containing 0.1 M NaOH and 30 mM EDTA. Emission was measured at 420 nm following excitation at 312 nm using a Perkin-Elmer MPF-44 spectrofluorimeter (Perkin-Elmer, Ueberlingen).
Cell Viability The viability
was tested
by using the method
of methylene
blue exclusion.
The
cells were incubated for 5 min at 37X in a buffer to which five drops of 1% methylene blue were added. Thereafter a microscopic determination of viability was performed. Dead cells cannot exclude the methylene blue.
Statistical Calculations All values are presented as mean + SD. Student’s t-test was used for significance calculation. Statistical significance was accepted at the 0.01 level of probability.
TABLE 2 Change in HzOz Production by Rat Pleural Macrophages with Increasing Time of Preincubation with HVA TIME [MINI N
[NMOL
*
(I@
CELLS
*
30
MIN)~‘]
INHIBITION
5
2.29 k 0.37
5
5
0.78 k 0.22**
66
10
5
0.63 k O.l**
72
20
5
0.78 " 0.16**
66
30
5
0.82 k 0.41**
64
0 (controls)
** Significantly
%
Hz02
(PREINCUBATIONWITH 200 JLMOL * L-' HVA)
different
from
controls;
p < 0.01
(Student’s
t-test)
63
64
C. Von Rein and R. Hirschelmann TABLE 3 HzOz Production by Rat Pleural Macrophages after Stimulation with 25 nM PMA (final concentration) HZOZ
RESULTS
AND
Despite
METHOD
N
calorimetric fluorimetric
10 6
* (I@’
CELLS
* 30
MIN)-‘I
3.99 k 1.01 0.77 k 0.28
DISCUSSION
the higher
nificantly lower assay (Table 3). Therefore
[NMOL
sensitivity
results
of the fluorimetric
for H,Oz
we determined
formation
the influence
HVA
than with
method
we obtained
the calorimetric
of HVA on the amount
The maximum
of inhibition
H202
production
tabolite
of the catecholamines.
is reached
sigred
of H202 generated
using the calorimetric method and we found a significant decrease mation. This impairment was dose-(Table 1) as well as time-dependent impairs
phenol
of H202 for(Table 2).
after 5 min (Table 2). Thus, HVA apparently
by rat pleural
macrophages.
Its concentration
HVA
is a physiological
me-
in rat plasma is in the range of 60-
70 nM (Seegal et al., 1986). In our experiments the HVA concentration of 200-300 FM (Tables 1 and 2) greatly exceeds physiological concentration. As there are no HVA receptors at the cell membrane (Watson and Abbot, 1990), a non-specific impairment of the cells by the high HVA concentrations is very likely. There may be an inhibition of the superoxide-generating membrane-bound NADPH oxidase, as H202 is formed mainly via dismutation of superoxide anion radicals lison, 1979), or even an impairment of a hitherto not characterized
(Segal and Alsystem for a
divalent oxygen reduction. Therefore, the fluorimetric assay described by Ruth et al. (1983) obviously has the disadvantage of HVA cytotoxicity against the cells, although we could not find a decreased viability of the cells after incubation with HVA. However, the time planation of cell damage
of incubation was rather short (maximal by HVA could be the rise in superoxide
centration after autoxidation sition metals would cause
of HVA in the buffer solution. generation of cell-damaging
Catalytic hydroxyl
30 min). An exand H202 contraces of tranradicals from
superoxide and H202 (Buettner, 1988; Halliwell, 1990). Thus the HVA method will produce inconsistent results unless transition metals are excluded, e.g., by chelation.
In any case, this method
should
be used with caution.
REFERENCES Arthur MJP (1988) Reactive oxygen intermediates and liver injury. / Hepato/6:125-131. Biemond P (1988) Superoxide dependent iron release from ferritin in inflammatory diseases. Free Radical
Biol Med
4:185-198.
Buettner GR (1988) In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals. / Biochem Biophys
Methods
16:27-40.
Ebermann R, Couperus A (1987) A nonenzymatic
Assays of Hz O2 Production by Rat leukocytes method for determination of hydrogen peroxide and organic peroxides. Anal Biochem 165:414419. Halliwell B (1990) How to characterize a biological antioxidant. Free Rad Res Commun 9:1-32. Hasui M, Hirabayashi Y, Kobayashi Y (1987) Simultaneous measurement by flow cytometry of phagocytosis and hydrogen peroxide production of neutrophils in whole blood. / lmmun Meth 117:53-58. Hool K, Nieman TA (1988) Immobilized luminol chemiluminescent reagent system for hydrogen peroxide determinations in flowing streams. Anal Chem 60:834-837. Hyslop PA, Sklar LA (1984) A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorometric assay of cellular activation processes. Anal Biochem 141: 280-286. Kedzioara j, Bartosz G (1988) Down’s syndrome: a pathology involving the lack of balance of reactive oxygen species. Free Radical Biol Med4:317330. Malehorn CL, Riehl TE, Hinze WL (1986) Improved determination of hydrogen peroxide or Iucigenin by measurement of lucigenin chemilumi-
nescence in 111:941-947.
organised
assemblies.
Analyst
Parks DA (1989) Oxygen radicals: mediators of gastrointestinal pathophysiology. CUT 30:293-298. Pick E, Keisari Y (1980) A simple calorimetric method for the measurement of hydrogen peroxide produced by cells in culture. / lmmunol Meth 38:161-170. Ruth W, Cooper PH, Baggiolini M (1983) Assay of H202 production by macrophages and neutrophils with homovanillic acid and horse radish peroxidase. / lmmunol Meth 63:347-357. Seegal RS, Brosch KO, Bush B (1986) High performance liquid chromatography of biogenic amines and metabolites in brain, cerebrospinal fluid, urine and plasma. / Chromat 377:131-144. Segal AW, Allison AC (1979) Oxygen consumption by stimulated human neutrophils. In: Oxygen free Radicals and Tissue Damage. Ciba Foundation Symposium 65 (new series); AmsterdamOxford-New York, Excerpta Medica, pp. 205219. Watson S, Abbot A (1990) Receptor Nomenclature Supplement. Tif?S 11. Wymann MP, von Tscharner V, Deranleau DA, Baggiolini M (1987) Chemiluminescence detection of H202 produced by human neutrophils during the respiratory burst. Anal Biochem 165:371-378.
65
