Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils

J BlOLUMlN CHEMILUMIN 1995; 10: 229-37

Fast and Sensitive Chemi I umi nescence Determination of H202 Concentration in Stimulated Human Neutrophils

S. Mueller" and J. Arnhold Institute of Medical Physics and Biophysics, School of Medicine, University of Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany

A fast and sensitive chemiluminescence assay for the determination of H 2 0 2 in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide.

Changes in H202 concentration in PMA-stimulated neutrophils were followed by injection of NaOCl to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H202 without influence of H202 decomposition by cellular enzymes or newly produced HzOz due to dismutation of superoxide anion radicals. Concentrations of H202 were diminished by catalase and enhanced by sodium azide owing t o inhibition of cellular catalase and myeloperoxidase. Changes in H2O2 concentration upon stimulation could be observed at 3000 cells/mL.

Keywords: Polymorphonuclear leukocytes; hydrogen peroxide; hypochlorous acid; respiratory burst; chemiluminescence; luminol; catalase

INTRODU CTlON

The generation of reactive oxygen species by stimulated polymorphonuclear leukocytes (PMN) and macrophages is considered to play an important role in antimicrobic and anticancerous activity, and also in tissue injury (1,2). Firstly, superoxide anion radicals are generated by NADPH oxidase during the respiratory burst (3,4) Their dismutation produces hydrogen peroxide (5). More reactive oxygen species including hypochlorous acid, hydroxyl radicals, singlet oxygen, and peroxynitrite are derived from reactions of both superoxide anion radicals and hydrogen peroxide (6-8).

Hydrogen peroxide is a central metabolite (4),

*Author for correspondence at: Department of Internal Medi- cine IV, University of Heidelberg, Bergheimer Strasse 58, 69 1 15 Heidelberg, Germany. Abbreviations: PMA, phorbol-12-myristate-13-acetate; PMN, polymorphonuclear leukocytes; SOD, superoxide dismutase.

and is the least reactive of all neutrophil-derived oxygen species. Since it easily permeates through biological membranes, it can cause oxidative con- versions far from its site of generation (5). There- fore, the concentration of hydrogen peroxide is of particular interest in investigations of the oxidative effects of PMN, e.g. in reperfusion injury of the liver (9) and of the myocardium in trans- plantation surgery (10). A sensitive H202 assay is also important for the characterization of the functional state of phagocytes in a number of pathologies (1 1).

Methods for determination of the concentration of H202 are usually based on enzymatic reactions using peroxidases and substrates to yield changes in absorbance, fluorescence or chemiluminescence. Methods using absorption spectrophoto- metry (12) are comparatively insensitive and subject to potential artefacts arising from changes in light transmission (3-7%) due to alteration in

Received 16 August 1994 Revised I2 January 1995

CCC 0884-3996195jO40229-09 0 1995 by John Wiley & Sons, Ltd.

230 S. MUELLER AND J. ARNHOLD

cell shape (1 3). Fluorescence or chemiluminescence assays for H202, such as scopoletin-horseradish peroxidase assay (14) or the luminol-peroxidase assay (1 5- 17) are more sensitive. Nevertheless these methods have several deficiences which are often overlooked. For example, in the scopoletin assay potential artefacts arise from interferences of other substrates, autoxidation of scopoletin, Raman scattering of the excitation light, and auto- fluorescence (14,17). Recently, the specifity of the luminol peroxidase assay was questioned (IS), since peroxidase-mediated oxidation of luminol can be significantly inhibited by superoxide dismutase (1 9,20). Furthermore, these methods are more suitable for the determination of the over- all H 2 0 2 production but not for the determination of actual H 2 0 2 concentrations in cell suspensions.

This paper presents a new method for determination of hydrogen peroxide concentration based on the oxidation of luminol by hypochlorous acid. According to the scheme of luminol oxidation by various substrates the yield of chemiluminescence depends on the presence of hydrogen peroxide (21). Using hypochlorous acid as oxidant, luminol is oxidized directly to a diazaquinone compound which is further converted by the hydrogen peroxide anion via an alpha-hydroxy-hydro- peroxide to an excited aminophthalate (21 -23). Although the amplification of this light emission due to coincubated hydrogen peroxide has been known for a long time (24-26), a linear relationship between light intensity and the concentration of hydrogen peroxide has been published only recently (27). The range of linearity can be extended down to the nanomolar region using a pretreatment of solutions with catalase (28,29).

We examined changes in the concentration of hydrogen peroxide in suspension of stimulated neutrophils as a function of time after stimulation. The injection of NaOCl to cell suspensions containing luminol results in a flash of light, and the intensity depends on the concentration of hydrogen peroxide. This experimental approach allows a rapid determination of actual concentrations of hydrogen peroxide, and measurements are possible even at very low cell numbers.

MATERIALS AND METHODS

Materials

The suppliers for chemicals were: Boehringer

(Mannheim, Germany) for luminol; Sigma (Deisenhofen, Germany) for phorbol-12-myristate-13-acetate (PMA), scopoletin, Ficoll-Hypa- que, and heparine; Serva (Heidelberg, Germany) for catalase, horseradish peroxidase, hydrogen peroxide, dextran, and superoxide dismutase; and Laborchemie Apolda (Apolda, Germany) for sodium hypochlorite.

Solutions

Hank’s balanced salt solution was prepared every day and used without phenol red. PMA (200pg/ mL) was dissolved in ethanol and further diluted with Hank’s solution.

Stock solutions of luminol were made in 140 mmol/L NaCl, 10 mmol/L phosphate buffer and adjusted to pH 7.4. Stock solutions of NaOCl and H 2 0 2 were prepared in water. Concentrations were determined spectrophotometricall immedi- ately prior to use (t290 = 350 M-’ cmPYat pH 12 (30) and €230 = 74 M-’ cm-’ (31) for NaOCl and H202, respectively). Solutions of NaOCl and H 2 0 2 were freshly prepared daily. The activity of catalase ( k ) was determined at pH 7.4 following the breakdown of hydrogen peroxide at 240 nm using the method described by Aebi (32).

Preparation of neutrophils

Polymorphonuclear neutrophils (PMN) were isolated from heparinized (10 I.E./mL) blood from healthy volunteers. The preparation included a dextran-enhanced sedimentation, Ficoll-Hypa- que density centrifugation, lysis of remaining red blood cells with distilled water and washing of cells with Hank’s balanced salt solution. PMN were stored in Hank’s medium at a concentration of 2 x lo6 cells/mL at 4°C. The cells were used within 2 h after preparation.

Chemi luminescence measurements

Luminescence measurements were performed using a AutoLumat LB 953 luminometer (Fa. Berthold, Wildbad, Germany) connected to a personal computer. The luminescence intensity for all experiments is given in relative light units (RLU) which are the internal standardized light units of Berthold luminometers. In cell-free experiments

NEUTROPHIL PEROXIDE ASSAY 231

50 pL of NaOCl was injected into 950 pL of a solution containing H202 and luminol. The kinetics of chemiluminescence were registered using intervals for integration of 50 ms. In some cases total counts over 4 s were determined.

PMNs were diluted with Hank’s medium and incubated with luminol (5 x 10-5mol/L) at 37°C. 100 pL of PMA (1.6 x mol/L, final concentration) was added to 850pL of cell suspension to stimulate neutrophils. In order to determine actual concentrations of hydrogen peroxide during the process of cell stimulation 50pL of NaOCl was injected using a second injector device located in the measuring position. The integral of peak luminescence was determined over 2 s.

The dependence of concentration of hydrogen peroxide on the time after cell stimulation was determined using up to 20 samples of the same cell population in a single batch varying intervals between the two injections. All operations were controlled by computer programs written in GW- Basic.

Solutions of NaOCl become unstable at low concentrations, thus care was taken to minimize its destruction. Vials with known concentrations of hydrogen peroxide were placed at the beginning and the end of each batch of samples to follow possible alterations in reagent concentrations.

Scopoletin assay

Hydrogen peroxide was determined in neutrophil suspensions by oxidation of scopoletin in the presence of horseradish peroxidase (14). Briefly, 100 pL of cell suspension or hydrogen peroxide solution of known concentrations were added to 1.5 mL sodium acetate (0.04 mol/L) containing 0.2 mmol/L Na2-EDTA, 9 pmol/L scopoletin, and 0.03 mmol/L horseradish peroxidase. After an incubation of 5 min, 1.5 mL 0.1 mol/L glycine- NaOH buffer (pH 10.4) was added. The fluorescence intensity was read at 460 nm (excitation, 390 nm).

RESULTS

Principle of H202 determination in a cell free system

The addition of sodium hypochlorite to a luminol solution in the presence of hydrogen peroxide

results in a strong chemiluminescence. Examples of intensity-time curves are shown in Fig. 1 (99% of the light was emitted within 2s). The exponen- tial decay of the chemiluminescence intensity does not depend on H202 in the concentration range indicated.

The luminescence intensity is linearly related to the concentration of H202 from nanomolar to micromolar concentrations (Fig. 2). For low concentrations of H202, these results were obtained using catalase-mediated decomposition of hydrogen peroxide. This procedure was necessary because water solutions contain small amounts of H202 which can be detected with the luminol hypochlorite assay. Since the H202 decomposition by catalase is a first-order reaction, the rate constant (k) is given by:

k = ( l /At ) ( ln S1/S2)

where At = t2 - t l is the measured time interval and SI and S2 represents H202 concentrations at times t l and t2, respectively (32). Consequently, samples with very low H 2 0 2 concentrations can be prepared and the H202 concentration can be calculated if catalase activity and initial H202 concentration are known.

The detection limit of H202 is 10p9mol/L. At concentrations of hydrogen peroxide higher than 5 x 10-6mol/L the dependence on chemilumi-

Figure 1. Chemiluminescence kinetics of the oxidation of luminol by NaOCl in the presence of different concentrations of hydrogen peroxide. 50pL of sodium hypochlorite was injected into 950 pL solution of luminol containing varying concentrations of hydrogen peroxide. The luminescence intensity was measured in intervals of 50ms from the beginning single photon counting. Final concentrations of H202 are indicated in the figure. Other conditions: 5 x 1 0-5 mol/L luminol, 1 0-6 mol/L NaOCI, 50mmol/L phosphate buffer, pH 7.4


% 10 5!

0 l o 4 : 0 C a,

C .-

I

I - = - - -

I - P

-2 CI

nescence intensity becomes nonlinear (data not shown).

In respect to chemiluminescence kinetics integration time can be limited to 2s. Under optimal conditions, the assay may be performed with a high degree of accuracy (SD<3%). Using other final concentrations of NaOCl similar curves were obtained (data not shown).

c .- E

E

* *OOOO~

3 80000- - .- Figure 2. Relationship between the luminescence intensities determined over 2 s and concentration of hydrogen peroxide. 50pL NaOCl (1 0-6 mol/L, final concentration) was added to

(5 x 10- mol/L) and hydrogen peroxide. Low concentrations of hydrogen peroxide were obtained after pretreatment of a 4 x 10-6mol/L H202 solution with catalase (1.48 x 1 O-' mol/L)

a, I: 950 pL ghosphate buffer, pH 7.4, containing luminol

Determination of the hydrogen peroxide concentration in suspensions of stimulated PMN

b

Examples of chemiluminescence peaks arising from the addition of NaOCl to suspensions of stimulated neutrophils are given in Figs 3(a) and 3(b). Four PMN samples were stimulated with PMA in the presence of luminol. Sodium hypochlorite was injected 60, 75, 90 or 360s after the stimulation of cells, respectively. Short bursts of luminescence result and these overlie the luminol-dependent chemiluminescence of these cells. This latter chemiluminescence appears only in Fig. 3(a) using a semilogarithmic plot since it is small in respect to the intense chemiluminescence from the luminol oxidation by NaOCl (an integration period of 50 ms was used to obtain this data).

The actual concentrations of H202 can be calculated from the integrals of luminescence peaks

time[s)

Figure 3. Chemiluminescence peaks arising in suspensions of stimulated neutrophils from injections of NaOCl in (a) a semilogarithmic and (b) a linear plot. The usual luminol- dependent chemiluminescence of PMN only appears in panel (A) because of the ordinate scaling. The intensity is low in comparison to intense luminescence peaks arising from injections of sodium hypochlorite to cell suspensions (arrows 1-4). The concentration of hydrogen peroxide can be calculated from the peak intensities. Final conditions: IOO,OOO cells/mL, 5 x 1 0 - ~ mol/L luminol, I O - ~ mol/L NaOCI, 10 ng/mL PMA, pH 7.4, 37°C.

(calibration curve: Fig. 2). The underlying luminol-dependent chemiluminescence is usually neglected or subtracted from the peak integral.

Peak integrals obtained after the addition of NaOCl to a cell suspension are plotted versus the time after stimulation of PMNs with PMA (Fig. 4). Three different final concentrations of NaOCl

5 x lop5, and 10-5mol/L) were used in these experiments. All curves exhibit a maximum at about 400s that is independent of the NaOCl concentration, and then the light emission decreases continuously in all samples. The actual concentrations of H202 can be calculated from cor- responding calibration curves. In Fig. 2 only the calibration curve using mol/L NaOCl is shown. Concentrations of hydrogen peroxide cal-


- 0 3 C B (I)

=L v

I

0 0 o a 0

time (sl

Figure 4. Chemiluminescence response upon injection of different amounts of NaOCl in stimulated neutrophils as a function of time after stimulation of cells with PMA (arrow). Final conditions: mol/L NaOCl (A), 5 x mol/L NaOCl kB), mol/L NaOCl (C), 100,000 cells/mL, 5 x 10- mol/L luminol, 10ng/mL PMA, pH 7.4

culated are independent of the amount of NaOCl added to cell suspensions in these experiments.

To prove the specificity of our method for hydrogen peroxide, PMN suspensions were stimulated in the presence of agents affecting the metabolism of H202. The incubation of cells with catalase markedly decrease both the initial concentration of H202 and also the changes in H2O2 during cell stimulation (Fig. 5). Only a slight increase in H202 concentration was observed in the presence of catalase (trace B) upon cell stimulation with PMA. These data confirm the efficient removal of H202 by catalase as shown in inhibition of lumi- no1 chemiluminescence in a cell-free system (28).

Sodium azide does not affect the chemiluminescence in the oxidation of luminol by NaOCl (28), but it efficiently inhibits haem-containing enzymes such as myeloperoxidase and catalase (33,34). The incubation of PMA-stimulated PMNs with sodium azide caused a continuous increase of the luminescence yield after the addition of NaOCl with increasing times after cell stimulation (Fig. 6). H202 is accumulated in stimulated cells because pathways destroying hydrogen peroxide were blocked by sodium azide. During the first three minutes after cell stimulation there were only small differences between the response curves in Fig. 6. This means that processes of hydrogen peroxide production prevail over its con- sumption in this period.

The effect of exogeneous superoxide dismutase on luminescence signals was also investigated. The incubation of PMN with SOD (1 18 U/mL)

time (s)

Figure 5. Effect of catalase on the H 2 0 2 concentration in stimulated neutrophils. Cells were stimulated in the absence (trace A) or presence (trace 6) of catalase (3.7 x 1 O-' mol/ L, k = 0.1 2 s-' ). Conditions: 20,000 cells/mL, 5 x I 0-5 mol/ L luminol, 10-5mol/L NaOCI, 10ng/mL PMA (arrow), pH 7.4

- 3 600000 LI a v

v) a .- C 0.4 E 200000

.- I a E

0 400 800 1200 O'O 0 time [sl

Figure 6. Effect of sodium a ide on the H202 concentration in stimulated neutrophils. Cells were stimulated in the absence (trace A) or presence (trace 6) of sodium azide

mol/ L luminol, 5 x mol/L NaOCI, 10ng/ml PMA (arrow), pH 7.4

mol/L). Conditions: 100,000 cells/mL, 5 x

produced no significant changes in hydrogen peroxide concentration (data not shown). If superoxide anions contributed to the chemiluminescence a strong inhibition would be expected on addition of SOD. However, our results demonstrate no interference of superoxide anion radicals on the assay. On the other hand, SOD efficiently converts superoxide anion radicals to hydrogen peroxide. The lack of an increase of luminescence in cell suspensions incubated with exogenous SOD may be due to a possible scavenging of added NaOCl by SOD (28). No enhancement of hydrogen peroxide concentrations after addition of SOD was observed by Test and Weiss (35).

HOCl/OCl- is known to react efficiently with


500000 -

000000 -

500000 ~

0-

many targets such as sulfhydryl and amino groups of proteins and other compounds (36-38) and a competition of these targets with luminol for HOCl/OCl- results (28). Therefore, influences of potential targets for HOCl/OCl- located in the plasma membrane of neutrophils or secreted during the process of cell stimulation into the surrounding medium would be expected. Fig. 7 shows an example where changes in the concentration of hydrogen peroxide in PMA-stimulated neutrophils were investigated in the presence of HSA. Increasing amounts of HSA lead to less intense chemiluminescence peaks and apparently lower values for H 2 0 2 . However, curve profiles

J J 1 9

6 400 8dO 1200 time(s1

Figure 7 . Effect of HSA on the cherniluminescence arising from the oxidation of lurninol by NaOCl in the presence of stimulated neutrophils. Cells were stimulated in the presence of 1 0-5 rnol/L HSA. Other conditions: 100,000 cells/mL, 5 x 1 0-5 mol/L lurninol, 5 x 1 0-6 mol/L NaOCI, 10 ng/ml PMA (arrow), pH 7.4

5 g 1200000 -J

a, 0 C

800000 a, C

- 5 400000

.-

.- E r

4 0 0 0

time[s) Figure 8. Concentration of hydrogen peroxide in PMA-stimulated neutrophils as a function of time after stimulation using very low cell numbers. Conditions: cell numbers: 12,000 cells/rnL (A) 6000 cells/mL (B), 3000 cells/mL (C), 5 x 1 0-5 rnol/L lurninol, 1 0-5 mol/L NaOCI, 10 ng/mL PMA (arrow), pH 7.4

were independent of the final concentration of HSA. This may be due to a partial scavenging of HOCl/OCl- by HSA according to our previous investigations (28). Other compounds such as alanine, cysteine and lysine were found to act in a similar way (data not shown).

In the next series of experiments the total cell number per vial was varied in order to test a possible scavenging of HOCl/OCl- by cell components and to examine detection limits of our method. Sodium hypochlorite was added to suspensions of unstimulated cells in the presence of luminol. A continuous decrease of light emission was observed at cell numbers higher than 5 x lo4 cells/ml with increasing amounts of neutrophils. No changes in chemiluminescence signal occurred at lower cell concentrations. On the basis of these data, it was concluded that a scavenging of HOCl/ OC1- by components of neutrophils occurs as in the case of HSA and other compounds (see above).

Examples of changes in the concentration of hydrogen peroxide upon stimulation in suspensions containing very low numbers of cells are given in Fig. 8. The initial value for H202 was about 1.1 x lo-’ mol/L and its concentration rises upon cell stimulation. Interestingly, an increase in H202 was detectable at 3000 cells/mL (trace C). At these low cell numbers the H202 concentration rises after a lag time of 200 s and without any maximum during the measuring time. With increasing cell numbers (> 5 x 104cells/mL) a maximum H202 concentration is expressed five to eight minutes after cell stimulation (see Figs 4, 6 and 7). The highest value of H202 was 2.8 x 10-6mol/L at 2 x 105cells/mL. At higher cell numbers no further increase in H202 could be detected under our experimental conditions (data not shown). We assume that this effect is caused by destruction of H202 by cellular enzymes such as catalase and myeloperoxidase (total cell content increases also at higher cell numbers).

Finally, concentrations of H202 obtained with injections of NaOCl were compared with results determined by an enzymatic assay for H202 using horseradish peroxidase and scopoletin (14). Both methods differ considerably in their incubation times of reactants with cell suspensions containing hydrogen peroxide. Therefore, we used for these experiments neutrophils which were stimulated in the presence of sodium azide. Here, continuous accumulation of H202 is observed (see Fig. 6). 20min after the addition of PMA only a smaller increase of the hydrogen peroxide concentration


Table 1. Comparative detection of H202 concentration in stimulated neutrophils. P M N (20,000 cells/mL) were stimulated by 10 ng/mL PMA in the presence of 10-4mol/L sodium azide. The determination of hydrogen peroxide (n = 5) was performed 20 min after stimulation (10ng/mL PMA).

Method H202 concentration (rnol/L)

Scopoletin and horseradish (8.3 f 0.3) XI 0-’

Lurninol and NaOCl (8.1 i 0 . 2 ) x l O - ’ peroxidase

occurs. Hydrogen peroxide concentrations determined with both methods under these conditions are given in Table 1. Slightly higher values were detected with the horseradish peroxidase scopoletin assay. This overestimation may be a conse- quence of longer incubation times (5 min) and the detection qf additionally generated H202 in this assay.

DISCUSSION

This paper presents a new method for the determination of hydrogen peroxide concentrations in suspensions of stimulated neutrophils by measuring the chemiluminescence yield of luminol oxidation by sodium hypochlorite. This approach allows calculation of actual H202 concentrations within a measuring time of 2s. Using computer- driven luminometers equipped with sample exchange devices or a microtitre plate luminometer and two injector devices (one for the injection of the cell stimulator, the other for the addition of NaOCl) changes in concentration of H202 during the process of cell stimulation can be followed. Moreover, the method is sensitive enough to detect alterations in H202 even at cell numbers lower than 5000 per vial.

The short integration time of 2 s allows measurement of the actual concentrations of H202 at a given time during the process of cell stimulation. Thus, changes in the H202 concentration due to destruction by cellular enzymes or dismutation of superoxide anion radicals are minimized. This is an important advantage in comparison to conven- tional enzymatic methods using horseradish peroxidase and various substrates (14- 17). These assays can overestimate H202 concentrations due to long

incubation times. Additionally, exogeneous added peroxidases and their substrates strongly influence the H202 metabolism of cells.

Experiments involving preincubation of cells with catalase or sodium azide indicate that the luminescence signals obtained after the injection of NaOCl depend on hydrogen peroxide concentration. The scheme of formation of excited aminophthalate during luminol oxidation includes a diazaquinone compound as an intermediate. This substance is unstable in aqueous solutions and yields dark products (39). It is converted to an excited aminophthalate in the presence of hydrogen peroxide anions (21).

Luminol is oxidized by various one-electron oxidants to semidiazaquinone radicals that dismu- tate to diazaquinone and luminol. In this process superoxide anion radicals appear (21,23). Using HOCl/OCl-, luminol is directly oxidized to diazaquinone (22). Therefore, superoxide anion radicals should not affect the yield of luminescence after the addition of NaOCl to luminol as confirmed in this and our previous work (28).

Based on these facts we assume that our method measures the dependence of changes in H202 concentrations in neutrophil suspensions on the time after cell stimulation without any influence of cellular metabolism. Some underestimation of H202 concentrations can result from a partial scavenging of added HOCl/OCl- by cellular components when using higher numbers of cells. Because such reactions affect only the first step of luminol oxidation by hypochlorous acid and not the subsequent conversion of diazaquinone (28), a correction of concentrations of hydrogen peroxide calculated from calibrations curves is possible.

Using the luminol hypochlorite assay, changes in the concentration of hydrogen peroxide could be detected at cell numbers of 3000 cells/mL. This cell sensitivity can be plausibly explained since leukocytes are known to groduce H202 in the order of nanomoles per 10 cells/minute (40,41). Thus hydrogen peroxide can rapidly accumulate and reach the detection limit of the assay.

At high cell numbers the rapid build-up of H202 is followed by a breakdown with a maximum 3-8 minutes after cell stimulation. This maximum does not exceed micromolar concentrations of H202 even at very high cell numbers, and since the breakdown can be inhibited by sodium azide, the H202 concentration seems to be controlled mainly by catalase. This enzyme decomposes H202 in a first-order reaction (32). Consequently,


the decomposition of H202 increases at higher H202 concentrations. This was recently confirmed in studies on the role of catalase in fibroblasts (42) and mouse erythrocytes (43). We presume that the H202 concentration is limited to micromolar concentrations as a self-protection mechanism. High concentrations of H202 favour the generation of other very reactive compounds such as hydroxyl radicals in a Fenton reaction (44), and hypochlorous acid via a myeloperoxidase-catalysed reaction (45,46).

Further investigations on the role of hydrogen peroxide in cellular metabolism are in progress. Our method appears to be a useful tool to study changes in H202 concentration of PMN and also other cells using different stimulators, and to examine its role in various pathologies.

Acknowledgements

This work was supported by the German Ministry of Research of Technology (Grant 01 ZZ 9103/9-R-6) and a grant from the German Academic Exchange Service (D AAD) .

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Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils