Presence Of Lucigenin Research Articles

Generation of the Reactive Oxygen Species on the surface of nanosized titanium(IV) oxides particles under UV-irradiation and their connection with photocatalytic properties

The chemiluminescence spectra of the samples with nanosized titanium(IV) oxide, both commercial – Hombifine N, Hombikat UV100, Degussa P25, «Anatase I» (Sigma Aldrich), «Anatase II» (Russia), and that prepared in present research by hydrothermal and sol-gel methods (anatase, X-ray amorphous titania, η-phase – TiO2-x×mH2O, and peroxide phase – TiOx(O2)2-x×mH2O) in aqueous media in the presence of luminol or lucigenin under UV-irradiation have been studied. The auxiliary program for data processing and calculating of the Reactive Oxygen Species (ROS: O2−, OH radicals, and H2O2) concentration, which correlated with the amount of photoluminescence photons (quantity hν, eV), has been developed. Photocatalytic activity (PCA) of prepared samples has been studied in the reaction of photodegradation of model dyes (methyl orange - MeO and rhodamine 6 G) and harmful organic pollutant (fungicide – difenoconazole, pesticide – thiamethoxam) under UV-irradiation. The connection between the rate constant (k, min-1) of photooxidation of MeO and OH radicals content (in the presence of luminol) and O2−, H2O2 species content (in the presence of lucigenin) has been described by a transition function of an aperiodic link of the 1st order and a polynomial of 2nd degree, respectively. The maximum k value of the reaction of MeO, difenoconazole, and thiamethoxam photodecomposition under UV irradiation has been achieved in the presence of commercial samples with nanosized anatase, characterized by the optimal crystallite size for photocatalysis (Anatase Sigma-Aldrich) and the maximum specific surface area (Hombifine N «Sachtleben Chemie GmbH»). The k value of the reaction of MeO, difenoconazole, and thiamethoxam photodecomposition under UV irradiation was minimal in the presence of samples containing X-ray amorphous titanium(IV) oxide or η-phase. The role of different factors (sample composition and phase modification, the content of reactive oxygen species, the size of nanoparticles at all levels, specific surface area, etc.) in the exhibition of photocatalytic activity has been discussed.

Mitochondria as a Source of Superoxide Anion Radical in Human Platelets

The radical-producing activity of human platelets has been studied using the enhanced chemiluminescence method. It is shown that chemiluminescence of isolated platelets is observed only in the presence of lucigenin, a selective probe for superoxide anion; the luminescence is amplified many times upon the addition of NADH and NADPH, the substrates of oxidative chains. The chemiluminescence is not affected by diphenyliodonium, an inhibitor of NADPH oxidase, but it is inhibited in a dose-dependent manner by the oxidative phosphorylation uncouplers dinitrophenol and rotenone. Thus, a superoxide anion radical is the main free radical generated by platelets, and mitochondria are one of the superoxide anion radical sources in platelets.

The Production of Reactive Oxygen and Halogen Species by Neutrophils in Response to Monomeric Forms of Myeloperoxidase

It was shown for the first time that myeloperoxidase, a homodimer that consists of two disulfidebonded identical protomers and catalyzes the formation of hypochlorous acid (HOCl), is decomposed by HOCl into monomers (MPO-Cl). Dimeric myeloperoxidase can also be converted into monomers (hemimyeloperoxidase) by reduction of the disulfide bond. In this study, the effects of two monomeric forms of myeloperoxidase, MPO-Cl and hemi-myeloperoxidase, and native dimeric myeloperoxidase on the production of reactive oxygen (•O 2 − and H2O2) and halogen (HOCl) species by neutrophils were compared. Neutrophil production of these species was monitored after addition of hemi-myeloperoxidase, MPO-Cl, or dimeric myeloperoxidase and also after the subsequent addition of activators, phorbol-12-myristate-13-acetate or N-formyl-Met-Leu-Phe. HOCl production was assessed by chemiluminescence in the presence of luminol; •O 2 − production was assessed by chemiluminescence in the presence of lucigenin and by cytochrome c reduction determined spectrophotometrically, and H2O2 production was measured using fluorimetry with scopoletin. The results indicate that MPO-Cl and hemi-myeloperoxidase, which can occur in blood under halogenative stress, do not prime neutrophil NADPH oxidase, and do not enhance the production of reactive oxygen (•O 2 − and H2O2) and halogen (HOCl) species.

New Analytical Method for Investigating the Antioxidant Power of Food Extracts on the Basis of Their Electron-Donating Ability: Comparison to the Ferric Reducing/Antioxidant Power (FRAP) Assay

Several analytical approaches are available for investigating the antioxidant power for antioxidants, and they are based on a variety of chemical principles, such as oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), and ferric reducing/antioxidant power (FRAP). This paper reports a new rapid method for investigating antioxidant power on the basis of the electron-donating ability. This method is called chemiluminescence analysis of antioxidant power (CAAP). The electrons donated from antioxidants are capable of inducing chemiluminescence in the presence of lucigenin and a base. Thus, the intensity of chemiluminescence induced by antioxidants is proportional to their electron-donating ability (antioxidant power). It was found that the correlation between CAAP and FRAP was positive (r = 0.959) and statistically significant (p < 0.05). In addition to the FRAP assay, the rapid CAAP assay is convenient for investigating the antioxidant power of herbal extracts.

Female gender is associated with decreased oxidative stress and increased insulin‐stimulated glucose uptake in skeletal muscle in TGR(mRen‐2)27 rats

The transgenic Ren‐2 rat is a model of enhanced Renin Angiotensin Aldosterone System activity, which exhibits insulin resistance, hypertension, and microalbuminuria. We investigated gender differences in the production of Reactive Oxygen Species (ROS) and NAPDH oxidase activity in 8 to 11 weeks old Ren 2 rats and age‐matched SD rats. ROS production was determined by photon emission in the presence of lucigenin and NADPH. Results are presented as mean ± SEM, and unpaired t test and ANOVA were used as appropriate. SBP was similar between males and females (170.5 ± 16.21 vs 180.4 ± 8.61 mmHg). In insulin‐sensitive soleus muscle, NAPDH oxidase activity was higher in Ren 2 males compared to females (6.378 ± 0.41 vs 4.98 ± 0.27 mOD/min/mg p &lt;0.05), while it did not differ between SD males and females (4.1 ± 0.24 vs 4.22 ± 0.47 mOD/min/mg p = 0.83). No significant differences in NAPDH oxidase activity were found in aorta, kidney or left ventricle. ROS production in soleus muscle was lower in Ren‐2 females compared to males (102.69 ± 4.48 RLU/s/mg vs 170.69 ± 11.33, respectively. p=0.025). Insulin stimulated glucose uptake was higher in Ren‐2 female compared to males (0.655 ± 0.057 vs 0.195 ± 0.0531 nmol/mg) Our findings suggest that female gender may be protective against oxidative stress and leads to increased insulin sensitivity in skeletal muscle in the Ren‐2 rat model.Funded by NIH 1R01‐HL073101‐02, VA Merit System 0018.

Antioxidant and pro‐oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions

To evaluate the antioxidant and pro-oxidant properties of chlorhexidine (CHX). The scavenging and generation of reactive oxygen species (ROS) by CHX in the presence or absence of saturated Ca(OH)(2) solutions was evaluated. The reaction emitted chemiluminescence in the presence of lucigenin thus was determined by a luminometer to evaluate the levels of ROS production. Changes in DNA conformation were analysed by agarose gel electrophoresis. Paired Student's t-test was used to compare the difference between groups. Chlorhexidine (0.00002-0.02%) effectively scavenged 56-88% of the superoxide radicals generated by the xanthine/xanthine oxidase reaction. Through analysis of PUC18 DNA conformation changes, CHX was shown to be a mild scavenger of hydroxyl radicals generated by H(2)O(2) plus FeCl(2). However, CHX (>0.083%) decreased the mobility of PUC18 plasmid DNA with potential production of DNA-DNA cross-link and severe DNA breaks (presence of DNA smear) at further higher concentrations. Furthermore, CHX induced ROS production including H(2)O(2) and superoxide radicals in 0.1N NaOH (pH = 12.76) or Ca(OH)(2) (pH = 12.5) solutions. Chlorhexidine exhibited both antioxidant and pro-oxidant properties under different conditions. These events are possibly involved in the killing of root canal and periodontal microorganisms when CHX and Ca(OH)(2) were used in combination or separately. Potential genotoxicity and tissue damage when extruded into the periradicular tissue and at higher concentrations should be considered during periodontal and endodontic practice.

Mechanism of superoxide anion generation in intact mitochondria in the presence of lucigenin and cyanide.

In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 microM; Luc2+) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F0F1-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F0F1-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc*+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc2+ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc*+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.

Lucigenin-derived chemiluminescence in intact isolated mitochondria.

There are many data both in favor and against the use of lucigenin as a probe for superoxide anion (SA) in mitochondria, cells, and simple enzymatic systems. In the present work high concentrations (50-400 micro M) of lucigenin were used for continuous recording of rapid and reversible changes in the SA level in intact isolated mitochondria. The SA level in the presence of lucigenin rapidly and reversibly changed during the transition of the mitochondria from one functional state to another: under conditions of ATP synthesis from ADP and Pi, of Ca2+ accumulation, and of reverse electron transfer. Induction of a Ca2+,cyclosporin A-sensitive pore in mitochondria completely suppressed the lucigenin-derived chemiluminescence (LDC). The electron transfer in the Q-cycle of the respiratory chain complex III and high electric potential difference across the inner membrane of mitochondria were obligatory conditions for generation of a SA-dependent chemiluminescent signal. Based on our own and literature data, a scheme of LDC generation is suggested. The origin of superoxide anion detected in intact mitochondria with lucigenin is discussed.

Dimethyldiselenide and Methylseleninic Acid Generate Superoxide in an In Vitro Chemiluminescence Assay in the Presence of Glutathione: Implications for the Anticarcinogenic Activity of L-Selenomethionine and L-Se-Methylselenocysteine

The reduction of cancer incidence by dietary supplementation with L-selenomethionine, L-Se-methylselenocysteine, and other methylated selenium compounds and metabolites is believed to be due to the metabolic generation of the monomethylated selenium species methylselenol. Dimethyldiselenide and methylseleninic acid were reduced by glutathione in an in vitro chemiluminescent assay in the presence of lucigenin for the detection of superoxide (O2 - ). The methylselenol produced on reduction of dimethyldiselenide and methylseleninic acid was found to be highly catalytic, continuously generating a steady state of O2 - . The O2 - detected by the chemiluminescence generated by methylselenol was fully quenched by superoxide dismutase, causing a complete cessation of chemiluminescence. In contrast, dimethyldisulfide in the presence of glutathione was not catalytic to any measurable extent and did not generate any superoxide. These in vitro results suggest that methylselenol catalysis is possible in vivo, and if metabolism generates sufficient concentrations of methlylselenol from L-selenomethionine or L-Se-methylselenocysteine in vivo, it could change the redox status of cells and oxidatively induce cellular apoptosis.

Mathematical treatment of chemiluminescence data allowing an optimised kinetic analysis of vascular NAD(P)H-dependent superoxide anion production

Regulation of vascular redox homeostasis plays a central role in the control of vascular tone (e.g.: redox modulation of endothelial NO synthase and soluble guanylate cyclase activities). Superoxide anion is one of the main reactive oxygen species involved in the modulation of the redox status of the vessel. It is crucial to know if pathological situations or drug treatments are able to modify NAD(P)H oxidase-dependent production of O 2 - . As a steady-state approach is the only way for a valid assessment of these parameters, a mathematical treatment allowing visualisation of the linear portion of the initial velocity of O 2 - production was developed. Using two vascular preparations (rat aortic rings and mouse thoracic aortae), the chemiluminescence (CL) in the presence of lucigenin was recorded every second in the 15-30 minute period after successive injections of NADPH and superoxide dismutase (SOD). Because, both O 2 - and photons are labile and evanescent products, visualisation of the steady-state requires calculation of their cumulative production by integral calculus using a first order integral equation. The cumulative production of CL and/or O 2 - was plotted as a function of the NADPH concentrations, were determined from the linear portion of these plots. For a precise molar quantification of O 2 - production, a calibration curve of initial velocities using xanthine oxidase + xanthine as a source of O 2 - was plotted by comparing CL and cytochrome C reduction. The NADPH-dependent CL production exihibited Michaelian behavior and SOD acted as a noncompetitive inhibitor. For example, the values for NADPH oxidase in rat aorta were: Km = 237 ± 2μM; Vmax = 500-600 pmol O 2 - /min/ring.

Oxidative activity of turkey monocytes, following the inoculation with Chlamydia psittaci

Chemiluminescence (CL) was used to investigate the competence of turkey monocytes to mount a respiratory burst response upon interaction with Chlamydia psittaci. The oxidative activity of purified turkey monocytes, following inoculation with the avian C. psittaci serovar D strain 92/1293, was studied using luminol- and lucigenin-enhanced CL. Purified turkey monocytes were inoculated with C. psittaci at multiplicity of infection (MOI) of approximately 100, 10 and 1. In the presence of luminol, no detectable CL or only a weak CL response was obtained, and if present it increased with increasing MOI. Either sham inoculated monocytes, or monocyte-free control assays supplemented with C. psittaci, gave no detectable luminol-enhanced CL responses. In the lucigenin-enhanced assays, monocytes inoculated with C. psittaci demonstrated an immediate CL peak, the height of which was proportional to the MOI used. Following inoculations at a MOI 1, a faint second peak was observed, when applying high concentrations of lucigenin. Sham inoculated monocytes gave no detectable lucigenin-enhanced CL responses. However, in the presence of lucigenin, the addition of C. psittaci to monocyte-free controls also resulted in an immediate CL peak, though no second peak was detected. This immediate lucigenin-dependent CL peak induced by C. psittaci was similar to the one observed in the presence of monocytes, and was not inhibited by superoxide dismutase. We demonstrated that this avian C. psittaci strain induces only a very weak respiratory burst response in turkey monocytes. In contrast, C. psittaci itself elicited an intense non-superoxide mediated lucigenin-dependent CL, indicating that in chlamydial research the detection of superoxide, using lucigenin, should be confirmed with a specific superoxide inhibitor.

Phenoxyl Radical-Induced Thiol-Dependent Generation of Reactive Oxygen Species: Implications for Benzene Toxicity

Mechanisms of phenoxyl radical-induced generation of oxygen radicals potentially involved in toxicity of benzene were studied. We hypothesized that phenoxyl radical intermediates formed from phenolic metabolites of benzene by oxidative enzymes (e.g., peroxidases, tyrosinase) are able to damage biomolecules via (i) oxidation of low-molecular-weight thiols and protein thiols and (ii) thioI-dependent generation of oxygen radicals and subsequent oxidation of DNA. Phenoxyl radicals were generated by the oxidation of phenol by myeloperoxidase + H2O2, horseradish peroxidase + H2O2, or tyrosinase. The reaction of phenol-phenoxyl radicals with GSH and dihydrolipoic acid was studied. Our HPLC measurements showed that both thiols reduced the phenoxyl radical back to phenol. This reaction was accompanied by the formation of thiyl radicals (detected by ESR as 5,5-dimethyl-1-pyrroline-N-oxide/glutathione thiyl radical spin adducts) and of superoxide radicals (measured by their chemiluminescence response in the presence of lucigenin). Hydroxylation of 2′-deoxyguanosine to 8-oxo-7,8-dihydro-2′deoxyguanosine was demonstrated in the course of the tyrosinase-catalyzed oxidation of phenol in the presence of dihydrolipoic acid and Fe(III)-EDTA. Redoxcycling of phenoxyl radicals by thiols produces oxygen radicals which can be responsible for the oxidative damage of DNA by radical intermediates of benzene metabolism.

Chemiluminescence and superoxide production in Acanthamoeba castellanii: free radicals generated during oxidative stress

The amoeba Acanthamoeba castellanii generated both luminol- and lucigenin-enhanced chemiluminescence upon addition of the respiratory inhibitor sodium cyanide, but not upon the addition of sodium azide. Photon emission in the presence of lucigenin was three- to fourfold greater than that measured in the presence of luminol, but both forms of chemiluminescence were strictly dependent upon the presence of O2, indicating the requirement for oxidative reactions in these processes. Lucigenin-chemilununescence measured during the phagocytosis of latex beads under identical conditions was, however, barely detectable above background levels. Cyanide similarly induced the formation of O 2 − , as indicated by the stimulation of superoxide-dismutase-inhibitable cytochrome c reduction, and the rate of production was similar to that previously observed during phagocytosis of latex particles or yeasts by these cells. Thus, in view of the similar rates of O 2 − production during cyanide exposure or phagocytosis, but disparate rates of lucigenin-chemiluminescence, these two treatments must activate different molecular processes leading to reactive oxidant production. The rates of cyanide-stimulated lucigenin-chemiluminescence were directly proportional to the O2 tensions in the medium from 0 to 300 μM, indicating that the rates of free-radical-generating reactions were directly related to the oxygen tensions in the environment. The superoxide dismutase inhibitor diethyldithiocarbamate similarly stimulated lucigenin-chemiluminescence, with photon emission again being dependent upon O2 tensions in the range 0–320 μM. A mechanism by which cells may limit O 2 − generating reactions, and so reduce damaging free radical reactions, was observed when anaerobic suspensions were reaerated. These data indicate that oxidative stress and phagocytosis provide two intracellular sources of free-radical generating reactions in these cells.

Effects of qing-fei-tang (seihai-to) and baicalein, its main component flavonoid, on lucigenin-dependent chemiluminescence and leukotriene B4 synthesis of human alveolar macrophages.

A traditional Chinese remedy, Qing-Fei-Tang (Seihai-to, T90), has been used for treatment of chronic respiratory diseases with long-lasting cough and sputum, e.g. chronic bronchitis. We examined the effect of T90 and its main component flavonoid, baicalein, on the lucigenin-dependent chemiluminescence (CL) and leukotriene B4 (LTB4) synthesis of human alveolar macrophages (AM). AM were obtained by bronchoalveolar lavage from patients with various respiratory diseases, including sarcoidosis, idiopathic pulmonary fibrosis, bronchial asthma, chronic bronchitis and lung cancer. CL were observed by stimulating 1 x 10(5) AM with phorbol myristate acetate in the presence of lucigenin. LTB4 were generated by incubating 1 x 10(6)/ml AM with Ca ionophore A23187 for 30 min and determined by reverse phase high performance liquid chromatography and radioimmunoassay. T90 (0.2-2.0 mg/ml) and baicalein (0.1-100 microM) inhibited both CL and LTB4 production of AM in a dose-dependent fashion. These inhibitory effects were not due to cytotoxic effects of the procedure because neither 2 mg/ml T90 nor 100 M baicalein affected the viability of AM nor lactate dehydrogenase release from AM. These results suggest that T90 exerts its effect on inflammatory lung diseases through the anti-inflammatory action, i.e. inhibiting the oxidative and arachidonate metabolism of local inflammatory lung cells.

Chemiluminescence response of human B-cell lines.

Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cells share certain properties with monocytes: they are capable of presenting protein antigens to antigen-specific T-lymphocytes and of releasing an Interleukin 1-like factor. It was our interest to study whether transformed B-cells resemble monocytes by generating toxic oxygen radicals. Human B-cell lines were developed from human peripheral blood lymphocytes by EBV-transformation. The induction of the respiratory burst in the B-cells was assessed by chemiluminescence (CL) in the presence of lucigenin. B-cells were stimulated with phorbol-myristate-acetate (PMA), zymosan particles, the chemotactic peptide f-met-phe, the complement split product C5a and with a recently described granulocyte activating cytokine (GRAM). Stimulation with PMA elicited a distinct CL-response in the tested B-cell lines. The CL-signal was significantly reduced by superoxide dismutase, but not by D-mannitol and catalase. No significant response to any of the other stimuli was detected. Furthermore, none of the stimuli induced a luminol-enhanced CL signal, which, in contrast to lucigenin, is dependent on the presence of peroxidase. Our results indicate that EBV infected B-cells were able to generate significant amounts of reactive oxygen species, particularly superoxide. It appears that virus transformation uncovers genetic information which is usually not expressed in non-transformed B-cells.

Effects of cadmium and lead on oxidative metabolism and phagocytosis by mouse peritoneal macrophages

We investigated the effects of cadmium and lead on the oxidative metabolism of mouse peritoneal macrophages induced either by phagocytosis of zymosan particles or by membrane perturbation with phorbol myristate acetate (PMA) without phagocytosis. Oxidative metabolism activity was measured by chemiluminescence in the presence of lucigenin. Furthermore, cell viability was tested by dye exclusion test and phagocytic activity was measured by ingestion of latex particles. Within the first hour of exposure to the metals the effects of cadmium and lead in peritoneal macrophages were the same. The oxidative metabolism induced by PMA was enhanced by both metals, the zymosan-induced oxidative metabolism was reduced, markedly by cadmium and weakly by lead. Cell viability was not affected. After 20 h of exposure the effects of cadmium and lead were different. Cadmium did not alter either the oxidative metabolism induced by zymosan and PMA or phagocytosis of latex-particles. Lead concentration-dependently suppressed the oxidative metabolism induced by zymosan and PMA as well as phagocytosis of latex-particles. However, cadmium reduced cell viability concentration-dependently in contrast to lead that reduced cell viability only slightly.

Lucigenin-dependent chemiluminescence as a new assay for NAD(P) H-oxidase activity in particulate fractions of human polymorphonuclear leukocytes

The enzyme responsible for the respiratory burst in human neutrophils is an oxidase that catalyzes the reduction of oxygen to superoxide anion (O 2 −). Superoxide anion production may be measured by chemiluminescence (CL) in the presence of lucigenin (10,10′-dimethyl-9,9′-biacridnium dinitrate). We established an assay of the oxidase, by measuring particulate fractions of PMN in the presence of lucigenin. This CL required the addition of NAD(P)H and was very low in fractions of resting cells. In particulate fractions of PMNs stimulated with PMA selectively, the NADPH-dependent CL was found to be increased. CL was linear with protein concentrations up to 100 μg and was shown to be at least 10 times more sensitive for the detection of O 2 − than the assay based on the spectrophotometric determination of superoxide mediated cytochrome c reduction. CL was abolished by inactivating the enzyme at 56°C.

C5a-Induced Chemiluminescence of Human Granulocytes and its Amplification by a Serum Factor

Purified human C5a elicits a fast chemiluminescence (CL) response from isolated human granulocytes in the presence of Lucigenin (bis-N-methylacridinium nitrate). The reaction is inhibitable to more than 90 % by superoxide dismutase (SOD) — final concentration 200 μg/ml —, to about 60 % by catalase — final concentration 10 mg/ml — and to 30 % by the hydroxyl radical scavenger D-mannit — final concentration 100 mM. Therefore O2− seems to be the oxygen radical responsible for most of the CL, while •OH and H2O2 are also involved.Addition of normal pool serum to the cells for 1–2 min before stimulation with C5a strongly enhances the effect in a dose and time-dependent manner. Therefore the existence of a «helper activity» in serum amplifying the C5a-induced CL of granulocytes is postulated. This «helper activity» is, however, not specific for C5a, since CL responses elicited with the chemotactic peptide f-met-phe or by phorbol-myristate-acetate (PMA) are also enhanced by preincubation with serum. In contrast, ConA-induced CL is not enhanced but decreased. Therefore, though not unique to C5a-induced CL, the «helper activity» seems not to represent a «general adjuvans» effect of serum on the granulocytes, but to be restricted to certain stimuli.