The activation of the early chemiluminescent response of human neutrophils by joint treatment with tumor necrosis factor (TNF-alpha) and the calcium ionophore A-23187]

The influence of recombinant human tumor necrosis factor-alpha (TNF-alpha) and calcium ionophore A23187 on luminol- and lucigenin-dependent chemiluminescence capacity (CL) of human polymorphonuclear leukocytes (PMN) has been studied. The CL response of TNF-alpha treated PMN is amplified by lucigenin, but not luminol. TNF-alpha and A23287 synergistically induced both the luminol- and lucigenin-dependent early CL response. The combination of A23187 and activator of protein kinase C--phorbol (myristoyl-13-acetyl)--also provoked early CL response. While the combination of TNF-alpha and A23187 decreased late CL response compared to A23187 alone. The obtained results suggests that synergistic CL response of PMN induced by TNF-alpha and A23187 is connected with activation of protein kinase by TNF-alpha.     

TLR-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor Alpha by Human Peripheral Blood Neutrophils

This paper presents the study on TLR-mediated production of reactive oxygen species and tumor necrosis factor alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand), and lipopolysaccharide (TLR4 ligand). Luminol- and lucigen-independent chemiluminescence was used to detect the production of reactive oxygen species. The concentration of tumor necrosis factor alpha was measured by enzyme immunoassay. The plots of dependence of the light sums of luminol- and lucigenin-dependent chemiluminescence on the concentration of each ligand were shaped as saturation curves. The comparison of the light sums of lucigenin-dependent chemiluminescence (the production of superoxide anion radical) and luminol-dependent chemiluminescence (the total production of reactive oxygen species) showed that the contribution of NADPH oxidase to the total TLR-mediated production of oxidants can reach 40–50%. Stimulation indices were calculated to compare the ability of TLR ligands to stimulate the production of reactive oxygen species and tumor necrosis factor alpha by neutrophils. It has been established that the activation of neutrophils with zymosan leads to higher (more than 8-fold) production of reactive oxygen species rather than production of tumor necrosis factor alpha. Unlike zymosan, lipopolysaccharide stimulated the production of tumor necrosis factor alpha to a greater extent (by more than 2 times) than the production of reactive oxygen species. Peptidoglycan takes an intermediate position between these ligands. Thus, the production of effector molecules (reactive oxygen species and tumor necrosis factor alpha) by human peripheral blood neutrophils depends on the nature of the TRL ligand.     

Chemiluminescence of mononuclear cells is enhanced during antigen recognition

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.     

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.     

What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence? 1. Investigations with hydrogen peroxide and sodium hypochlorite

Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H₂O₂ is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe²⁺ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co²⁺ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co²⁺. Cu²⁺ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration–reaction curve is not as steep. NaOCl enhances H₂O₂/Fe²⁺-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10⁴ (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity. © 1998 John Wiley & Sons, Ltd.     

Oxidative response and membrane modification of diabetic platelets challenged with PAF

Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study [table: see text] demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.     

Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism

The EPR signal recorded in reaction medium containing L-lysine and methylglyoxal is supposed to come from the anion radical (semidione) of methylglyoxal and cation radical of methylglyoxal dialkylimine. These free radical inter-mediates might be formed as a result of electron transfer from dialkylimine to methylglyoxal. The EPR signal was observed in a nitrogen atmosphere, whereas only trace amounts of free radicals were registered under aerobic conditions. It has been established that the decay of methylglyoxal anion radical on aeration of the medium is inhibited by superoxide dismutase. Using the methods of EPR spectroscopy and lucigenin-dependent chemiluminescence, it has been shown that nonenzymatic generation of free radicals including superoxide anion radical takes place during the interaction of L-lysine with methylglyoxal — an intermediate of carbonyl stress — at different (including physiological) pH values. In the course of analogous reaction of L-lysine with malondialdehyde (the secondary product of the free radical derived oxidation of lipids), the formation of organic free radicals or superoxide radical was not observed.     

Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.     

Neutrophil effector functions triggered by Fc-gamma and/or complement receptors are dependent on B-ring hydroxylation pattern and physicochemical properties of flavonols

Tissue damage in autoimmune diseases involves excessive production of reactive oxygen species (ROS) triggered by immune complexes (IC) and neutrophil (PMN) interactions via receptors for the Fc portion of IgG (FcgammaR) and complement receptors (CR). Modulation of both the effector potential of these receptors and ROS generation may be relevant to the maintenance of body homeostasis. In the present study, the modulatory effect of four flavonols (myricetin, quercetin, kaempferol, galangin) on rabbit PMN oxidative metabolism, specifically stimulated via FcgammaR, CR or both classes of receptors, was evaluated by luminol- and lucigenin-dependent chemiluminescence assays. Results showed that flavonol inhibitory effect was not dependent on the cell membrane receptor class stimulated but related to the lipophilicity of the compounds (their apparent partition coefficient values were obtained by high-performance liquid chromatography), and was also inversely related to the number of hydroxyl groups in the flavonol B ring and the ROS-scavenger activity (assessed by the luminol--H2O2--horseradish peroxidase reaction). Under the experimental conditions the flavonols tested were not toxic to PMNs (evaluated by lactate dehydrogenase release and trypan blue exclusion) and did not interfere with IC-induced phagocytosis (evaluated by transmission electron microscopy). Our results suggested that inhibition of IC-stimulated PMNs effector functions by the flavonols tested herein was the result of cooperation of different cellular mechanisms.     

Systematic study on ROS production induced by oleic, linoleic, and gamma-linolenic acids in human and rat neutrophils

The effects of oleic, linoleic, and gamma-linolenic acids on the production of ROS by unstimulated and PMA-stimulated neutrophils were investigated by using five techniques: luminol- and lucigenin-amplified chemiluminescence, cytochrome c, hydroethidine, and phenol red reduction. Using lucigenin-amplified chemiluminescence, an increase in extracellular superoxide levels was observed by the treatment of neutrophils with the fatty acids. There was also an increase in intracellular ROS levels under similar conditions as measured by the hydroethidine technique. An increment in the intra- and extracellular levels of H2O2 was also observed in neutrophils treated with oleic acid as measured by phenol red reduction assay. In the luminol technique, peroxidase activity is required in the reaction of luminol with ROS for light generation. Oleic, linoleic, and gamma-linolenic acids inhibited the myeloperoxidase activity in stimulated neutrophils. So, these fatty acids jeopardize the results of ROS content measured by this technique. Oleic, linoleic, and gamma-linolenic acids per se led to cytochrome c reduction and so this method also cannot be used to measure ROS production induced by fatty acids. Oleic, linoleic, and gamma-linolenic acids do stimulate ROS production by neutrophils; however, measurements using the luminol-amplified chemiluminescence and cytochrome c reduction techniques require further analysis.     

Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence

Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan.     

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.     

Chitosan Oligosaccharides Attenuates Oxidative-Stress Related Retinal Degeneration in Rats

This study investigated the therapeutic potential and mechanisms of chitosan oligosaccharides (COS) for oxidative stress-induced retinal diseases. Retinal oxidative damage was induced in Sprague-Dawley rats by intravitreal injection of paraquat (PQ). Low-dose (5 mg/kg) or high-dose (10 mg/kg) COS or PBS was intragastrically given for 14 days after PQ injection. Electroretinograms were performed to determine the functionality of the retinas. The surviving neurons in the retinal ganglion cell layer and retinal apoptosis were determined by counting Neu N-positive cells in whole-mounted retinas and TUNEL staining, respectively. The generation of reactive oxygen species (ROS) was determined by lucigenin- and luminol-enhanced chemiluminescence. Retinal oxidative damages were assessed by staining with nitrotyrosine, acrolein, and 8-hydroxy-2''-deoxyguanosine (8-OHdG). Immunohistochemical studies were used to demonstrate the expression of nuclear factor-kappa B (NF-κB) p65 in retinas. An in vitro study using RGC-5 cells was performed to verify the results. We demonstrated COS significantly enhanced the recovery of retinal function, preserved inner retinal thickness, and decreased retinal neurons loss in a dose-dependent manner. COS administration demonstrated anti-oxidative effects by reducing luminol- and lucigenin-dependent chemiluminenscense levels and activating superoxide dismutase and catalase, leading to decreased retinal apoptosis. COS markedly reduced retinal NF-κB p65. An in vitro study demonstrated COS increased IκB expression, attenuated the increase of p65 and thus decreased NF-κB/DNA binding activity in PQ-stimulated RGC-5 cells. In conclusion, COS attenuates oxidative stress-induced retinal damages, probably by decreasing free radicals, maintaining the activities of anti-oxidative enzymes, and inhibiting the activation of NF-κB.     

The mechanism of superoxide anion generation by the interaction of phenylhydrazine with hemoglobin.

The mechanism by which superoxide anion is generated by the interaction of phenylhydrazine with either oxy- or methemoglobin was investigated. Rather than superoxide anion generation resulting from an accelerated autooxidation of oxyhemoglobin, it was found that both oxy- and methemoglobin function as peroxidases toward phenylhydrazine with the resultant oxidation of this compound to phenyldiazine. Generation of phenyldiazine from the oxidation of phenylhydrazine by hemoglobin or by the hydrolysis and subsequent decarboxylation of methyl phenylazoformate (C6H5N=NCOOCH3) resulted in the production of superoxide anion. It is suggested that under certain conditions hemoglobin may function as a drug-metabolizing peroxidase.     

Oxidation of glutathione and superoxide generation by inorganic and organic selenium compounds

The carcinostatic activities of selenium (Se) compounds have been shown to be composition and concentration dependent. Several studies have indicated that the ratios between glutathione (GSH) and Se may play an important role in Se catalysis and toxicity. The present study examined the catalytic effect of three selenium compounds on GSH oxidation using lucigenin-dependent chemiluminescence (CL) as an indirect measure of superoxide generation. Various GSH:Se ratios were assayed for the glutathione oxidase activity of selenite, selenocystamine and diselenodipropionic acid. CL emitted from the reaction of selenite with GSH increased more rapidly and was greater than those from the diselenides, but the diselenide CL reactions were sustainable. Both selenite- and diselenide-induced CL were markedly suppressed by superoxide dismutase (SOD). Iodoacetic acid (IAc) effectively inhibited CL generated from selenite-, selenocystamine- and diselenodipropionic acid-catalyzed GSH oxidation. These results suggest that GSH oxidation catalyzed by selenite, and the diselenides selenocystamine and diselenodipropionic acid, generated the superoxide radical in which the CL was inhibited by SOD. Furthermore, CL inhibition by IAc suggests that the catalytic species producing superoxide were the GSSe(-) or RSe(-) anion. This redox chemistry may be responsible for selenite and organoselenium toxicity and apoptosis, making possible the design and synthesis of organoselenium-containing pharmaceuticals.     

The effect of carnosine and trolox on cellular chemiluminescence of leukocytes]

The effects of hydrophilic antioxidant carnosine, trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid), and superoxide dismutase on the myeloperoxidase activity of leukocytes, superoxide anion and active oxygen species generation have been studied. Physiological concentrations of carnosine have been shown to decrease the ability of human leukocytes to produce chemiluminescence as a result of myeloperoxidase activation. However, the chemiluminescence induced by the generation of the superoxide or its derivatives is unaffected by this process. Trolox does not inhibit the induction of superoxide-dependent chemiluminescence of leukocytes either.     

Lucigenin-enhanced chemiluminescence of the animal tissues

Lucigenin-enhanced chemiluminescence (LcCL) allows one to investigate the reactions of superoxide anion radical (*O2-) generated by mitochondria and is applied to study the superoxide production in enzymatic and membrane systems by isolated mitochondria and cells, and in whole organs. The application of lucigenin-enhanced chemiluminescence to estimate the respiration of human tissues involves the use of small tissue pieces, which can be obtained, for instance, by biopsia; however, no systematic investigations have been performed on these objects. In the present paper, a comparative study of lucigenin-enhanced chemiluminescence of tissues isolated from different organs of the rat was carried out to elucidate its dependence on the extent of tissue defragmentation, storage time, and access for oxygen. It was shown that the addition of lucigenin to a piece of tissue, a suspension of fine tissue fragments, and homogenates greatly enhanced chemiluminescence, and a whole piece of tissue possessed a much lesser (by 1-1.5 order of magnitude) intensity of chemiluminescence than homogenate or gruel. In the absence of stirring of the surrounding solution, the lucigenin-enhanced chemiluminescence of tissue quickly decreased, apparently due to a decrease in the level of oxygen in the tissue, as the result of its consumption. The chemiluminescence consisted of two components: a lucigenin-dependent and lucigenin-independent one (intrinsic chemiluminescence). Thus, the tissue was a source of lucigenin-enhanced chemiluminescence, and this luminescence was observed only at a sufficient access for oxygen. The lucigenin-independent component did not practically depend on oxygen and was determined by the components coming out of the tissue into the surrounding solution. Nitric oxide (NO) inhibited chemiluminescence as its concentration increased and did not affect considerably the rate of oxygen consumption by the tissue. The results obtained allow one to conclude that lucigenin can be used as a rather effective chemiluminescent probe for the production of superoxide radicals by tissue pieces.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2.. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Effect of adherence to plastic on peripheral blood monocyte and alveolar macrophage chemiluminescence

The chemiluminescence of peripheral blood monocytes and alveolar macrophages was determined in the presence of luminol and lucigenin, either before or after the cell adherence to the luminometer curvettes. In the case of monocytes, cell adherence induces an increase of luminol-dependent chemiluminescence and has almost no effect on the lucigenin-dependent chemiluminescence. However, it shows a strong inhibition of the lucigenin-dependent chemiluminescence and almost no effect on luminol-dependent chemiluminescence, in the case of alveolar macrophages. These results show that adhesion to plastic alters the metabolic burst of both monocytes and alveolar macrophages. Although the mechanisms are poorly understood, they seem to be related to the modifications that take place during the differentiation of peripheral monocytes to alveolar macrophages.