Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase.

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H2O2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.

[Reconstruction of osteochondral defects with a collagen I hydrogel. Results of a prospective multicenter study]. / Rekonstruktion von Gelenkknorpeldefekten mit einem Kollagen-I-Hydrogel. Ergebnisse einer prospektiven Multicenterstudie.

STUDY GOALS: The aim of the study was to evaluate the therapeutic benefit of CaReS®, a type I collagen hydrogel-based autologous chondrocyte implantation technique, for the treatment of osteochondral defects of the knee (Outerbridge grades III and IV) within a prospective multicenter study. MATERIAL AND METHODS: A total of 116 patients in 9 clinical centers were treated with CaReS between 2003 and 2008. The Cartilage Injury Evaluation Package 2000 of the International Cartilage Repair Society (ICRS) was employed for data acquisition and included the subjective International Knee Documentation Committee score (IKDC score), the pain level (visual analog scale, VAS), the physical and mental SF-36 score, the overall treatment satisfaction and the functional IKDC status of the indexed knee. Follow-up evaluation was performed 3, 6 and 12 months after surgery and annually thereafter. RESULTS: The mean defect size treated was 5.4 ± 2.7 cm(2) with 30% of the cartilage defects being ≤4 cm(2) and 70% ≥4 cm(2). The mean follow-up period was 30.2 ± 17.4 months (minimum 12 months and maximum 60 months). The mean IKDC score significantly improved from 42.4 ± 13.8 preoperatively to 70.5 ± 18.7 (p < 0.01) in the mean follow-up period. Global pain level significantly decreased (p < 0.001) from 6.7 ± 2.2 preoperatively to 3.2 ± 3.1 at the latest follow-up. Both the physical and mental components of the SF-36 score significantly increased. At the latest follow-up 80% of the patients rated the overall treatment satisfaction as either good or very good. The functional IKDC knee status clearly improved from preoperative to the latest follow-up when 23.4% of the patients reported having no restriction of knee function (I), 56.3% had mild restriction (II), 17,2% had moderate restriction (III) and 3.1% revealed severe restriction (IV). CONCLUSIONS: The CaReS technique is a clinically effective and safe method for the reconstruction of isolated osteochondral defects of the knee joint and reveals promising clinical outcome up to 5 years after surgery. A longer follow-up period and larger patient cohorts are needed to evaluate the sustainability of CaReS treatment.

alpha1-antitrypsin prevents polymorphonuclear leucocyte-elastase effects on spermatozoa quality.

Elevated levels of polymorphonuclear leucocyte (PMN)-derived elastase, which is suggested as marker for inflammations in the male genital tract, correlate well with spermatozoa deterioration. PMN elastase caused a time- and concentration-dependent (up to a elastase concentration of 0.5 microg/mL) externalization of phosphatidylserine and intercalation of propidium iodide on human spermatozoa. There are apparently a limited number of target sites for elastase on spermatozoa surface, because the further enhancement of elastase amount did not fasten alterations in spermatozoa parameters. Analysis of flow cytometry data revealed that most spermatozoa were in a necrotic state after an exposure with elastase for 22 h. Some apoptotic cells were only detected at shorter incubation periods. Seminal plasma prevented in a concentration-dependent manner the PMN elastase-mediated loss of vitality of spermatozoa. We detected by blotting techniques large amounts of alpha(1)-antitrypsin in seminal plasma. This antiproteinase is known to inactivate elastase at inflammatory sites. Increasing concentrations of alpha(1)-antitrypsin prevented gradually spermatozoa deterioration induced by elastase. Thus, alpha(1)-antitrypsin contributes to an efficient protease/antiproteinase balance in seminal plasma. A disturbed balance will promote the development of chronic inflammations which can also be the reason for male infertility problems.

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research.

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.

Inhibition of the heme-induced hemolysis of red blood cells by thechlorite-based drug WF10.

Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.

Impact of cyanogen iodide in killing of Escherichia coli by the lactoperoxidase-hydrogen peroxide-(pseudo)halide system.

In the presence of hydrogen peroxide, the heme protein lactoperoxidase is able to oxidize thiocyanate and iodide to hypothiocyanite, reactive iodine species, and the inter(pseudo)halogen cyanogen iodide. The killing efficiency of these oxidants and of the lactoperoxidase-H2O2-SCN-/I- system was investigated on the bioluminescent Escherichia coli K12 strain that allows time-resolved determination of cell viability. Among the tested oxidants, cyanogen iodide was most efficient in killing E. coli, followed by reactive iodine species and hypothiocyanite. Thereby, the killing activity of the LPO-H2O2-SCN-/I- system was greatly enhanced in comparison to the sole application of iodide when I- was applied in two- to twenty-fold excess over SCN-. Further evidence for the contribution of cyanogen iodide in killing of E. coli was obtained by applying methionine. This amino acid disturbed the killing of E. coli mediated by reactive iodine species (partial inhibition) and cyanogen iodide (total inhibition), but not by hypothiocyanite. Changes in luminescence of E. coli cells correlate with measurements of colony forming units after incubation of cells with the LPO-H2O2-SCN-/I- system or with cyanogen iodide. Taken together, these results are important for the future optimization of the use of lactoperoxidase in biotechnological applications.

Role of functional groups of human plasma and luminol in scavenging of NaOCl and neutrophil-derived hypochlorous acid.

Hypochlorous acid HOCl/OCl- and other oxidants derived from stimulated polymorphonuclear leukocytes are involved in tissue damage during a number of pathological processes. In order to obtain more detailed information on possible reactions of HOCl/OCl- the effects of both NaOCl and PMN-derived hypochlorous acid on functional groups of amino acid solutions and human plasma are studied. In valine and lysine solutions NaOCl diminishes the number of amino groups in a molar ratio of 1:1 between NaOCl and amino groups. In cysteine and methionine samples the decrease of amino groups starts only after all sulfhydryl or thioether groups are oxidized by NaOCl. If freshly prepared human plasma is treated with increasing amounts of NaOCl all plasma SH groups are oxidized first, then probably the thioether groups and only after this the amino groups are affected. Furthermore, it was found, that the reactivity of luminol against NaOCl is similar to that of amino groups. Increasing amounts of SH groups of components of human plasma are oxidized by incubation with PMA-stimulated polymorphonuclear leukocytes dependent on the incubation time. Plasma amino groups are not affected under the same experimental conditions. The addition of plasma to FMLP-stimulated PMN in the presence of luminol decreases that part of chemiluminescence caused by extracellularly generated hypochlorous acid. Plasma samples pretreated with NaOCl cause a lower inhibition of light generation in FMLP-stimulated PMN only when more than 4.10(-8) mol NaOCl per mg protein are used to pretreat plasma. It is assumed that in the development of tissue injuries caused by infiltrated PMN the following sequence of damage occurs in accessible tissue regions. First, the sulfhydryl groups are oxidized, then the thioether groups, and only after this amino and other target groups are affected.

Quenching and dequenching of octadecyl Rhodamine B chloride fluorescence in Ca(2+)-induced fusion of phosphatidylserine vesicles: effects of poly(ethylene glycol).

Ca(2+)-induced fusion of SUV and LUV composed of ox brain phosphatidylserine (PS) was studied as a function of temperature and concentration of Ca2+ using octadecyl Rhodamine B chloride (R-18). Ca2+ was added to a 1:1 mixture of labelled (8 mol%) and unlabelled vesicles (assay conditions) or to samples containing only labelled liposomes (control conditions). Both, in SUV and LUV the dependence of differences in fluorescence between assay and control samples on temperature can be divided into three regions. At temperatures lower than 20 degrees C the differences in fluorescence increase only slightly in SUV or remain unchanged in LUV after the addition of Ca2+. At 28 degrees C and higher temperatures the differences of fluorescence intensities increase much more drastically, whereby SUV exhibit higher fusion rates than LUV. Between 20 degrees C and 28 degrees C exists an intermediate region for both SUV and LUV. Here the fluorescence changes continuously from one behaviour to the other independent of the concentration of Ca2+. A drastic quenching of R-18 fluorescence occurs in LUV composed of PS below 10 degrees C, where the lipids are in the gel state. In SUV the fluorescence is only weakly changed in this temperature region. It is assumed that a demixing between dye and phospholipid molecules occurs below phase transition. During fusion the phase transition of PS is shifted from 8-10 degrees C to about 24-28 degrees C as revealed by polarization measurements using diphenylhexatriene. Because the differences in R-18 fluorescence between assay and control samples depend strongly on temperature we assume that the shift in phase transition temperature of PS occurs immediately after the addition of Ca2+ to SUV or LUV. Poly(ethylene glycol) 6000 accelerates fusion in both SUV and LUV under all conditions where a fusion takes place. Further, the threshold concentration of Ca2+ to induce fusion is diminished from about 1 mmol/l without polymers to about 0.5 mmol/l in the presence of 10% (w/v) PEG 6000. The intermediate region of changes in fluorescence properties of R-18 in the Ca(2+)-induced fusion of PS is not changed by PEG.

Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid.

The formation of lysophosphatidylcholines from unsaturated phosphatidylcholines upon treatment with hypochlorous acid was evaluated by means of MALDI-TOF mass spectrometry and 31P NMR spectroscopy. With an increasing number of double bonds in a fatty acid residue, the yield of lysophosphatidylcholines with a saturated fatty acid residue increased considerably in comparison to the total amount of higher molecular weight products like chlorohydrins and glycols. High amounts of lysophosphatidylcholines were formed from phospholipids containing arachidonic or docosahexaenoic acid residues. In phospholipids with monounsaturated fatty acid residues, the position of the double bond did not influence the yield of lyso-products. Besides the exclusive formation of chlorohydrin and glycol, hypochlorous acid caused the cleavage of the unsaturated fatty acid residue independent of its location at the first or second position of the glycerol backbone. In contrast, strong alkaline conditions, i.e. saponification led also to a hydrolysis of the saturated fatty acid residue from phosphatidylcholines. It is concluded that both MALDI-TOF mass spectrometry and 31P NMR spectroscopy are able to detect the formation of lysophosphatidylcholines. We conclude also that the formation of lysophospholipids from unsaturated phosphatidylcholines by hypochlorous acid can be relevant in vivo under acute inflammatory conditions.

Peroxidation of egg yolk phosphatidylcholine liposomes by hypochlorous acid.

The powerful neutrophil-derived oxidant hypochlorous acid HOCl/OCl- is assumed to contribute to tissue injury in a number of pathological states accompanied by massive accumulation of neutrophils. The production of malondialdehyde to indicate lipid peroxidation was studied in egg yolk phosphatidylcholine liposomes upon treatment with NaOCl as a source for hypochlorous acid. Its accumulation was inhibited by alpha-tocopherol and butylated hydroxytoluene. Singlet oxygen, hydroxyl radicals or superoxide anion radicals derived from direct reactions of hypochlorous acid seem not to be involved in initiation of lipid peroxidation because the malondialdehyde accumulation was unaffected by hydrogen peroxide, catalase, superoxide dismutase, ferrous sulphate or ferric chloride. Double bonds of fatty acid residues seem to be the primary target for NaOCl. Their number is continuously diminished in liposomes (2 mg lipids/ml) after incubation with increasing amounts of NaOCl at 37 degrees C for 40 min as detected by two independent methods (iodine bromide reduction and 1H-NMR spectroscopy). A 1:1 molar ratio between the loss of double bonds and NaOCl added was found only at low NaOCl concentrations. Then double bonds are decreased with a lower efficiency. A continuous increase of lipid peroxidation products was only observed up to 0.5-0.7 mmol/l NaOCl. The yield of lipid hydroperoxides kept constant at higher NaOCl concentrations. However, diene conjugates and malondialdehyde exhibit a maximum at 0.7-1 mmol/l or 0.5 mmol/l NaOCl, respectively, while the concentration of these products decreases at higher doses of NaOCl. The decrease of malondialdehyde was more pronounced than for diene conjugates. These results were discussed from the background that at minimum two (diene conjugates) or three (malondialdehyde) double bonds in a fatty acid residue are necessary for formation of lipid peroxidation products.

Cartilage degradation by stimulated human neutrophils: reactive oxygen species decrease markedly the activity of proteolytic enzymes.

BACKGROUND: Although neutrophilic granulocytes clearly contribute to cartilage degradation in rheumatic diseases, it is unclear if reactive oxygen species (ROS) or proteolytic enzymes are the most important components in cartilage degradation and how they interact. RESULTS: Neutrophils were stimulated by chemicals conferring a different degree of ROS formation and enzyme release. Supernatants of neutrophils were incubated with thin slices of pig articular cartilage. Supernatants of cartilage were assayed by NMR spectroscopy, MALDI-TOF mass spectrometry and relevant biochemical methods. Stimulation conditions of neutrophils correlated well with the extent of cartilage degradation. Due to the release of different enzymes, cartilage degradation could be best monitored by NMR since mainly low-mass degradation products were formed. Astonishingly, the suppression of the formation of ROS resulted in decreased cartilage degradation. CONCLUSION: ROS formed by neutrophils are not directly involved in cartilage degradation but influence the activity of proteolytic enzymes, which are the main effectors of cartilage degradation.

Oxidative activity of human polymorphonuclear leukocytes stimulated by the long-chain phosphatidic acids.

It has already been suggested that phosphatidic acids (PAs) play an important role in the regulation of signaling pathways involved in the production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs). The present study was performed to elucidate the effects of extracellularly added PA-- 1,2-distearoyl- (DSPA) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphate (SAPA)--on the ROS production and on the elastase release by human PMNs. ROS production was monitored by luminol-amplified chemiluminescence and the elastase activity was measured in the supernatant of the PA-stimulated human PMNs by colorimetric assay. Obtained effects were compared with those of cells stimulated by either a chemotactic tripeptide, phorbol ester or calcium ionophore. Our results show that long-chain PAs at concentrations higher than 3 x 10(-5) mol/l stimulate the ROS production by human PMNs, whereas they were ineffective in promoting the elastase release. The chemiluminescence pattern of the SAPA-stimulated cells exhibited a biphasic curve, whereas cell stimulation with DSPA resulted in a monophasic chemiluminescence curve. Stimulation of the ROS production by PAs in dependence of the fatty acid composition required the activity of protein kinases.

Detection of the halogenating activity of heme peroxidases in leukocytes by aminophenyl fluorescein.

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.

Reactions of pholasin with peroxidases and hypochlorous acid.

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl(-) and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H(2)O(2) in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H(2)O(2)/Cl(-) system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.

Effects of hypochlorous acid on unsaturated phosphatidylcholines.

Effects of hypochlorous acid and of the myeloperoxidase-hydrogen peroxide-chloride system on mono- and polyunsaturated phosphatidylcholines were analyzed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Chlorohydrins and glycols were detected as main products according to the characteristic shift of molecular masses. Mainly mono-chlorohydrins result upon the incubation of HOCl/(-)OCl with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, whereas only traces of mono-glycols were detected. 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine yielded a complex mixture of products. Mono-chlorohydrins and glycols dominated only at short incubation, while bis-chlorohydrins as well as products containing one chlorohydrin and one glycol moiety appeared after longer incubation. Similarly, a complex product mixture resulted upon incubation of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine with hypochlorous acid. Additionally, tris-chlorohydrins, products with two chlorohydrin and one glycol moiety, as well as lysophosphatidylcholines and fragmentation products of the arachidonoyl side chain were detectable. Mono-chlorohydrins of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were detected after the incubation of the latter phospholipid with the myeloperoxidase-hydrogen peroxide-chloride system at pH 6.0. These chlorohydrins were not observed in the absence of chloride, hydrogen peroxide, or myeloperoxidase as well as in the presence of methionine, taurine, or sodium azide. Thus, mono-chlorohydrins in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine produced by hypochlorous acid from the myeloperoxidase-hydrogen peroxide-chloride system can also be detected by means of MALDI-TOF MS.

Contribution of reactive oxygen species to cartilage degradation in rheumatic diseases: molecular pathways, diagnosis and potential therapeutic strategies.

Inflammatory joint diseases are of considerable socio-economic significance. However, mechanisms of cartilage destruction are so far only poorly understood. This review is dedicated to reactive oxygen species (ROS) like superoxide anion radicals, hydrogen peroxide, singlet oxygen, hypochlorous acid, hydroxyl radicals and nitric oxide that are generated under inflammatory conditions and also to their potential contribution to cartilage degradation. First, the relevance of rheumatic diseases and potential mechanisms of cartilage degradation are discussed in this review, followed by the description of the chemical constituents and the molecular architecture of articular cartilage as well as the different cell types that play a role in inflammation and cartilage destruction. Methods of the assessment of cartilage degeneration are also shortly discussed. In the main chapter of this review the characteristics of individual ROS, their generation under in vivo conditions as well as their reactivities with individual cartilage components are discussed. Because of the low selectivity of ROS, useful "markers" of cartilage degradation allowing the differentiation of effects induced by individual ROS are also discussed. In the last chapter current therapeutic concepts of the treatment of rheumatic diseases are reviewed. The recently developed "anti-TNF-alpha" therapy that is primarily directed against neutrophilic granulocytes that are powerful sources of ROS and, therefore, important mediators of joint degeneration are emphasised.

Linoleic acid hydroperoxide favours hypochlorite- and myeloperoxidase-induced lipid peroxidation.

Liposomes composed of soybean phosphatidylcholine were peroxidized using the reagent sodium hypochlorite or the myeloperoxidase-hydrogen peroxide-Cl- system. Linoleic acid hydroperoxide previously prepared from linoleic acid by means of lipoxidase was incorporated into liposomes. The yield of thiobarbituric acid reactive substances (TBARS) continuously increased with higher amounts of hydroperoxide groups after the initiation of lipid peroxidation by hypochlorous acid producing systems. The accumulation of TBARS was inhibited by scavengers of free radicals such as butylated hydroxytoluene and by the scavengers of hypochlorous acid, taurine and methionine. Lipid peroxidation was also prevented by sodium azide or chloride free medium in the myeloperoxidase-hydrogen peroxide-Cl- system. Here we show for the first time that the reaction of hypochlorous acid with a biologically relevant hydroperoxide yields free radicals able to cause further oxidation of lipid molecules.

Interaction of tert-butyl hydroperoxide with hypochlorous acid. A spin trapping and chemiluminescence study.

The formation of radical species during the reaction of ter-tbutyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (aN = 1.537 mT, aH beta = 0.148 mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (aN = 1.484 mT, aH beta = 0.233 mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl-alpha-phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe2+ or Ce4+ in the presence of POBN. Using Fe2+ a spin adduct with a N = 1.633 mT and aH beta = 0.276 mT was observed. The major spin adduct formed with Ce4+ was characterised by a N = 1.480 mT and aH beta = 0.233 mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce4+. The addition of Fe2+ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce4+ the same spin adduct and the same luminescence profile. Because Ce4+ is known to oxidize organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils.

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils. Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.

Hypochlorite-induced peroxidation of egg yolk phosphatidylcholine is mediated by hydroperoxides.

Using a chemiluminescent method, the consumption of HOCl/OCl- was investigated during interaction with liposomes prepared from dimyristoylphosphatidylcholine (DMPC) or egg yolk phosphatidylcholine (EYPC). The concentration of HOCl/OCl-decreased with time in the suspension of EYPC that contain unsaturated lipids and did not change in DMPC liposome suspensions. HOCl/OCl- was consumed more rapidly in peroxidized EYPC. The amount of double bonds was lowered by 40% in peroxidized liposomes and decreased by approximately one-third under the action of HOCl/OCl- in both native and peroxidized EYPC samples. Second-order rate constants for the interaction between HOCl and phospholipid double bonds of 0.50 M-1 s-1 were calculated for native EYPC on basis of the consumption of HOCl/OCl- or from the decrease in concentration of double bonds. In peroxidized EYPC this reaction constant was similar as determined following changes in double bonds. It is concluded that the consumption of HOCl/OCl- increased in peroxidized liposomes due to additional reactions with lipid peroxidation products. tert-Butyl hydroperoxide and cumene hydroperoxide, or organic peroxides or epoxides (cis-9,10-epoxystearic acid; cholesterol-5 alpha,6 alpha-epoxide; trans-2,3-epoxy-butane; cis-2,3-epoxy-butane) were incorporated into liposomes and investigated in respect to their ability (1) to increase the consumption of HOCl/OCl- in DMPC liposomes, (2) to generate a non-enhanced chemiluminescence with HOCl/OCl- and (3) to evoke an accumulation of lipid peroxidation products (TBARS) in EYPC liposomes in the absence and presence of NaOCl. None of peroxides or epoxides tested showed any effect on the consumption of HOCl/OCl- or the generation of chemiluminescence. Nor increase of TBARS both in the absence or presence of HOCl/OCl-. In contrast, tert-butyl hydroperoxide and cumene hydroperoxide increased the consumption of HOCl/OCl- in DMPC liposomes and mediated a higher accumulation of TBARS in EYPC liposomes in the presence of HOCl/OCL- over the control. These data suggest that lipid peroxidation in EYPC can be initiated by the reaction of HOCl/OCL- with organic hydroperoxides.