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.

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.

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.

[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.

Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase.

In Mediterranean folk medicine Olea europaea L. leaf (Ph.Eur.) preparations are used as a common remedy for gout. In this in vitro study kinetic measurements were performed on both an 80% ethanolic (v/v) Olea europaea leaf dry extract (OLE) as well as on nine of its typical phenolic constituents in order to investigate its possible inhibitory effects on xanthine oxidase (XO), an enzyme well known to contribute significantly to this pathological process. Dixon and Lineweaver-Burk plot analysis were used to determine K(i) values and the inhibition mode for the isolated phenolics, which were analysed by RP-HPLC for standardisation of OLE. The standardised OLE as well as some of the tested phenolics significantly inhibited the activity of XO. Among these, the flavone aglycone apigenin exhibited by far the strongest effect on XO with a K(i) value of 0.52 µM. In comparison, the known synthetic XO inhibitor allopurinol, used as a reference standard, showed a K(i) of 7.3 µM. Although the phenolic secoiridoid oleuropein, the main ingredient of the extract (24.8%), had a considerable higher K(i) value of 53.0 µM, it still displayed a significant inhibition of XO. Furthermore, caffeic acid (K(i) of 11.5 µM; 1.89% of the extract), luteolin-7-O-ß-D-glucoside (K(i) of 15.0 µM; 0.86%) and luteolin (K(i) of 2.9 µM; 0.086%) also contributed significantly to the XO inhibiting effect of OLE. For oleuropein, a competitive mode of inhibition was found, while all other active substances displayed a mixed mode of inhibition. Tyrosol, hydroxytyrosol, verbascoside, and apigenin-7-O-ß-D-glucoside, which makes up for 0.3% of the extract, were inactive in all tested concentrations. Regarding the pharmacological in vitro effect of apigenin-7-O-ß-D-glucoside, it has to be considered that it is transformed into the active apigenin aglycone in the mammalian body, thus also contributing substantially to the anti-gout activity of olive leaves. For the first time, this study provides a rational basis for the traditional use of olive leaves against gout in Mediterranean folk medicine.

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.

pH-dependent regulation of myeloperoxidase activity.

The balance between peroxidase and chlorinating activities of myeloperoxidase (MPO) is very important for the enhancement of antimicrobial action and prevention of damage caused by hypochlorite. In the present paper, the peroxidase and chlorinating activities have been studied at various pH values. The possibility of using neutrophil protein solution for the evaluation of MPO activity has been demonstrated. It is shown that at neutral pH MPO had higher affinity to peroxidase substrate guaiacol: at pH 7.4, chloride ions did not compete with guaiacol up to the concentration of 150 mM. At acidic pH, chlorinating activity of MPO dominates: only hypochlorite production can be detected at equal chloride and guaiacol concentrations of 15 mM. However, horseradish peroxidase does not exhibit any difference in activity in the presence of chloride ions even at acidic pH values. It was demonstrated by MALDI-TOF mass-spectrometry that the amount of hypochlorite produced is sufficient to modify phospholipids (with formation of Cl- and Br-hydrins and lyso-derivatives) only at acidic pH (5.0). Thus, in the presence of phenolic peroxidase substrate, MPO chlorinating activity can be displayed at acidic pH only. It can lead to elimination of hypochlorite production in normal tissues at neutral pH (7.4) and its enhancement in phagosomes where the pH range is 4.7-6.0.

Leukocytic myeloperoxidase-mediated formation of bromohydrins and lysophospholipids from unsaturated phosphatidylcholines.

Using MALDI-TOF mass spectrometry, we have shown that leukocytic myeloperoxidase (MPO) in the presence of its substrates (H2O2 and Br?) does not induce any changes in saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Incubation of liposomes prepared from mono-unsaturated phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) with the (MPO + H2O2 + Br-) system resulted in formation of bromohydrins as the main products. 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysophosphatidylcholine) was the main product of the reaction of polyunsaturated phosphatidylcholine (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) with the (MPO + H2O2 + Br-) system. The formation of lysophospholipids as well as of bromohydrins was not observed when the enzyme or one of its substrates (H2O2 or Br-) was absent from the incubation medium, or if an inhibitor of MPO (sodium azide) or hypobromite scavengers (taurine or methionine) were added. Thus, it can be postulated that the formation of bromohydrins as well as lysophospholipids by the (MPO + H2O2 + Br-) system results from reactions of hypobromite formed during MPO catalysis with double bonds of acyl chains of phosphatidylcholine. Such destructive processes may take place in vivo in membrane- or lipoprotein-associated unsaturated lipids in centers of inflammation.

Degradative action of reactive oxygen species on hyaluronan.

Many human diseases are associated with harmful action of reactive oxygen species (ROS). These species are involved in the degradation of essential tissue or related components. One of such components is synovial fluid that contains a high-molecular-weight polymer--hyaluronan (HA). Uninhibited and/or inhibited hyaluronan degradation by the action of various ROS has been studied in many in vitro models. In these studies, the change of the molecular weight of HA or a related parameter, such as HA solution viscosity, has been used as a marker of inflicted damage. The aim of the presented review is to briefly summarize the available data. Their correct interpretation could contribute to the implementation of modern methods of evaluation of the antioxidative capacity of natural and synthetic substances and prospective drugs--potential inflammatory disease modifying agents. Another focus of this review is to evaluate briefly the impact of different available analytical techniques currently used to investigate the structure of native high-molecular-weight hyaluronan and/or of its fragments.

Destabilization of the acrosome results in release of phospholipase A2 from human spermatozoa and subsequent formation of lysophospholipids.

Phospholipase A(2) controls the phospholipid composition in spermatozoal membranes and is released from the acrosome of human spermatozoa. The extracellular phospholipase A(2) activity of human spermatozoa was determined by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry after destabilization of acrosome by the calcium-ionophore calcimycin. MALDI-TOF mass spectrometry allowed the monitoring of changes in both substrate and products of spermatozoal phospholipase A(2) (PLA(2)) without the use of labelled phospholipids. The spermatozoal PLA(2) was characterized as a secretory one (sPLA(2)). Secretory PLA(2) exhibited a high substrate specificity for 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), the most abundant spermatozoal phospholipid. A time- and cell number-dependent formation of the lysophospholipid PDPC was observed following incubation of extracellular medium of calcimycin-treated spermatozoa (CTS) with PDPC. Antibodies against sPLA(2), specific inhibitors of sPLA(2) and Ca(2+)-chelators could inhibit its generation. An antibody against lysophospholipase enhanced the lysoproduct concentration in the extracellular medium of CTS containing sPLA(2) because further metabolization of these products was blocked. The results demonstrated that destabilization of the acrosome is able to induce a release of secretory phospholipase A(2) from human spermatozoa with subsequent generation of lysophosphocholine in the surrounding of spermatozoa.

Generation of free radicals during decomposition of hydroperoxide in the presence of myeloperoxidase or activated neutrophils.

It was shown with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone that myeloperoxidase (MPO) in the presence of its substrates H2O2 and Cl- as well as activated neutrophils destroy tert-butyl hydroperoxide producing two adducts of O-centered radicals which were identified as peroxyl and alcoxyl radicals. Inhibitory analysis performed with traps of hypochlorite (taurine and methionine), free radical scavengers (2,6-di-tret-butyl-4-methylphenol and mannitol), and MPO inhibitors (salicylhydroxamic acid and 4-aminobenzoic acid hydrazide) revealed that the destruction of the hydroperoxide group in the presence of isolated MPO or activated neutrophils was directly caused by the activity of MPO: some radical intermediates appeared as a result of the chlorination cycle of MPO at the stage of hypochlorite generation, whereas the other radicals were produced independently of hypochlorite, presumably with involvement of the peroxidase cycle of MPO. The data suggest that the activated neutrophils located in the inflammatory foci and secreting MPO into the extracellular space can convert hydroperoxides into free radicals initiating lipid peroxidation and other free radical reactions and, thus, promoting destruction of protein-lipid complexes (biological membranes, blood lipoproteins, etc.).

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.

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.

Radiation-induced fragmentation of cardiolipin in a model membrane.

PURPOSE: To obtain evidence for the possibility of free-radical fragmentation of cardiolipin under the action of ionizing radiation as measured by its aqueous dispersion from liposomes. MATERIALS AND METHODS: Liposomes of tetramyristoylcardiolipin (TMCL) were exposed to gamma-rays from 60Co or 137Cs sources at doses between 1 and 24kGy. Fragmentation products were identified using thin-layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Using MALDI-TOF MS and thin-layer chromatography, it was shown that gamma-irradiation of liposomes consisting of TMCL was accompanied by free-radical fragmentation of the lipid to form dimiristoylphosphatidic acid and dimiristoylphosphatidyl hydroxyacetone. The yields of dimiristoylphosphatidic acid were greater than those of dimiristoylphosphatidyl hydroxyacetone, and formation of the named compounds was inhibited by dissolved oxygen. CONCLUSION: It is shown for the first time that on gamma-irradiation, cardiolipin can undergo free-radical fragmentation in its polar component.

Properties, functions, and secretion of human myeloperoxidase.

The heme-containing protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes at sites of inflammation. It is involved in the generation of reactive oxygen and nitrogen species and tissue damage. The general properties and functional aspects of this enzyme are reviewed. Special attention is given to luminescence methods for investigating the release of myeloperoxidase from stimulated cells.

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.

Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidylcholines.

Spermatozoa with plasma membranes that lost their asymmetry or permeability for larger molecules can be identified by binding of annexin V to membrane phosphatidylserine (PS). Paramagnetic annexin-V-conjugated microbeads (AN-MB) can be used to eliminate these spermatozoa by magnetic activated cell sorting (MACS). Semen samples of six healthy volunteers with normal spermiogram parameters were divided into two sperm fractions by MACS as a function of bound AN-MB, and their individual lipid compositions were examined by matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS). As a model system, liposomes composed of phosphatidylcholines (PC) from egg yolk were digested by phospholipase A2 (PLA2). The MALDI-TOF mass spectra of organic extracts of both sperm subpopulations differed significantly. The ratio between lyso-phosphatidylcholine LPC 16 : 0 and PC 16 : 0/22 : 6 was approximately 2.5-4.7-fold higher (median 2.9) in the sperm group binding AN-MB than in spermatozoa with intact membrane unable to bind AN-MB. The ratio between LPC 22 : 6 and PC 16 : 0/22 : 6 was also enhanced in the spermatozoa with impaired membrane structure (factor in the range: 1.9-3.9; median 2.6). These alterations corresponded to the effects of PLA2 on artificial phospholipids. It is concluded that spermatozoa with deteriorated membrane and exposed PS are characterized by an increased lyso-phosphatidylcholine content that is likely generated by phospholipases.

[Interaction of tert-butyl hydroperoxide with hypochlorite induces peroxyl radicals. A chemiluminescence study]. / Vzaimodeistvie tret-butilgidroperoksida s gipokhloritom privodit k obrazovaniiu perekisnykh radikalov. Issledovanie metodom khemiliuminestsentsii.

The interaction of hypochlorite (HOCl/OCl-) with tert-butyl hydroperoxide ((CH3)3COOH) was investigated by chemiluminescence. It was shown that the addition of HOCl/OCl- to (CH3)3COOH induces a fast chemiluminescent flash. The intensity of this flash increases with the increase in both HOCl/OCl- and (CH3)3COOH concentration. The chemiluminescence is quenched in a concentration-dependent manner in the presence of free radical spin traps N-tert-butyl nitrone and alpha-(4-pyridyl-1-oxyl)-N-tert-butyl nitrone. This fact proves that free radicals take part in the interaction of HOCl/OCl- and (CH3)3COOH. Hypochlorite yielded a very similar chemiluminescence spectrum in its reaction with (CH3)3COOH as Ce4+. It differed considerably from the spectrum in the system H2O2 and HOCl/OCl-. It is well known that the interaction of Ce4+ and (CH3)3COOH produces peroxyl radicals. These results confirm the hyothesis that the interaction of HOCl/OCl- and (CH3)3COOH is mediated by peroxyl radicals. Thus, organic hydroperoxides always present in unsaturated lipids can induce lipid peroxidation processes in the reaction with HOCl/OCl-.

Peroxyl radical is produced upon the interaction of hypochlorite with tert-butyl hydroperoxide.

As we reported previously, hypochlorite interacting with organic hydroperoxides causes their decomposition ((1995) Biochemistry (Moscow), 60, 1079-1086). This interaction was supposed to be a free-radical process and serve as a source of free radicals initiating lipid peroxidation (LP). The present study is the first attempt to detect and identify free radicals produced in the reaction of hypochlorite with tert-butyl hydroperoxide, (CH3)3COOH, which we have used as an example of organic hydroperoxides. We have used a direct method for free radical detection, EPR of spin trapping, and the following spin traps: N-tert-butyl-alpha-phenylnitrone (PBN) and alpha-(4-pyridyl-1-oxyl)-N-tert-butylnitrone (4-POBN). When hypochlorite was added to (CH3)3COOH in the presence of a spin trap, an EPR spectrum appeared representing a superposition of two signals. One of them belonged to a spin adduct formed as a result of direct interaction of hypochlorite with the spin trap (hyperfine splitting constants were: abetaH = 0.148 mT; aN = 1.537 mT; and deltaHPP = 0.042 mT for 4-POBN and abetaH = 0.190 mT; aN = 1.558 mT; and deltaHPP = 0.074 mT for PBN). The other signal was produced by hypochlorite interactions with (CH3)3COOH itself (hyperfine splitting constants were: abetaH = 0.233 mT; aN = 1.484 mT; deltaHPP = 0.063 mT and abetaH = 0.360 mT; aN = 1.547 mT; deltaHPP = 0.063 mT for 4-POBN and PBN, respectively). Comparison of spectral characteristics of this spin adduct with those of tert-butoxyl or tert-butyl peroxyl radicals produced in known reactions of (CH3)3COOH with Fe2+ and Ce4+, respectively, showed that the radical (CH3)3COO* is produced from the interaction of hypochlorite with (CH3)3COOH. Like Ce4+ but not Fe2+, hypochlorite addition to (CH3)3COOH was accompanied by a bright flash of chemiluminescence characteristic of the reactions in which peroxyl radicals are produced. Thus, all these results suggest peroxyl radical production in the reaction of hypochlorite with hydroperoxide. This reaction is one of the most possible ways for the initiation of free-radical LP that occurs in vivo, when hypochlorite interacts with unsaturated lipids comprising natural protein-lipid complexes, such as lipoproteins and biological membranes.