Isolation and characterization of an apoprotein from the d less than 1.006 lipoproteins of human and canine lymph homologous with the rat A-IV apoprotein.

The singlet oxygen traps, 2,5-diphenylfurane and 1,3-diphenylisobenzofurane were oxidized to cis-benzoylethylene and o-dibenzoylbenzene during the decomposition of diisopropyl-N-nitrosamine catalyzed by peroxidase. Singlet oxygen quenchers inhibited this conversion and also the chemiluminescence accompaying the catalyzed reaction. The chemiluminescence is enhanced by 1,4-diazobicyclo (2.2.2) octane, fluorescein, eosin rhodamine B and rose bengal but little effect was detected in the presence of 9,10-dibromoanthracene-2-sulfonate, 9,10-diphenylanthracene-2-sulfonate and anthracene-2-sulfonate. An emission spectrum of the unsensitized reaction in 560 – 600 nm region was observed. It is concluded that singlet oxygen is formed during peroxidase catalyzed degradation of diisopropyl-N-nitrosamine.     

Singlet oxygen formation by a peroxidase, H2O2 and halide system.

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3-diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme. Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.     

Chlorophyll destruction in the presence of bisulfite and linoleic acid hydroperoxide

Chlorophyll was rapidly destroyed in the presence of bisulfite and linoleic acid hydroperoxide. Both bisulfite and linoleic acid hydroperoxide were required for chlorophyll destruction and both were consumed in the reaction; however, there was no oxygen requirement. Chlorophyll destruction occurred most readily in the slightly acidic pH region with little destruction occurring above pH 8. The free radical scavengers, hydroquinone and α-tocopherol, were very effective at inhibiting chlorophyll destruction, but the singlet oxygen quenchers, β-carotene, 2,5-dimethylfuran and 1,3-diphenylisobenzofuran, were only slightly effective. The addition of metal chelators indicated that metals were not participating in the reaction. The evidence indicates that chlorophyll was destroyed by a free radical mechanism. Based on the present results and that of others, it is suggested that chlorophyll was destroyed via oxidation by the alkoxy radical which was produced during the decomposition of linoleic acid hydroperoxide by bisulfite.     

Singlet oxygen production by soybean lipoxygenase isozymes

The oxidation of linoleic acid catalyzed by soybean lipoxygenase isozymes was accompanied by 1268 nm chemiluminescence characteristic of singlet oxygen. The recombination of peroxy radicals as first proposed by Russell (Russell, G.A. (1957) J. Am. Chem. Soc. 79, 3871-3877) is a plausible mechanism for the observed singlet oxygen production. Lipoxygenase-3 was the most active isozyme. Under the optimal aerobic conditions of p2H 7, 100 micrograms/ml lipoxygenase-3, 100 microM linoleic acid, 100 microM 13-hydroperoxylinoleic acid, and air-saturated buffer, the yield of singlet oxygen was 12 +/- 0.4 microM or 12% of the amount predicted by the Russell mechanism. High yields of singlet oxygen required the presence of 13-hydroperoxylinoleic acid. Systems containing lipoxygenase-2 and lipoxygenase-3 produced comparable yields of singlet oxygen without added 13-hydroperoxylinoleic acid, since the lipoxygenase-2 served as an in situ source of hydroperoxide. Lipoxygenase-1 was active only at low oxygen concentrations. Its singlet oxygen-producing capacity was greatly increased by the addition of acetone to the system. Lipoxygenase-2 did not produce detectable quantities of singlet oxygen.     

Mitoxantrone and ametantrone inhibit hydroperoxide-dependent initiation and propagation reactions in fatty acid peroxidation

The anthracenedione antineoplastic agents mitoxantrone and ametantrone are potent inhibitors of basal and drug-stimulated lipid peroxidation in a variety of subcellular systems (Kharasch, E. D., and Novak, R. F. (1983) J. Pharmacol. Exp. Ther. 226, 500-506). The mechanism by which these compounds function as antioxidants has been investigated using enzymic and chemical systems. Mitoxantrone and ametantrone inhibited NADPH-cytochrome P-450 reductase- and xanthine oxidase-catalyzed conjugated diene formation from linoleic acid in a concentration-dependent manner with half-maximal inhibition achieved at approximately 0.5 microM anthracenedione. Inhibition of linoleic acid peroxidation was not attributable to a decrease in P-450 reductase activity, hydroxyl radical scavenging, or iron chelation by the anthracenediones. Nonenzymic fatty acid peroxidation was also inhibited by the anthracenediones. Linoleic acid oxidation initiated by superoxide (ferrous iron autoxidation) or by hydroxyl radicals (Fenton's reagent) was diminished by mitoxantrone and ametantrone after a brief delay, suggesting an effect subsequent to activated oxygen-dependent initiation. In contrast, linoleic acid oxidation initiated by iron-dependent hydroperoxide decomposition was inhibited immediately. Reinitiation of linoleic acid oxidation in an anthracenedione-inhibited system was accomplished only by superoxide generation, but not by fatty acid hydroperoxide decomposition. These results suggest the anthracenediones diminished neither oxygen radical formation nor oxygen radical-dependent initiation of peroxidation. Rather, inhibition of fatty acid peroxidation by mitoxantrone and ametantrone results from the inhibition of hydroperoxide-dependent initiation and propagation reactions.     

Singlet oxygen production by bleomycin. A comparison with heme-containing compounds

Fe(III)-bleomycin catalyzes the decomposition of 13-hydroperoxylinoleic acid and of 15-hydroperoxyarachidonic acid to produce small quantities of singlet oxygen. No singlet oxygen is produced when hydrogen peroxide, ethyl hydroperoxide, cumene hydroperoxide, and t-butyl hydroperoxide are used as substrates. The heme-containing catalysts, methemoglobin and hematin, have identical hydroperoxide substrate requirements for singlet oxygen production. The hydroperoxide requirements for singlet oxygen production correlate with those reported by Dix et al. (Dix, T.A., Fontana, R., Panthani, A., and Marnett, L.J. (1985) J. Biol. Chem. 260, 5358-5365) for the production of peroxyl radicals in the hematin-catalyzed decomposition of hydroperoxides. The bimolecular reaction of peroxyl radicals is a plausible reaction mechanism for the singlet oxygen production in the systems studied.     

The oxidation of bilayer dispersions of unsaturated phosphatidylcholines by decomposing potassium peroxychromate

The oxidation of aqueous dispersions of unsaturated phosphatidylcholines by products released during the decomposition of potassium peroxychromate has been investigated. The rate and extent of oxidation have been measured by loss of unsaturated fatty acids and related to the rate of decomposition of peroxychromate as monitored by pH titrimetry and chromate analysis. The loss of oleic and linoleic acid from egg lecithin dispersions was similar in systems containing between 0.062 and 2 g peroxychromate and was limited to less than 50% of the total unsaturated residues of the substrate. Studies of the rate of oxidation suggested that the mechanism of reaction involved the progressive oxidation of the substrate dependent on the continuous supply of relatively short-lived oxidising species. The use of azide as a singlet oxygen quencher and 2,5-dimethyl- and 2,5-diphenylfurans as singlet oxygen traps did not prevent oxidation of the phospholipid.     

The effects of alpha-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.     

Lipid peroxidation induced by cercosporin as a possible determinant of its toxicity

The photodynamic action of cercosporin was assayed in various kinds of natural and artificial membranes. Cerosporin induces lipoperoxidation of liposomes, rat liver and pea internode mitochondria and microsomes, estimated both as malondialdehyde (MDA) formation and O2 consumption. Cercosporin-induced lipoperoxidation is inhibited by either singlet oxygen quenchers, free radical trapping agents or EDTA. Superoxide anion (O2-), hydrogen peroxide and hydroxyl radicals (.OH) are not involved in the activity of cercosporin. In addition cercosporin, by chelating iron, lowers the lipoperoxidation induced by such a metal. Therefore cercosporin stimulates, through singlet oxygen production, the hydroperoxide formation but, at the same time, it inhibits the continuation of the iron-mediated free radical chain. The present results suggest that cellular lipid peroxidation has a certain relevance to toxic activity of cercosporin.     

Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation

Chinese hamster ovary cells (CHO-K1) photosensitized with 12-(1'-pyrene)dodecanoic acid (P12) are killed when exposed to long wavelength ultraviolet (UV) light (greater than 300 nm). Mutants deficient in plasmalogen biosynthesis are hypersensitive to this treatment. We now demonstrate that plasmenylethanolamine is rapidly and preferentially destroyed when CHO-K1 cells, photosensitized either with P12 or merocyanine 540, are irradiated with light of the appropriate wavelength. Using [2-14C]ethanolamine, [1-14C]hexadecanol, or [U-14C]hexadecanol to follow the turnover of plasmenylethanolamine, we show that 2-monoacylglycerophosphoethanolamine, formic acid, and pentadecanal are formed during P12/UV treatment of CHO-K1 cells, but not of mutant cells deficient in plasmalogen synthesis. The decomposition of plasmenylethanolamine is O2-dependent, is enhanced in D2O, and is reduced in the presence of sodium azide. The process may be explained, in part, by the cycloaddition of singlet oxygen to the vinyl ether linkage of plasmenylethanolamine, generating a dioxetane intermediate that would be expected to decompose under physiological conditions to the observed products. An additional possibility is the formation of an allylic hydroperoxide at the 1'-carbon of the alkyl moiety by an "ene" reaction of singlet oxygen, or by radical-mediated oxidation, followed by metabolism or chemical decomposition of the hydroperoxide. Given the P12/UV hypersensitivity of plasmalogen-deficient mutants, we suggest that plasmalogens might protect animal cell membranes from singlet oxygen and/or radical-initiated oxidation by functioning as scavengers and decomposing to products that can be reutilized.     

A linoleic acid (8R)-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis. Biosynthesis of (8R)-hydroxylinoleic acid and (7S,8S)-dihydroxylinoleic acid from (8R)-hydroperoxylinoleic acid.

The fungus Gaeumannomyces graminis metabolized linoleic acid extensively to (8R)-hydroperoxylinoleic acid, (8R)-hydroxylinoleic acid, and threo-(7S,8S)-dihydroxylinoleic acid. When G. graminis was incubated with linoleic acid under an atmosphere of oxygen-18, the isotope was incorporated into (8R)-hydroxylinoleic acid and 7,8-dihydroxylinoleic acid. The two hydroxyls of the latter contained either two oxygen-18 or two oxygen-16 atoms, whereas a molecular species that contained both oxygen isotopes was formed in negligible amounts. Glutathione peroxidase inhibited the biosynthesis of 7,8-dihydroxylinoleic acid. These findings demonstrated that the diol was formed from (8R)-hydroperoxylinoleic acid by intramolecular hydroxylation at carbon 7, catalyzed by a hydroperoxide isomerase. The (8R)-dioxygenase appeared to metabolize substrates with a saturated carboxylic side chain and a 9Z-double bond. G. graminis also formed omega 2- and omega 3-hydroxy metabolites of the fatty acids. In addition, linoleic acid was converted to small amounts of nearly (65% R) racemic 10-hydroxy-8,12-octadecadienoic acid by incorporation of atmospheric oxygen. An unstable metabolite, 11-hydroxylinoleic acid, could also be isolated as well as (13R,13S)-hydroxy-(9E,9Z), (11E)-octadecadienoic acids and (9R,9S)-hydroxy-(10E), (12E,12Z)-octadecadienoic acids. In summary, G. graminis contains a prominent linoleic acid (8R)-dioxygenase, which differs from the lipoxygenase family of dioxygenases by catalyzing the formation of a hydroperoxide without affecting the double bonds of the substrate.     

Studies on hydroperoxide-dependent folic acid degradation by hemin

Hemin (ferric protoporphyrin IX chloride) in the presence of hydrogen peroxide or tert-butyl hydroperoxide was found to cleave folic acid at the C9-N10 bond. The ferrous form of hemin was not involved in hydroperoxide-dependent folic acid degradation, as indicated by the lack of inhibition by carbon monoxide. Molecular oxygen was not required for the degradation. GSH-Mn(II) or NAD(P)H in the presence of molecular oxygen did not support hemin-mediated folic acid degradation. The degradation increased as the temperature was elevated from 10 to 70 degrees C. Ascorbic acid and azide were potent inhibitors. Superoxide dismutase and hydroxyl radical quenchers, such as ethanol, mannitol, benzoate, and dimethyl sulfoxide did not inhibit the reaction. Catalase inhibited hydrogen peroxide-supported degradation but not the tert-butyl hydroperoxide-dependent one. Thiol compounds, such as thioglycolic acid, thiourea, glutathione, cysteine, and 2-mercaptoethanol, inhibited the hydrogen peroxide-dependent degradation but supported the tert-butyl hydroperoxide-mediated one. N5-formyl tetrahydrofolic acid, but not N10-formyl folic acid, was degraded by hemin in the presence of H2O2 or TBHP. The data obtained are suggestive of a mechanism similar to N-demethylation reactions catalyzed by cytochrome P-450 and some peroxidases.     

Ubiquinol and the papaverine derivative caroverine prevent the expression of tumour- promoting factors in adenoma and carcinoma colon cancer cells induced by dietary fat

High consumption of dietary fat promotes colon carcinogenesis. While this effect is well known the underlying mechanism is not understood. Fatty acid hydroperoxides (LOOH) arise from unsaturated fatty acids in the presence of oxygen and elevated temperature during food processing. An approach was made starting from the assumption that LOOH are present in dietary fats as a result of boiling. LOOH undergoes homolytic cleavage in the presence of iron. We studied their effects on gene expression in colorectal tumour cells using linoleic acid hydroperoxide (LOOH) as model compound. Addition to the medium of LT97 adenoma and SW480 carcinoma cells enhanced the production of hydrogen peroxide. Both cell lines were observed to increase VEGF and COX-II expression based on mRNA. Expression of VEGF was inhibited by caroverine and ubiquinon.     

Activation of Molecular Oxygen by Infrared Laser Radiation in Pigment-Free Aerobic Systems

With the goal of mimicking the mechanisms of the biological effects of low energy laser irradiation, we have shown that infrared low intensity laser radiation causes oxygenation of the chemical traps of singlet oxygen dissolved in organic media and water saturated by air at normal atmospheric pressure. The photooxygenation rate was directly proportional to the oxygen concentration and strongly inhibited by the singlet oxygen quenchers. The maximum of the photooxygenation action spectrum coincided with the maximum of the oxygen absorption band at 1270 nm. The data provide unambiguous evidence that photooxygenation is determined by the reactive singlet ¹_g state formed as a result of direct laser excitation of molecular oxygen. Hence, activation of oxygen caused by its direct photoexcitation may occur in natural systems.     

Activatin of guanylate cyclase during the oxidation of arylamine carcinogens.

When added alone, the arylamine procarcinogens N-acetyl-aminofluorene, 4-acetyl-aminobiphenyl or their N-hydroxy derivatives failed to alter partially purified soluble guanylate cyclase from rat liver or particulate guanylate cyclase activity from colonic mucosa. However, addition of linoleic acid hydroperoxide to the enzyme preparation in the presence N-OH-acetyl-aminofluorene or N-OH-acetyl-aminobiphenyl significantly increased guanylate cyclase activity. With linoleic acid hydroperoxide plus N-OH-acetyl-aminofluorene, both the activation of hepatic guanylate cyclase and the formation of the carcinogen oxidation product 2-nitrosofluorene required hematin but not molecular O₂. Both processes were inhibited by ascorbic acid. These data strongly imply that guanylate cyclase activation was dependent upon hematin catalyzed oxidation of N-OH-acetyl-aminofluorene by the lipid peroxide. The results provide the first evidence that guanylate cyclase activation can occur during the conversion of a procarcinogen to a more reactive chemical species, and thereby emphasize the importance of examining carcinogen interaction with the GC system under conditions which permit such chemical conversion.     

Formation of superoxide anion during ferrous ion-induced decomposition of linoleic acid hydroperoxide under aerobic conditions

We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2*-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2*-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O*)R2] is converted into the hydroxylcarbon radical [R1C*(OH)R2] in water, and that this carbon radical reacts with oxygen to give the alpha-hydroxyperoxyl radical [R1R2C(OH)OO*], which decomposes into the carbocation [R1C+(OH)R2] and O2*-.     

Singlet oxygen as a mediator in the hematoporphyrin-catalyzed photooxidation of NADPH to NADP+ in deuterium oxide.

The oxygen-dependent photooxidation of NADPH in the presence of hematoporphyrin in D2O results in the production of enzymatically active NADP+. The reaction is not inhibited by benzoate, mannitol, superoxide dismutase, or catalase. Moreover, addition of either potassium superoxide or H2O2 does not potentiate the reaction. This suggests OH-, H2O2, and O-2 are not likely to be the reactive oxygen species in this system. The oxidation is inhibited by various singlet oxygen quenchers and inhibitors such as 1,4-diazabicyclo[2.2.2]octane, 2,5-dimethylfuran plus methanol, histidine, and methionine. In addition, the rate of oxidation in H2O is less than one-fifth of that in D2O. The results suggest a singlet oxygen-mediated process. During the oxidation, no superoxide radical production could be detected with either ferricytochrome c or nitroblue tetrazolium. However, H2O2 has been found as one of the products. These observations are consistent with an oxidation-reduction reaction between singlet oxygen and NADPH to form H2O2 and NADP+, catalyzed by the light-activated photosensitizer hematoporphyrin.     

Induction of cytotoxicity and mutagenesis is facilitated by fatty acid hydroperoxidase activity in Chinese hamster lung fibroblasts (V79 cells)

The metabolic activation of benzo[a]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was studied in V79 Chinese hamster fibroblasts after supplementations with arachidonic acid or treatments with linoleic acid hydroperoxide. The extent of metabolic activation was estimated using cytotoxicity and mutagenesis as endpoints. Pretreatment of cells with arachidonic acid for 24 h resulted in significant elevations in the content of this fatty acid in cell phospholipids and increased prostaglandin synthesis. Arachidonic acid and linoleic acid hydroperoxide facilitated 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene cytotoxicity and mutagenesis, and to a lesser extent increased the cytotoxicity and mutagenicity of benzo[a]pyrene. No other compounds tested were mutagenic under these conditions, however, linoleic acid hydroperoxide markedly increased their cytotoxicity. Arachidonic acid-facilitated toxicity and mutagenesis was inhibited by indomethacin, whereas no inhibition was seen when linoleic acid hydroperoxide was used. Nordihyroquairaretic acid abolished the cytotoxicity and mutagenesis facilitated by arachidonic acid and linoleic acid hydroperoxide. Our findings demonstrate that induction of cytotoxicity and mutagenesis following treatment of V79 cells with carcinogens may be limited by low levels of arachidonic acid in these cells. A peroxidatic mechanism is proposed, with limited substrate specificity, for the metabolic activation of chemicals in V79 cells.     

Water-soluble inhibitor(s) of tumor respiration formed from ultraviolet-induced oxidation of linoleic and linolenic acids

Inhibition of Ehrlich ascites carcinoma respiration by aqueous extracts of oxidized linoleic or linolenic acid (aqueous emulsions UV-irradiated, 90 min) was associated entirely with relatively involatile compounds which were both thiobarbituric acid (TBA)-reactive and peroxidase-reactive. Inhibitory compounds were heat stable and migrated in thin-layer chromatography with aldehydes, "hydroperoxides," and TBA-reactive compounds. Peroxidase-catalyzed reduction of the "hydroperoxide" diminished the inhibition. At 4.7 x 10(-5) M "hydroperoxide" concentration, the residues from both linoleic and linolenic acid inhibited tumor oxygen consumption to a similar degree. However, at this concentration of "hydroperoxide" only the dried extract from linolenic acid was able to produce inhibition (100%) of aerobic glucose utilization by tumor cells. No glycolytic inhibition by the dried residue of oxidized linoleic acid was observed. At least 12 compounds (approximate chain length, 7C-13C) containing alpha,Beta-unsaturated carbonyl groups were isolated by gas-liquid chromatography (GLC) of dried extracts of oxidized linolenic acid. No single fraction inhibited tumor respiration, but the recombined mixture of all compounds caused complete respiratory inhibition of ascites tumor cells. Less material was required to inhibit oxygen consumption before than after GLC presumably because the more highly inhibitory components of the extract (along with "hydroperoxides" and TBA-reactive compounds) were lost during GLC. Extracts from oxidized linolenic acid were found to produce in all tumor cells cytoplasmic evaginations which were readily detected by phase microscopy.     

Singlet oxygen generation from the decomposition of alpha-linolenic acid hydroperoxide by cytochrome c and lactoperoxidase

Generation of singlet oxygen is first investigated in the decomposition of polyunsaturated lipid peroxide, alpha-linolenic acid hydroperoxide (LAOOH), by heme-proteins such as cytochrome c and lactoperoxidase. Chemiluminescence and electron spin resonance methods are used to confirm the singlet oxygen generation and quantify its yield. Decomposition products of LAOOH are characterized by HPLC-ESI-MS, which suggests that singlet oxygen is produced via the decomposition of a linear tetraoxide intermediate (Russell's mechanism). Free radicals formed in the decomposition are also identified by the electron spin resonance technique, and the results show that peroxyl, alkyl, and epoxyalkyl radicals are involved. The changes of cytochrome c and lactoperoxidase in the reaction are monitored by UV-visible spectroscopy, revealing the action of a monoelectronic and two-electronic oxidation for cytochrome c and lactoperoxidase, respectively. These results suggest that cytochrome c causes a homolytic reaction of LAOOH, generating alkoxyl radical and then peroxyl radical, which in turn releases singlet oxygen following the Russell mechanism, whereas lactoperoxidase leads to a heterolytic reaction of LAOOH, and the resulting ferryl porphyryl radical of lactoperoxidase abstracts the hydrogen atom from LAOOH to give peroxyl radical and then singlet oxygen. This observation would be important for a better understanding of the damage mechanism of cell membrane or lipoprotein by singlet oxygen and various radicals generated in the peroxidation and decomposition of lipids induced by heme-proteins.