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W Korytowski

Publications and source records attributed to W Korytowski.

36 records · Page 2Linked to original sources
PubMed1994

Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells.

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits < 0.5 pmol for cholesterol hydroperoxides and < 50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm2 (which reduced clonally assessed survival by 30%) showed 12-15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (< 12 min) were assigned to the following species by virtue of comigration with authentic standards: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxycholest-5-ene-7 alpha/7 beta-hydroperoxide (7 alpha/7 beta-OOH). Formation of 5 alpha-OOH and 6 beta-OOH (single oxygen adducts) was confirmed by subjecting [14C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18-22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18-22 min peak during Ca2+/phospholipase A2 treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts of PCOOH and ChOOH at a light fluence that clonally inactivated < 10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing.

Animals↗
PubMed1993

Analysis of cholesterol and phospholipid hydroperoxides by high-performance liquid chromatography with mercury drop electrochemical detection.

High-performance liquid chromatography (HPLC) with reductive mode electrochemical detection on a mercury drop has been employed for the separation and determination of lipid hydroperoxides. Under the conditions used, baseline separation is achieved for three cholesterol hydroperoxide (ChOOH) standards, not only from one another, but also from two different phosphatidylcholine hydroperoxide (PCOOH) standards. Applying this method to a test system, photodynamically treated murine leukemia cells, we have identified and quantified a major family of overlapping PCOOHs and three ChOOHs, two of which are characteristic singlet oxygen adducts. In a typical separation, the detection limit is < 0.5 pmol for ChOOHs and < 50 pmol for more slowly eluting PCOOHs. In this respect, mercury drop detection outperforms all previously described electrochemical detection methods for lipid hydroperoxides and compares favorably with other HPLC-based approaches. However, in terms of equipment cost, relative simplicity of operation, and fewer potential artifacts, this method has a clear advantage over all other existing high-sensitivity methods.

Animals↗
PubMed1992

Photoperoxidation of cholesterol in homogeneous solution, isolated membranes, and cells: comparison of the 5 alpha- and 6 beta-hydroperoxides as indicators of singlet oxygen intermediacy.

Singlet oxygen (1O2) can react with cholesterol (Ch) to give three possible ene-addition hydroperoxides: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), and 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH). The rates of dye-sensitized photogeneration and also the fates of 5 alpha-OOH and 6 beta-OOH in membrane bilayers have been studied and compared. Irradiation of unilamellar [14C]Ch/phospholipid vesicles in the presence of aluminum phthalocyanine tetrasulfonate or merocyanine 540 resulted in formation of 5 alpha-OOH and 6 beta-OOH, as determined by high performance liquid chromatography with radiochemical or electrochemical detection. The initial rate of 6 beta-OOH formation was 30-35% that of 5 alpha-OOH in a variety of liposomal systems. However, after a lag, 5 alpha-OOH invariably decayed via allylic rearrangement to 7 alpha-OOH (also known to be a free radical product), whereas 6 beta-OOH accumulated in unabated fashion until Ch depletion became limiting. Photooxidation of Ch in an isolated natural membrane (erythrocyte ghost) or in L1210 leukemia cells gave similar results. When the reaction was carried out in pyridine or methanol, the rate of 6 beta-OOH formation relative to 5 alpha-OOH was reduced by approximately half, with essentially no isomerization of the latter to 7 alpha-OOH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Chromatographic separation and electrochemical determination of cholesterol hydroperoxides generated by photodynamic action.

Reverse-phase HPLC with electrochemical detection (HPLC-EC) was used to separate and quantitate photochemically generated cholesterol hydroperoxides. The EC measurements were performed in the reduction mode under anaerobic conditions. When cholesterol-containing liposomes were irradiated in the presence of a phthalocyanine dye, at least four major oxidation products of cholesterol were detected by HPLC-EC:5 alpha-hydroperoxide (5 alpha-OOH), 6 beta-hydroperoxide (6 beta-OOH), 7 alpha-hydroperoxide (7 alpha-OOH), and 7 beta-hydroperoxide (7 beta-OOH). The detection limit for each compound was found to be approximately 25 pmol. Product identification was based on matching HPLC and TLC behavior of standards and on physical indicators (melting points and NMR chemical shifts). The cholesterol hydroperoxides were barely separated from EC-silent diol derivatives, which could be detected by 210 nm absorbance after reduction of the hydroperoxides with triphenylphosphine. Irradiation of a dye-sensitized natural membrane, the human erythrocyte ghost, also resulted in formation of 5 alpha-OOH, 6-OOH, and 7-OOH, as evidenced by HPLC-EC. Under the chromatographic conditions used, these species were well separated not only from one another but also from a family of at least six phospholipid hydroperoxides. These results illustrate the strengths of HPLC-EC as a relatively convenient, sensitive, and selective means of analyzing cholesterol hydroperoxides in biological samples.

Cholesterol↗
PubMed1990

Bleaching of melanin pigments. Role of copper ions and hydrogen peroxide in autooxidation and photooxidation of synthetic dopa-melanin.

Bleaching of eumelanin has been studied in model systems consisting of synthetic dopa-melanin and various concentrations of hydrogen peroxide, molecular oxygen, and copper(II) ions at neutral and alkaline pH. The data show that at neutral pH, in the dark, metal-ion-free melanin is very resistant to oxidation by hydrogen peroxide. However, the rate of bleaching of melanin, induced by H2O2 is significantly accelerated by illumination from UVA-visible light. Bound-to-melanin copper(II) also accelerates the bleaching of melanin with the efficiency dependent on concentration of H2O2 and oxygen. It suggests possible involvement of melanin-copper complexes in Fenton-like processes. The formation of hydroxyl radicals during melanin bleaching has been concluded on the basis of the electrochemical detection of hydroxylation products of salicylate used as OH scavenger. Redox conversion of bound-to-melanin copper ions was monitored by EPR spectroscopy and direct measurement of melanin-Cu(II) complexes. It has been shown that melanin-copper(I) complexes were readily oxidized by either oxygen or hydrogen peroxide. The data indicate that bleaching of melanin is a complex process with two distinct stages, reversible oxidation of the hydroquinone moieties of melanin followed by irreversible reactions of the monomers that lead to degradation of the melanin polymer.

Copper↗
PubMed1990

Interactions of flavins with melanin. Studies on equilibrium binding of riboflavin to dopa-melanin and some spectroscopic characteristics of flavin-melanin complex.

Natural melanins are photoprotective pigments that in mammals are principally found in the skin, hair, and eyes. Although the molecular mechanism of photoprotection of pigmented cells has not yet been established, several hypotheses have been proposed with melanin acting as a light filter, free radical scavenger, and quencher of electronically excited states of reactive intermediates. It can be expected that the detoxicating efficiency of melanin should be enhanced if the melanin and potentially cytotoxic species are brought close together. Such a situation may occur for a number of photosensitizing dyes that have the ability to bind to melanin. The interaction of melanin with flavins has been studied under strictly controlled experimental conditions. The equilibrium dialysis method has been employed to determine dissociation constants and the number of binding sites in melanin at pH 5-9. The data reveal that synthetic DOPA-melanin has two different classes of binding sites with dissociation constants of 10(-6) and 10(-5) M, respectively. The overall binding capacity of melanin, at pH 7, is 250 nmol RF/mg melanin. The amount of bound-to-melanin RF increases with pH. The absorption spectra of melanin complexes with RF and lumiflavin indicate that hydrophobic interaction may be involved in the binding of these flavins by melanin. No changes in flavin fluorescence have been detected after binding of flavin to melanin. It appears that, contrary to cationic photosensitizing dyes, the singlet excited state of flavin molecules is not quenched by melanin.

Binding Sites↗
PubMed1988

Oxygen activation during the interaction between MPTP metabolites and synthetic neuromelanin--an ESR-spin trapping, optical, and oxidase electrode study.

Oxidase electrode measurements as well as optical and electron spin resonance spectroscopic data have shown that synthetic neuromelanin oxidizes the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium in a dose-dependent manner forming 1-methyl-4-phenylpyridinium and hydrogen peroxide. Hydroxyl radicals are formed in this reaction which is promoted by iron chelates. In contrast, neither 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nor 1-methyl-4-phenylpyridinium reacts with synthetic neuromelanin in a similar fashion. The mechanism of selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in pigmented neuronal cells is discussed in the light of these findings.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1988

Reaction between ortho-semiquinones and oxygen: pulse radiolysis, electron spin resonance, and oxygen uptake studies.

The cytotoxicity to tumor cells or cardiotoxic side effects of certain para-quinone antitumor drugs have been attributed to the corresponding semiquinones and derived superoxide and hydroxyl radicals. It has also been suggested that ortho-semiquinones, including those that arise during melanogenesis, produced via either the one-electron oxidation of catechol(amine)s or the one-electron reduction of the corresponding quinones, react with molecular oxygen to give superoxide and hydrogen peroxide. Furthermore it has been shown that catechol(amine)s which form noncyclizable quinones are more cytotoxic toward melanogenic cells than those forming cyclizable quinones. In order to provide further kinetic information on the interaction of oxygen with ortho-semiquinones, using pulse radiolysis we directly measured the rates of reaction of various ortho-semiquinones with molecular oxygen. The semiquinones of the corresponding catechol(amine)s were also produced by the horseradish peroxidase/hydrogen peroxide system, and detected by electron spin resonance spectroscopy using the spin stabilization method. Oxygen consumption was monitored using a standard Clark oxygen electrode. Our data indicate that while ortho-semiquinones from catechol(amine)s and catechol estrogens do not react with molecular oxygen at a rate equal to or greater than k less than or equal to 10(5) M-1 s-1, semiquinones from hydroxy-substituted catechol(amine)s react with dioxygen with rates in the range k = 10(6)-10(7) M-1 s-1.

Antineoplastic Agents↗
PubMed1987

Evidence for the one-electron oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+).

Optical data have shown that the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium undergoes one-electron oxidation/reduction in the presence of iron chelates. The activation energy for one-electron oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium is less than that for two-electron oxidation. Horseradish peroxidase catalyzes the oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium. Reactivity of 1-methyl-4-phenylpyridinyl radical is discussed in relation to the well-known pyridinyl radicals.

Free Radicals↗
PubMed1987

Tyrosinase-catalyzed oxidation of dopa and related catechol(amine)s: a kinetic electron spin resonance investigation using spin-stabilization and spin label oximetry.

The oxidation of four catechol(amine)s by tyrosinase has been studied by electron spin resonance and optical methods. Rates of oxygen consumption and of dopaquinone and dopachrome formation during the oxidation of dopa have been measured, and compared with rates of dopasemiquinone production measured using spin-stabilization procedures. In the presence of spin-stabilizing metal ions, production of semiquinone is approximately quantitative. Time-dependent ESR spectra obtained from dopa and dopamine show a slow regeneration of semiquinone, suggesting that a semiquinone precursor is slowly reformed. In contrast, time-dependent spectra for 4-methylcatechol and N-acetyldopamine show decay of the primary semiquinone together with buildup of a secondary semiquinone apparently derived from the corresponding 6-hydroxy-catechol(amine). Thus, catecholamines that give rise to a cyclizable quinone show a pattern of behavior that differs from those that produce a non-cyclizable quinone. These results are discussed in terms of their possible significance to melanogenesis and the toxicity of catechol(amine)s, which has been attributed to production of semiquinones and/or other oxygen radicals.

Benzoquinones↗
PubMed1987

Mechanism of oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+).

Oxidase electrode measurements have shown that the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium autoxidizes to hydrogen peroxide and 1-methyl-4-phenylpyridinium in a reaction promoted by iron chelates. The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity is discussed in the light of these findings.

Deferoxamine↗
PubMed1986

Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies.

Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin.

Cations, Divalent↗
PubMed1985

Mechanism of dismutation of superoxide produced during autoxidation of melanin pigments.

The hydrogen peroxide produced during the autoxidation of melanin pigments has been measured using an oxidase electrode. The autoxidation has been shown to occur via the superoxide intermediate. The melanin pigment competes with superoxide dismutase for the scavenging of superoxide radicals. However, superoxide dismutase at high concentrations caused a substantial increase in the production of hydrogen peroxide, formed during melanin autoxidation. The implications of this finding are discussed in light of melanin's ability to function as a pseudo-dismutase.

Animals↗
PubMed1985

Transverse motion of spin-labeled 3,3',5-triiodo-L-thyronine in phospholipid bilayers.

Using electron spin resonance stop-flow technique, the transverse motion (flip-flop) of 3-([alpha-carboxy-4-(4-hydroxy-3-iodophenoxy)-3,5- diiodophenethyl]carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolin (T3-SL) in dipalmitoyl L-alpha-phosphosphatidylcholine (DPPC) membranes was evaluated. At 22 degrees C, the electron spin resonance spectra of T3-SL in DPPC vesicles were compared before and after the addition of sodium ascorbate, a membrane impermeable reducing agent. The addition of ascorbate reduces the signal amplitude by 67% in 3 min but yields no further reduction for at least 60 min. These results indicate that T3-SL does not flip-flop at any appreciable rate in the membranes. This finding suggests that once partitioned into the membrane, T3 remains in the outer half of the lipid bilayer, thus reducing the possibility that T3 enters the cell by passive diffusion.

Ascorbic Acid↗
PubMed1985

Nitroxides as redox probes of melanins: dark-induced and photoinduced changes in redox equilibria.

The interaction of nitroxide free radicals and their reduced products (hydroxylamines) with synthetic and natural melanins has been studied. Electron spin resonance spectroscopy was used to measure changes in radical concentration in the dark and during irradiation with visible or uv light. Some reduction of nitroxide occurs in the dark, and is reversible: the nitroxide can be completely regenerated by the one-electron oxidant ferricyanide. The kinetics of the process depend strongly on radical charge and pH. For positively charged nitroxides the rate is much faster than for either neutral or anionic radicals. At pH 10 the rate is about 20 times faster than at pH 5. Oxidation of hydroxylamine also can occur so that a redox equilibrium is established. The equilibrium constant has been estimated for the reaction between a nitroxide and melanin from autoxidation of 3,4-dihydroxyphenylalanine. Results are also dependent upon the type of melanin used and chemical modification (oxidation or reduction) of the melanin. Redox equilibria are altered during irradiation with either visible or uv light. Rapid oxidation of hydroxylamine to nitroxide is apparent, together with a slower reduction of nitroxide. Action spectra for these processes are related to those for melanin radical production and oxygen consumption in nitroxide-free melanin systems. Reduction of nitroxide is inhibited by oxygen, suggesting a competition between nitroxide and oxygen for photoinduced reducing equivalents.

Darkness↗