Inhibition of 3,3",5,5"-Tetramethylbenzidine and o-Phenylenediamine Peroxidation with 1-Amino-2-Naphtol-4-Sulfonic Acid and Its Polydisulfide

The kinetics of coupled peroxidation of 3,3",5,5"-tetramethylbenzidine (TMB) and 1-amino-2-naphtol-4-sulfonic acid (ANSA) or its polydisulfide (poly(ADSNSA)) was studied in a 0.01 M phosphate buffer (pH 6.4) at 20°C. Both ANSA and poly(ADSNSA) strongly inhibited the TMB oxidation resulting in a marked delay in the product formation. Stoichiometric inhibition coefficients f, i. e., the average numbers of free-radical particles terminated by one inhibitor molecule, were estimated. The free-radical trapping effect of poly(ADSNSA) was 7.5 times greater than that of ANSA. Kinetics of coupled o-phenylenediamine (PhDA) and ANSA or poly(ADSNSA) oxidation was studied in phosphate–citrate buffers at pH 3 to 7. No lag periods in oxidation product accumulation were observed under any of the reaction conditions. A weak activation of PhDA conversion depending on pH and PhDA/ANSA ratios was observed at low ANSA concentrations, whereas increased ANSA or poly(ADSNSA) concentrations were inhibitory. The degree of PhDA-inhibition was maximal in acid media, reached minimum at pH 5 to 6, and than again increased at pH above 6. A tentative mechanism of coupled aromatic amine–phenol bi-substrate system peroxidation is discussed.     

Influence of pH on the reactivity of diphenyl ditelluride with thiols and anti-oxidant potential in rat brain

Thiol oxidation by diphenyl ditelluride is a favorable reaction and may be responsible for alteration in regulatory or signaling pathways. We have measured rate constants for reactions of diphenyl ditelluride with cysteine, dimercaptosuccinic acid, glutathione and dithiothreitol in phosphate buffer. The relative reactivities of the different thiols with diphenyl ditelluride were independent of the pK_a of the thiol group, such that at pH 7.4, cysteine and dithiothreitol were the most reactive and low reactivity was observed with glutathione and dimercaptosuccinic acid. The reactivity of diphenyl ditelluride was not modified by change in pH. Rate of oxidation increased with increasing pH for all thiols except dimercaptosuccinic acid, where the rate of oxidation was faster at low pH. The lipid peroxidation product malonaldehyde (MDA) was measured in rat brain homogenate and phospholipids extract from egg yolk after incubation in phosphate buffer at various pHs ranging from 7.4 to 5.4. TBARS production increased when homogenates were incubated in the pH (5.4-6.8) medium both in the absence and presence of Fe(II). These data indicate that lipid peroxidation processes, mediated by iron, are enhanced with decreasing pH. The iron mobilization may come from reserves where it is weakly bound. Diphenyl ditelluride significantly protected TBARS production at all studied pH values in a concentration dependent manner in brain homogenate. This study provides in vitro evidence for acidosis induced oxidative stress and anti-oxidant action of diphenyl ditelluride.     

Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins

By application of pulse radiolysis it was demonstrated that nitrogen dioxide (NO2.) oxidizes Gly-Tyr in aqueous solution with a strongly pH-dependent rate constant (k6 = 3.2 X 10(5) M-1 S-1 at pH 7.5 and k6 = 2.0 X 10(7) M-1 S-1 at pH 11.3), primarily generating phenoxyl radicals. The phenoxyl can react further with NO2. (k7 approximately 3 X 10(9) M-1 S-1) to form nitrotyrosine, which is the predominant final product in neutral solution and at low tyrosyl concentrations under gamma-radiolysis conditions. Tyrosine nitration is less efficient in acidic solution, due to the natural disproportionation of NO2., and in alkaline solutions and at high tyrosyl concentrations due to enhanced tyrosyl dimerization. Selective tyrosine nitration by interaction of NO2. with proteins (at pH 7 to 9) was demonstrated in the case of histone, lysozyme, ribonuclease A, and subtilisin Carlsberg. Nitrotyrosine developed slowly also under incubation of Gly-Tyr with nitrite at pH 4 to 5, where NO2. is formed by acid decomposition of HONO. It is recalled in this context that NO2.-induced oxidations, by regenerating NO2-, can propagate NO2./NO2- redox cycling under acidic conditions. Even faster than with tyrosine is the NO2.-induced oxidation of cysteine-thiolate (k9 = 2.4 X 10(8) M-1 S-1 at pH 9.2), involving the transient formation of cystinyl radical anions. The interaction of NO2. with Gly-Trp was comparably slow (k approximately 10(6) M-1 S-1), and no reaction was detectable by pulse radiolysis with Met-Gly and (Cys-Gly)2, or with DNA. Slow reactions of NO2. were observed with arachidonic acid (k approximately 10(6) M-1 S-1 at pH 9.0) and with linoleate (k approximately 2 X 10(5) M-1 S-1 at pH 9.4), indicating that NO2. is capable of initiating lipid peroxidation even in an aqueous environment. NO2.-Induced tyrosine nitration, using 50 microM Gly-Tyr at pH 8.2, was hardly inhibited, however, in the presence of 1 mM linoleate, and was not affected at all in the presence of 5 mM dimethylamine (a nitrosamine precursor). It is concluded that protein modifications, and particularly phenol and thiol oxidation, may be an important mechanism, as well as initiation of lipid peroxidation, of action of NO2. in biological systems.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC0.08) or high (PC0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38 degrees C in buffer-containing (control) liposomes prepared from PC0.08, whereas it was slightly increased in those prepared from PC0.66. Encapsulated hemolysate had no effect in PC0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.     

A method for obtaining linear reciprocal plots with caeruloplasmin and its application in a study of the kinetic parameters of caeruloplasmin substrates

1. The curved plots of 1/v against 1/[S] obtained when caeruloplasmin oxidizes NN-dimethyl-p-phenylenediamine were investigated. The first free-radical oxidation product of caeruloplasmin oxidation of NN-dimethyl-p-phenylenediamine is required for curvature, as straight-line plots were obtained when activities were measured either before appreciable free-radical product had appeared or in the presence of ascorbate, which reduced it back to NN-dimethyl-p-phenylenediamine. 2. In the presence of ascorbate linear reciprocal-plots were obtained with all of the 37 substrates tested. V(max.) values varied over only an eightfold range and those for the 20 p-amino compounds over only a twofold range. K(m) values, however, varied over a 10(4)-fold range. The small range of V(max.) values indicates that the rate-limiting step in caeruloplasmin action is relatively independent of the nature of the substrate. K(m) values suggest that substrates bind primarily by ring electrons, although certain side-chain groups increased the K(m) in a manner unrelated to likely changes of ring-electron densities. A mechanism involving repulsion between negative charges on the substrate and the enzyme was supported by the variation of the K(m) of 5-hydroxyindol-3-ylacetic acid with pH.     

Ascorbic acid and copper in linoleate oxidation. 3. Catalysts in combination

In promoting oxidation of 0.02 m potassium linoleate in a buffered (pH 7.0) aqueous dispersion at 37 degrees C, ascorbic acid at low concentrations (1.8 x 10(-6) and 1.8 x 10(-5) m) in combination with copper (1.3 x 10(-7) to 1.3 x 10(-3) m) had greater catalytic activity than the additive activity of the two catalysts individually. Possible explanations for the enhanced catalysis include reduction of copper by ascorbic acid to the cuprous form, increased concentration of semidehydroascorbic acid radical, and formation of a metal-ascorbic acid-oxygen complex. Some combinations of ascorbic acid (1.8 x 10(-4) and 1.8 x 10(-3) m) and copper (1.3 x 10(-6) and 1.3 x 10(-3) m) inhibited the formation of conjugated dienes but not the oxidation of ascorbic acid, and caused rapid loss of part of the conjugated dienes that were already present. It is suggested that free-radical inhibitors formed by the combination of catalysts inhibit initiation of lipid oxidation but not copper-catalyzed oxidation of ascorbic acid. Effects of the inhibitory combinations on changes in UV absorption by conjugated dienes, and absorbance in the TBA test, indicate the presence of at least two conjugated dienes that differ in stability.     

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.     

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.     

Peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of 2,4-dinitrosoresorcinol and polydisulfide derivatives of resorcinol and 2,4-dinitrosoresorcinol

A comparative study of the kinetics of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of 2,4-dinitrosoresorcinol (DNR), its polydisulfide derivative [poly(DNRDS)], and resorcinol polydisulfide [poly(RDS)], substances that competitively inhibit the formation of TMB conversion product, was carried out. The inhibition constants, Ki for DNR, poly(DNRDS), and poly(RSD) were determined at 20 degrees C and pH 6.4 to be 110, 13.5, and 0.78 microM, respectively. The stoichiometric coefficients of inhibition were calculated to be 0.38 and 76 for poly(DNRDS) and poly(RDS), respectively. In the pH range 6.4-7.0, the initial rates of the peroxidative oxidation of TMB, and its mixtures with DNR and poly(DNRDS) and the Ki value for poly(RDS) substantially decreased with increasing pH. The kinetic parameters of poly(RDS) (Ki 0.22-0.78 microM and f76) suggest that it is the most efficient inhibitor of peroxidase oxidation of TMB: in micromolar concentrations, it completely stops this process and can be used in EIA.     

A comparison of the kinetics of low-density lipoprotein oxidation induced by copper or by gamma-rays: influence of radiation dose-rate and copper concentration

The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis. The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or by oxygen free radicals generated by 60Co gamma-rays. The effects of copper concentration and irradiation dose-rate on LDL peroxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene, hydroperoxides, and thiobarbituric acid reactive substance formation as well as alpha-tocopherol disappearance. In the case of gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiation dose-rate. The radiation chemical rates (nu) increased with increasing dose-rate. Copper-induced LDL peroxidation followed two kinetic patterns: a slow kinetic for copper concentrations between 5-20 microM, and a fast kinetic for a copper concentration of 40 microM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copper binding site on apo-B. When compared with gamma-rays, copper ions act as drastic and powerful oxidants only at higher concentrations (> or = 40 microM).     

Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.     

Echinochrome, a naturally occurring iron chelator and free radical scavenger in artificial and natural membrane systems

Echinochrome, or 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone, possesses cardioprotective activity, and diminishes the myocardial ischemia/reperfusion injury that is known to be accompanied by free-radical oxidative damage and calcium overload. In this study, we investigated the lipophilicity of echinochrome, its ability to inhibit free-radical oxidation both in the bulk organic phase and in an artificial membrane system (liposomes), and to prevent the ferrous/ascorbate-induced leakage of calcium from the isolated sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The experimentally-determined octanol/water partition coefficient (LogP) of echinochrome was +3.11, and the distribution coefficient (LogD) was +2.58 at pH 6.0 and -0.15 at pH 8.0. Echinochrome displayed high scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals with a stoichiometry of about 1:7. Echinochrome was more effective in inhibiting the phosphatidyl choline liposome peroxidation induced by Fe2+/ascorbate than that induced by hemin. The iron chelating ability of echinochrome was estimated spectrophotometrically. In isolated SR, echinochrome protected the ATP-dependent Ca2+-pump system from damage by Fe2+/ascorbate. It was concluded that iron chelation predominates in the overall antioxidant potential of echinochrome.     

Evaluation of the antioxidant properties of thyroid hormones and propylthiouracil in the brain-homogenate autoxidation system and in the free radical-mediated oxidation of erythrocyte membranes

The antioxidant capacity of thyroid hormones and the antithyroid drug propylthiouracil was studied in three model systems, namely, autoxidation of rat brain homogenates and oxidation of rat erythrocyte plasma membranes (EPM) induced by either 2,2'-azobis-(2-amidinopropane) (AAP) thermolysis or by gamma irradiation. Thyroid hormones significantly inhibited the development of lipid peroxidation in these systems at micromolar concentrations, as assessed either by visible light emission, thiobarbituric acid reactive substances accumulation or oxygen uptake. This behaviour was not observed when L-3,3',5-triiodothyronine (T3) and L-thyroxine (T4) were assayed at nanomolar concentrations. In EPM exposed to AAP or gamma irradiation, propylthiouracil inhibited the induced lipid peroxidation, with Q1/2 values of 112-150 microM. It is concluded that the antioxidant capacity of thyroid hormones found in vitro may not be of relevance in physiological conditions, which exhibit variations of T3 and T4 levels in the nanomolar range. On the other hand, the behaviour of propylthiouracil as an inhibitor of EPM lipid peroxidation is observed at concentrations close to the therapeutic levels, thus representing a possible complementary action to its antithyroid activity.     

In vitro effects of alloxan/copper combinations on lipid peroxidation, protein oxidation and antioxidant enzymes

The in vitro effects of alloxan and the product of its reduction dialuric acid (alone or in combination with copper ions) on lipid peroxidation, carbonyl content, GSH level and antioxidant enzyme activities in rat liver and kidney have been studied. The effects of Cu2+/alloxan and Cu2+/dialuric acid were compared with those of Fe3+/alloxan and Fe3+/dialuric acid. Unlike alloxan, dialuric acid increased liver and kidney lipid peroxidation; similar effects were registered in the presence of Fe3+. In the presence of Cu2+/dialuric acid, the lipid peroxidation was strongly inhibited and vice versa--the liver protein oxidation was increased. Alloxan and dialuric acid, as well as their combinations with Fe3+ had no effect on the total GSH level. Both substances did not affect the Cu2+-induced changes in GSH level, glucose-6-phosphate dehydrogenase and gluthatione reductase activities. In contrast, Cu2+ had no effect on dialuric-acid induced changes in gluthatione peroxidase and superoxide dismutase activities. The present in vitro results, concerning the metal dependence of the effects of alloxan and dialuric acid, are a premise for in vivo study of alloxan effects in metal-loaded animals.     

NADPH-dependent lipid peroxidation capacity in unfixed tissue sections: characterization of the pro-oxidizing conditions and optimization of the histochemical detection

Summary Factors which influence the iron-stimulated lipid peroxidation in rat liver have been studied by incubating unfixed cryostat sections with a pro-oxidant system and using an optimized histochemical detection method for lipid peroxidation products with 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B. We used a method that was slightly different from the one described previously. The final reaction product was exclusively localized in the cytoplasm of liver parenchymal cells with a homogeneous distribution within the liver lobule. The absorbance maximum, as measured cytophotometrically, was found to be 550 nm. Maximum lipid peroxidation was observed when the pro-oxidant system contained 0.2 mm NADPH, 1 mm ADP and 15 μm FeCl₂. Some reaction product was found when NADPH was omitted. Iron concentrations higher than 180 μm prevented the formation of lipid peroxidation products in certain areas of the sections, whereas ADP concentrations higher than 1 mm inhibited the reaction in the whole section. A pH dependency was also observed, with the highest lipid peroxidation at pH 7.2. Optimum lipid peroxidation was induced by incubating for 30 min at 37°C with the pro-oxidant system. A linear relationship was found between the thickness of the sections (up to 20 μm) and the amount of lipid peroxidation products. The addition of scavengers of O2- (superoxide dismutase), hydrogen peroxide (catalase) and OH · (mannitol) to the first step medium did not affect the amount of final reaction product. These findings appear to confirm the hypothesis proposed for events occurring in isolated microsomes, leading to the formation of hydroperoxides and ultimately lipid peroxidation-derived carbonyls. The present method is a useful tool for studying the capacity of lipid peroxidation in tissues under different (patho)physiological conditions.     

Stimulation and inhibition of iron-dependent lipid peroxidation by desferrioxamine

Peroxidation of rat brain synaptosomes was assessed by the formation of thiobarbituric acid reactive products in either 50 mM potassium phosphate buffer (pH 7.4) or pH adjusted saline. In phosphate, addition of Fe2+ resulted in a dose-related increase in lipid peroxidation. In saline, stimulation of lipid peroxidation by Fe2+ was maximal at 30 uM, and was less at concentrations of 100 uM and above. Whereas desferrioxamine caused a dose-related inhibition of iron-dependent lipid peroxidation in phosphate, it stimulated lipid peroxidation with Fe2+ by as much as 7-fold in saline. The effects of desferrioxamine depended upon the oxidation state of iron, and the concentration of desferrioxamine and lipid. The results suggest that lipid and desferrioxamine compete for available iron. The data are consistent with the hypothesis that either phosphate or desferrioxamine may stimulate iron-dependent lipid peroxidation under certain circumstances by favoring formation of Fe2+/Fe3+ ratios.     

Copper-induced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we have studied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes (small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were prepared by sonication and exposed to CuCl(2) in the absence or presence of naturally occurring reductants (ascorbic acid (AA) and/or alpha-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). The resultant oxidation process was monitored by recording the time-dependence of the absorbance at several wavelengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively high copper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentrations. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introduced into the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions, ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol. Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to the high oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biological significance of these results has yet to be evaluated.     

Kinetic analysis of copper-induced peroxidation of HDL, autoaccelerated and tocopherol-mediated peroxidation

Comparison of the kinetic profiles of copper-induced peroxidation of HDL and LDL at different copper concentrations reveals that under all the studied experimental conditions HDL is more susceptible to oxidation than LDL. The mechanism responsible for HDL oxidation is a complex function of the copper/HDL ratio and of the tocopherol content of the HDL. At high copper concentrations, the kinetic profiles were similar to those observed for LDL oxidation, namely, relatively rapid accumulation of oxidation products, via an autoaccelerated, noninhibited mechanism, was preceded by an initial "lag phase." Under these conditions, the maximal peroxidation rate (V(max)) of HDL and LDL depended similarly on the molar ratio of bound copper/lipoprotein. Analysis of this dependency in terms of the binding characteristics of copper to lipoprotein, yielded similar dissociation constant (K = 10(-6) M) but different maximal binding capacities for the two lipoproteins (8 Cu(+2)/HDL as compared to 17 Cu(+2)/LDL). Given the size difference between HDL and LDL, these results imply that the maximal surface density of bound copper is at least 2-fold higher for HDL than for LDL. This difference may be responsible for the higher susceptibility of HDL to copper-induced oxidation in the presence of high copper concentrations. At relatively low copper concentrations, the kinetic profile of HDL oxidation was biphasic, similar to but more pronounced than the biphasic kinetics observed for the oxidation of LDL lipids at the same concentration of copper. Our results are consistent with the hypothesis that the first phase of rapid oxidation occurs via a tocopherol-mediated-peroxidation (TMP) mechanism. Accordingly, enrichment of HDL with tocopherol resulted in enhanced accumulation of hydroperoxides during the first phase of copper-induced oxidation. Notably, the maximal accumulation during the first phase decreased upon increasing the ratio of bound copper/HDL. This behavior can be predicted theoretically for peroxidation via a TMP mechanism, in opposition to autoaccelerated peroxidation. The possible pathophysiological significance of these findings is discussed.     

Metmyoglobin promotes arachidonic acid peroxidation at acid pH

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.