Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase

The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.     

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.     

Detection of Picomole Levels in Lipid Hydroperoxides by a Chemiluminescence Assay

A highly sensitive and simple chemiluminescent method for the quantitation of lipid hydroperoxides at the picomole level is described. The method is based on detecting the chemiluminescence generated during the oxidation of luminol by the reaction with hydroperoxide and cytochrome c under mild conditions. A semilogarithmic relationship was observed between the hydroperoxide added and the chemiluminescence produced. For lipid hydroperoxides, cytochrome c was a most favorable catalyst for generating the chemiluminescence, rather than cytochrome c heme peptide and horseradish peroxidase. This method had high sensitivity to methyl linoleate hydroperoxide, arachidonic acid hydroperoxide and cholesterol hydroperoxide, but low to /-butyl hydroperoxide, J-butyl perbenzoate, diacyl peroxides (lauroyl peroxode and benzoyl peroxide) and dialkyl peroxides (di-/-butyl peroxide and dicumyl peroxide).     

The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress.

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human alpha-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (kcat/Km) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s-1 mM-1, respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s-1 mM-1, respectively. Immunotitration studies with human liver extracts showed that the antibodies against human alpha-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the alpha-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetraenoic acid, an important intermediate in the 5-lipoxygenase pathway.     

Spectrophotometric measurement of hydroperoxides at increased sensitivity by oxidation of Fe2+ in the presence of xylenol orange

The method, developed by modifying the FOX methods described by Wolff (Methods Enzymol. 233, 182-189, 1994), involves the oxidation of Fe²⁺ by peroxides at low pH in the presence of both the ferric-complexing dye xylenol orange and sucrose, the amplifier of the reaction. The method proved to be a convenient, simple and efficient assay for the direct measurement of both water and lipid soluble peroxides. In fact it improves by about 60% the sensitivity of the FOX1 method for water soluble peroxides, and by 7-8 times that of the FOX2 method for lipid soluble peroxides. It allows the detection of 0.1 μM peroxide in the test solution. The method is suitable to measure the lipid hydroperoxides present in phosphatidylcholine liposomes and in human LDL. The data obtained allowed us to define a mathematical expression to calculate the lipid hydroperoxide content of liposomes knowing their oxidation index.     

Hydroperoxide reduction by thioredoxin-specific glutathione peroxidase isoenzymes of Arabidopsis thaliana

Arabidopsis thaliana contains eight glutathione peroxidase (GPX) homologs (AtGPX1-8). Four mature GPX isoenzymes with different subcellular distributions, AtGPX1, -2, -5 and -6, were overexpressed in Escherichia coli and characterized. Interestingly, these recombinant proteins were able to reduce H2O2, cumene hydroperoxide, phosphatidylcholine and linoleic acid hydroperoxides using thioredoxin but not glutathione or NADPH as an electron donor. The reduction activities of the recombinant proteins with H2O2 were 2-7 times higher than those with cumene hydroperoxide. Km values for thioredoxin and H2O2 were 2.2-4.0 and 14.0-25.4 microM, respectively. These finding suggest that GPX isoenzymes may function to detoxify H2O2 and organic hydroperoxides using thioredoxin in vivo and may also be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP balance.     

Iodometric measurement of lipid hydroperoxides in human plasma

Many assay techniques have been used to measure lipid hydroperoxides in plasma, including absorbance of conjugated dienes and reactivity with thiobarbituric acid. Because these measurements are not specific for lipid hydroperoxides, we modified an exisiting iodometric method to correct for interfering phenomena and to provide a more specific measurement of the lipid hydroperoxide content of plasma. To ensure reproducible extraction of hydroperoxides from the many possible forms in plasma, the plasma was treated to hydrolyze enzymatically cholesterol ester, triglycerides, and phospholipids, and the nonesterified fatty acid peroxides were then extracted with ethyl acetate. Extracted lipids were reacted with potassium iodide in acetic acid and methylene chloride, and the resulting triiodide ion (I3-) was measured spectrophotometrically. Correction for nonoxidizing chromophores was made after back-titration of the triiodide ion to iodide with sodium thiosulfate and other non-peroxide oxidants were estimated by their resistance to reduction with glutathione peroxidase. Recovery of added hydroperoxide standards provided routine validations of the procedure's efficiency. The method indicated that insignificant amounts of hydroperoxide may be in the less polar lipids, but the total amount of lipid hydroperoxide esterfied in the plasma lipids of apparently healthy humans may be as much as 4.0 +/- 1.7 microM.     

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.     

A critical evaluation of the effect of sorbitol on the ferric-xylenol orange hydroperoxide assay

Measurement of hydroperoxide concentration by the ferric-xylenol orange assay has the advantages of simplicity, convenience, and inertness to oxygen (Gay, C., et al. (1999) Anal. Biochem. 273, 149-155). However, its sensitivity is limited by the molar absorption coefficients, which are less than 50,000 M(-1) cm(-1) for most hydroperoxides. An earlier report showed that this could be significantly enhanced by the inclusion of 100 mM sorbitol in the assay solution, resulting in an increase of the apparent absorption coefficient for H(2)O(2) from 4.46 x 10(4) to 2.24 x 10(5) M(-1) cm(-1) (Wolff, S. P. (1994) Methods Enzymol. 233, 182-189). It was claimed that the technique was also valid for other hydroperoxides, such as butyl and cumyl. In an attempt to extend this modification to a wider range of hydroperoxides, we have confirmed the enhancement of the assay of H2O2 by sorbitol. However, the sensitivity of the measurements of butyl, cumyl, amino acid, protein, and human blood serum hydroperoxides was only approximately doubled by the inclusion of sorbitol. A mechanism explaining the difference in the assay of H2O2 and the organic hydroperoxides is proposed.     

Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein.

A simple and sensitive method for the direct measurement of lipid peroxides in lipoprotein and liposomes is described. The method is based on the principle of the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fe(3+)-xylenol orange complex which can be measured spectrophotometrically at 560 nm. Calibration with standard peroxides, such as hydrogen peroxide, linoleic hydroperoxide, t-butyl hydroperoxide, and cumene hydroperoxide gives a mean apparent extinction coefficient of 4.52 x 10(4) M-1 cm-1 consistent with a chain length of approximately 3 for ferrous ion oxidation by hydroperoxides. Endoperoxides are less reactive or unreactive in the assay. The assay has been validated in the study of lipid peroxidation of low density lipoprotein and phosphatidyl choline liposomes. By pretreatment with enzymes known to metabolize peroxides, we have shown that the assay measures lipid hydroperoxides specifically. Other methods for measuring peroxidation, such as the assessment of conjugated diene, thiobarbituric acid reactive substances and an iodometric assay have been compared with the ferrous oxidation-xylenol orange assay.     

Glutathione transferases: a possible role in the detoxication and repair of DNA and lipid hydroperoxides

Two types of GSH peroxidase occur in the cell both of which detoxify fatty acid hydroperoxides, thymine hydroperoxide and DNA hydroperoxides. One is a Se-dependent enzyme which also detoxifies H2O2. The other contains members of the GSH transferase supergene family. These non-selenium dependent GSH peroxidases do not detoxify H2O2 and have substrate specificities varying markedly with the isoenzyme. Of particular interest is GSH transferase 5*-5* an enzyme extracted from the nucleus with urea which has a relatively high activity towards DNA hydroperoxide. The possible role of these enzymes in the detoxication of lipid and DNA hydroperoxides is discussed and it is pointed out that they may be important participants in mechanism for the repair of free-radical damage.     

Biological hydroperoxides and singlet molecular oxygen generation

The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is a potential source of singlet molecular oxygen ((1)O(2)) in biological systems. Recently, we have clearly demonstrated the generation of (1)O(2) in the reaction of lipid hydroperoxides with biologically important oxidants such as metal ions, peroxynitrite and hypochlorous acid. The approach used to unequivocally demonstrate the generation of (1)O(2) in these reactions was the use of an isotopic labeled hydroperoxide, the (18)O-labeled linoleic acid hydroperoxide, the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) and the direct spectroscopic detection and characterization of (1)O(2) light emission. Using this approach we have observed the formation of (18)O-labeled (1)O(2) by chemical trapping of (1)O(2) with anthracene derivatives and detection of the corresponding labeled endoperoxide by HPLC-MS/MS. The generation of (1)O(2) was also demonstrated by direct spectral characterization of (1)O(2) monomol light emission in the near-infrared region (lambda = 1270 nm). In summary, our studies demonstrated that LOOH can originate (1)O(2). The experimental evidences indicate that (1)O(2) is generated at a yield close to 10% by the Russell mechanism, where a linear tetraoxide intermediate is formed in the combination of two peroxyl radicals. In addition to LOOH, other biological hydroperoxides, including hydroperoxides formed in proteins and nucleic acids, may also participate in reactions leading to the generation (1)O(2). This hypothesis is currently being investigated in our laboratory.     

Selective microdetermination of lipid hydroperoxides

A sensitive and selective assay for lipid hydroperoxides was developed based upon the activation by hydroperoxides of the cyclooxygenase activity of prostaglandin H synthase. The assay measures hydroperoxides directly by their stimulatory action on the cyclooxygenase and thus differs from the methods used currently which rely on the measurement of secondary products to estimate the amount of hydroperoxide. The present assay of enzymatic response was approximately linear in the range 10 to 150 pmol of added lipid hydroperoxide. This sensitivity for lipid peroxides is about 50-fold greater than that of the thiobarbiturate assay with fluorescence detection. When applied to samples of human plasma, the enzymatic assay indicated that the concentration of lipid hydroperoxides in normal subjects is 0.5 microM, more than 50-fold lower than estimated by the thiobarbiturate assay (30-50 microM). Nevertheless, the circulating concentration of 0.5 microM lipid hydroperoxide approaches that reported to have deleterious effects upon vascular prostacyclin synthase.     

Organic hydroperoxide resistance gene encodes a thiol-dependent peroxidase

ohr (organic hydroperoxide resistance gene) is present in several species of bacteria, and its deletion renders cells specifically sensitive to organic peroxides. The goal of this work was to determine the biochemical function of Ohr from Xylella fastidiosa. All of the Ohr homologues possess two cysteine residues, one of them located in a VCP motif, which is also present in all of the proteins from the peroxiredoxin family. Therefore, we have investigated whether Ohr possesses thiol-dependent peroxidase activity. The ohr gene from X. fastidiosa was expressed in Escherichia coli, and the recombinant Ohr decomposed hydroperoxides in a dithiothreitol-dependent manner. Ohr was about twenty times more efficient to remove organic hydroperoxides than to remove H(2)O(2). This result is consistent with the organic hydroperoxide sensitivity of Delta ohr strains. The dependence of Ohr on thiol compounds was ascertained by glutamine synthetase protection assays. Approximately two thiol equivalents were consumed per peroxide removed indicating that Ohr catalyzes the following reaction: 2RSH + ROOH --> RSSR + ROH + H(2)O. Pretreatment of Ohr with N-ethyl maleimide and substitution of cysteine residues by serines inhibited this peroxidase activity indicating that both of the Ohr cysteines are important to the decomposition of peroxides. C125S still had a residual enzymatic activity indicating that Cys-61 is directly involved in peroxide removal. Monothiol compounds do not support the peroxidase activity of Ohr as well as thioredoxin from Saccharomyces cerevisiae and from Spirulina. Interestingly, dithiothreitol and dyhydrolipoic acid, which possess two sulfhydryl groups, do support the peroxidase activity of Ohr. Taken together our results unequivocally demonstrated that Ohr is a thiol-dependent peroxidase.     

Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

Oxygenation of anaerobically isolated brain and liver homogenates is associated with chemiluminescence and formation of lipid hydroperoxides, the latter determined by the thiobarbituric acid assay. Light emission and formation of malonaldehyde are 20-fold higher in the brain than in liver; chemiluminescence of both decays when accumulation of malonaldehyde ceases. Exogenous organic peroxides, such as t-butyl hydroperoxide, inhibit the light-emission response to oxygenation by brain homogenate, whereas they enhance that of liver homogenate. t-Butyl hydroperoxide-induced photoemission of liver homogenate shows a polyphasic kinetic pattern that is O2-dependent. The spectral analysis of chemiluminescence arising from brain and liver homogenates on oxygenation shows a spectrum with five emission bands at 420-450, 475-485, 510-540, 560-580 and 625-640 nm. These bands are subjected to intensity changes or shifts of the wavelength whenever t-butyl hydroperoxide is present, either inhibiting or stimulating light emission. The blue-band chemiluminescence, around 435 nm, is possibly due to the weak light emission arising from excited carbonyl compounds [Lloyd (1965) J. Chem. Soc. Faraday Trans. 61, 2182-2193; Vassil'ev (1965) Opt. Spectrosc. (USSR) 18, 131-135], whereas the presence of other bands suggests generation of singlet molecular oxygen either in the process triggered on oxygenation (lipid oxygenation) or after supplementation with organic hydroperoxides. We offer several explanations for the spectral analysis presented here.     

Induction of vascular relaxation by hydroperoxides

Hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, and 3-chloroperoxybenzoic acid (CPB) and 15-HPETE relaxed, in a concentration dependent manner rat aortic rings contracted with PGF2 alpha (1 X 10(-5)). Relaxation is not inhibited by either indomethacin (2 X 10(-5) M), a cyclo-oxygenase inhibitor or eicosatetraynoic acid (1 X 10(-5) M), a dual cyclo-oxygenase and lipoxygenase inhibitor. Rings with intact endothelium relaxed to a greater degree on exposure to CPB and 15-HPETE. Methylene blue, a soluble guanylate cyclase inhibitor (1 X 10(-5) M) blocked the relaxation elicited by the five peroxides, whereas both superoxide dismutase (scavenger of superoxide anion) and mannitol (scavenger of hydroxyl radical) have no effect. We conclude that relaxation of vascular smooth muscle is a general property of peroxides and that the endothelium may in some instances facilitate this effect.     

A nonenzymatic method for determination of hydrogen peroxide and organic peroxides

Reduction of hydrogen peroxide and organic peroxides (t-butyl hydroperoxide and linoleic acid hydroperoxide) was achieved with homovanillic acid as hydrogen donor in the presence of the triethylenetetramine-Fe3+ complex. By the catalytic action of this complex, homovanillic acid is oxidized to its fluorescent dimer. Based on this reaction a fluorometric method for the measurement of the hydroperoxides mentioned above is described. The method can be extended to the determination of substrate-enzyme systems that produce hydrogen peroxide, e.g., glucose-glucose oxidase. The method allows the determination of substances such as hydrogen peroxide and t-butyl hydroperoxide with an accuracy and precision of less than 3%. Glucose can be determined with similar precision and an accuracy of 4.7%.     

Kinetic study by pulse radiolysis of the lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by HO2 and O2-RADICALS.

The lactate dehydrogenase-catalyzed chain oxidation of NADH (LDH-NADH) by the superoxide radicals, HO2 and O2, has been studied with pulse radiolysis in the pH range between 4.5 and 9.0. The rate constants for the oxidation of the LDH-NADH by HO2 and O2 determined at 23 degrees are 1.2 times 10-6 M(-1) s(-1) and 3.6 times 10-4 M(-1) s(-1), respectively. The latter represents an activation of over 1000-fold by the enzyme. A chain reaction mechanism consistent with the results from these kinetic studies has been proposed.     

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.