Radiation induced peroxidation of polyunsaturated fatty acids: recent results on formation of hydroperoxides

Aqueous solutions of linoleic acid were irradiated in air with gamma-rays of 137Cs. High pressure liquid chromatography (HPLC) was been used to separate and measure the production of hydroperoxides. The results obtained after reverse phase chromatography, associated with a microperoxydase for hydroperoxide detection, indicate the presence of two different hydroperoxides. One type of hydroperoxide was the major product obtained when the initial linoleic concentrations were below the critical micellar concentration (2 mM), and the second type was produced when the concentrations were above 2 mM. A further separation carried out on the second hydroperoxide by direct phase HPLC showed that it contains three compounds, mainly HPODE 9 and 13.     

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.     

Evidence of the formation of different hydroperoxides in irradiated gamma-linolenate solutions: effect of micelle formation

Peroxidation of unconjugated polyunsaturated fatty acids such as linolenic acid proceeds through a free radical chain mechanism and is accompanied by the formation of conjugated dienes such as hydroperoxides. In an investigation of radiation-induced oxidation of aqueous linolenate, we have measured two indexes of peroxidation: (1) conjugated dienes by means of absorption spectroscopy and (2) hydroperoxides by high-pressure liquid chromatography using detection of chemiluminescence. The experimental results indicate a strong effect of the concentration of linolenate on the yields of oxidized products. In addition, this work shows the quantitative production of two kinds of hydroperoxides. The ratio of these hydroperoxides is independent of the radiation dose but is dependent on the linolenate concentration. One hydroperoxide is formed predominantly below the critical micellar concentration (3 mM under our conditions), while the second is observed predominantly when micelles are formed in the aqueous medium. The influence of the composition of the medium on the nature of both hydroperoxides is discussed. [bj163]     

Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase

Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action.     

Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase

Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action.     

Selective quantification of arachidonic acid hydroperoxides and their hydroxy derivatives in reverse-phase high performance liquid chromatography

For the quantification of lipid hydroperoxides by high performance liquid chromatography (HPLC), it has been necessary to improve the detection system specific to the hydroperoxy group. We first developed a technique which combined detection by uv absorption due to conjugated diene and detection based on electrochemical (EC) reduction in reverse-phase HPLC for the selective determination of arachidonic acid hydroperoxides (hydroperoxyeicosatetraenoic acid, HPETE) and its reduced derivative, hydroxyeicosatetraenoic acid (HETE). 15-HPETE was quantified selectively by EC detection, although both 15-HPETE and 15-HETE were detected by uv absorption and were hardly resolved in the chromatogram. Isomers in HPETE obtained from autoxidized arachidonic acid were partially separated in the chromatogram and seem to have been quantified similarly to 15-HPETE. The application of this analytical system to the analysis of 15-HPETE added in human plasma has demonstrated that the recovery of HPETE extracted from human plasma is much lower than that from normal saline and that HPETE is reduced to HETE by incubation at 37 degrees C. The fact that a high concentration of glutathione accelerated this reduction may indicate that human plasma possesses a glutathione-dependent HPETE-reducing ability as a defense system against excess accumulation of lipid hydroperoxides. Blood plasma effectively suppressed the decomposition of HPETE induced by ferrous ion indicating the presence of factors which prevent the action of ferrous ion on HPETE.     

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (α-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%～5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.     

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas.

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%-5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.     

Measurement of Plasma Hydroperoxide Concentrations by the Ferrous Oxidation-Xylenol Orange Assay in Conjunction with Triphenylphosphine

We describe the application of the FOX2 (ferrous oxidation in xylenol orange, version 2) method to the measurement of hydroperoxides in plasma. Authentic plasma hydroperoxides can be determined by a strategy in which the hydroperoxide reductant, triphenylphosphine, is used to discriminate between the background signal generated by ferric ions present in plasma and that generated by hydroperoxide in plasma. The approach was validated by extraction of total lipids from plasma using ethyl acetate prior to assay with the FOX2 reagent. Plasma from 23 normal individuals contained hydroperoxide in the range of 0.22 to 7.8 μM with a mean of 3.02 μM and a population standard deviation of 1.85 μM. After partitioning with ethyl acetate, plasma hydroperoxide levels ranged from 0.22 to 6.22 μM, with a mean value of 2.52 μM and a population standard deviation of 1.65 μM.     

Peroxidation stimulated by lipid hydroperoxides on bovine retinal pigment epithelium mitochondria: effect of cellular retinol-binding protein

This study analyzes the effect of cellular retinol-binding protein (CRBP), partially purified from retinal pigment epithelium (RPE) cytosol, on the non-enzymatic lipid peroxidation induced by fatty acid hydroperoxides of mitochondrial membranes isolated from bovine RPE. The effect of different amounts (50, 75 and 100 nmol) of linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) on the lipid peroxidation of RPE mitochondria was studied; RPE mitochondria deprived of exogenously added hydroperoxide was utilized as control. The process was measured simultaneously by determining chemiluminescence as well as polyunsaturated fatty acid (PUFA) degradation of total lipids isolated from RPE mitochondria. The addition of hydroperoxides to RPE mitochondria produces a marked increase in light emission that was hydroperoxide concentration dependent. The highest value of activation was produced by LHP. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized RPE mitochondria incubated with and without hydroperoxides was found in the docosahexaenoic acid content, this decreased 40.90+/-3.01% in the peroxidized group compared to native RPE mitochondria. The decrease was significantly high: 86.32+/-2.57% when the lipid peroxidation was stimulated by 100 nmol of LHP. Inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (100-600 microg) of CRBP to RPE mitochondria. The inhibitory effect reaches the highest values in the presence of LHP.     

High-performance liquid chromatography with mercury cathode electrochemical detection: application to lipid hydroperoxide analysis

Lipid hydroperoxide species can be analyzed with high sensitivity and specificity, using reversed-phase high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop cathode [HPLC-ED(Hg)]. The purpose of this study was to examine different variables in the operation of HPLC-ED(Hg) and to select optimal conditions for the analysis of several biologically relevant peroxides, including species derived from cholesterol, cholesteryl linoleate, oleate, linoleate, and two synthetic phosphatidylcholines. Parameters such as operating potential and mobile-phase solvent proportions, electrolyte composition, and ionic strength were evaluated for each peroxide class. Under optimal conditions, we have achieved baseline separation of four cholesterol hydroperoxide species, not only from one another, but also from phospholipid hydroperoxides; detection limits were <0.3 pmol and <30 pmol for the cholesterol and phospholipid hydroperoxides, respectively.     

Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.     

Some characteristics of hydrogen- and alkylhydroperoxides metabolizing systems in cardiac tissue

The effect of hydroperoxides on the cardiac tissue was studied by using hemoglobin-free perfused rat heart. Ethylhydroperoxide was degraded mainly through the glutathione peroxidase system of the heart at a maximal rate of about 1.2 mumol/min per g wet wt. When ethylhydroperoxide infused was not degraded completely, the hydroperoxide concentration in the effluent perfusate paralleled the formation of ferrylmyoglobin in the heart. The infusion of ethylhydroperoxide caused release of oxidized glutathione into the effluent perfusate as a result of the enhancement of the cytosolic glutathione peroxidase reaction. The leakage of oxidized glutathione reached the maximal rate of 3.5 nmol/min per g wet wt with the infusion of 175 microM ethylhydroperoxide. At hydroperoxide concentrations above 150 microM, oxidations of pyridine nucleotides and of cytochrome a + a3 occurred, probably through a stimulation of the mitochondrial glutathione peroxidase reaction, and resulted in sudden failure of the heart function. The infusion of t-butyl- and cumene-hydroperoxides, which are unable to react with myoglobin, also caused the oxidations of pyridine nucleotides and cytochrome a + a3, the inhibition of oxygen consumption and the failure of heart function. The results indicate that the cardiac toxicity of hydroperoxides is due mainly to their effect on mitochondrial metabolism.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

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.     

Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.     

Inhibition of UV-induced accumulation of lipid peroxides by melanins and ommochromes

The effect of screening pigments--melanin and ommochromes--on the UV-induced accumulation of lipid hydroperoxides by cardiolipin was studied. It was shown that the time dependence of lipid hydroperoxide accumulation at different UV irradiation intensities is linear. Addition of melanin or ommochromes results in a significant decrease of the lipid hydroperoxide accumulation rate. The kinetic curve rapidly reaches the saturation values, which is suggestive of intensive chemical inhibition of the reaction. The screens transmitting UV irradiation in the same degree as do melanin and ommochromes have a far less pronounced inhibiting effect. The inhibitory mechanism of screening pigments based on their antiradical activity is discussed.     

Photoinduced DNA cleavage by naphthalimide hydroperoxide-specific strand scission at -GG- site of DNA

We have prepared a series of naphthalene hydroperoxides (1-3) which possess hydroperoxy group at gamma-position of imide carbonyl. Upon photoirradiation (greater than 350 nm) hydroperoxides (1-3) decomposed with efficient generation of hydroxyl radical, which was confirmed by esr spin trapping technique using dimethylpyrroline oxide as a spin trapper. All these hydroperoxides induced DNA strand scission upon photoirradiation (greater than 350 nm), especially hydroperoxide 3 cleaved plasmid phi X 174 DNA (Form I) to give nicked (Form II) and linear (Form III) DNA even at 1 microM concentration. Further, it was observed that 3 exclusively cleaved DNA at the 5'-G site of -GG-sequence.     

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.     

The relative effectiveness of human plasma glutathione peroxidase as a catalyst for the reduction of hydroperoxides by glutathione

To reveal clues to the function of human plasma glutathione peroxidase (GPx), we investigated its catalytic effectiveness with a variety of hydroperoxides. Comparisons of hydroperoxides as substrates for plasma GPx based on the ratio ofV _max /K _m were blocked by the limited solubility of the organic hydroperoxides, which prevented kinetic saturation of the enzyme at the chosen glutathione concentration. Therefore, we compared the hydroperoxides by the fold increase in the apparent first-order rate constants of their reactions with glutathione owing to catalysis by plasma GPx. The reductions of aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,t-amyl hydroperoxide,t-butyl hydroperoxide, paramenthane hydroperoxide) were better catalyzed by plasma GPx than were reductions of the more “physiological” substrates (linoleic acid hydroperoxide, hydrogen peroxide, peroxidized plasma lipids, and oxidized cholesterol).