Autoxidation and Photosensitized Oxidation of Methyl Eicosapentaenoate: Secondary Oxidation Products

Methyl eicosapentaenoate (methyl 5,8,11,14,17-eicosapentaenoate) was subjected to autoxidation and methylene blue-sensitized photooxidation. The secondary oxidation products were separated, and characterized by gas chromatography-mass spectrometry. The autoxidation products included hydroperoxy endoperoxide isomers, prostaglandin-like hydroperoxy bicyclic endoperoxide isomers and 5,18-dihydroperoxide. The photosensitized oxidation products included only dihydroperoxide isomers as the secondary products.     

Mutagenic activities of hydroperoxythymine derivatives, products of radiation and oxidation reactions.

By using a bacterial test system, it has been shown that hydroperoxy derivatives of thymine and thymidine produced by ionizing radiation, near-UV radiation, and certain oxidation reactions are highly mutagenic. Considering that hydroperoxy derivatives of biomolecules have been implicated widely as likely candidates causing mutagenesis, carcinogenesis, and aging, it would be advantageous to screen these compounds when they can be isolated in pure state in order to assess their potential hazards to human health. The findings from these assays would provide information to further our understanding of the mechanism of their mutagenic action.     

Prostaglandins and cancer: A review of tumor initiation through tumor metastasis

Prostaglandin is a term applied to a series of compounds derived enzymatically and nonenzymatically from 20 carbon fatty acids such as arachidonic acid (20:4). Research over the past 20 years has identified the prostaglandins as products of almost every cell although the amount and class of prostaglandin produced varies considerably with cell type. Within the last ten years additional, related products of 20:4 metabolism (operationally termed eicosanoids) have been discovered, eg. prostacyclin, thromboxanes, malondialdehyde, leukotrienes, hydroperoxy and hydroxy fatty acids. The role of these 20:4 metabolites in physiological and pathological states is currently under intensive investigation and the biological action of these compounds have been implicated in many key regulatory processes. Therefore, it is not unreasonable to predict that these potent bioregulatory compounds may have a central role in the initiation and regulation (positive and negative) of the spectrum of diseases which we functionally term “cancer”.     

Lipid hydroperoxy and hydroxy derivatives in copper-catalyzed oxidation of low density lipoprotein

Oxidation of low density lipoprotein (LDL) causes changes in the biological properties of LDL that may be important in atherogenesis. That LDL oxidation is accompanied by lipid peroxidation has been demonstrated, but previous analyses of the products of LDL oxidation have not included measurement of specific lipid hydroperoxy and hydroxy derivatives. In this study, LDL was isolated from plasma of normal volunteers and exposed to oxygenated buffer and 5 microM CuSO4 for 24 h. Oxidized LDL showed decreased linoleate (18:2) and arachidonate (20:4) content with increased concentrations of thiobarbituric acid reactive substances (TBARS) and hydroxy and hydroperoxy 18:2 and 20:4. The electrophoretic mobility of the LDL protein also was increased by oxidation. After reduction, the hydroxy fatty acids were characterized by gas chromatography-mass spectrometric analysis of the trimethylsilyl ether methyl ester derivatives. The hydroperoxy and hydroxy derivatives accounted for approximately 70% of the linoleate consumed during LDL oxidation and represented 45-fold more product than was measured by TBARS analysis. Numerous biological properties, including cytotoxic and chemoattractant activities of hydroperoxy and hydroxy fatty acids, have been reported, but the manner in which they may contribute to atherogenesis requires further study.     

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.     

Similarity of the oxidation products of L-cystathionine by L-amino acid oxidase to those excreted by cystathioninuric patients

L-Cystathionine is oxidized by snake venom L-amino acid oxidase at a rate about half that with L-leucine at pH 8.5. The appearance of an absorbance at 296 nm and quantitation of the products of oxidation in the presence of catalase indicate formation in the solutions of a seven-membered ketimine ring produced by cyclization of the monoamino monoketo derivative of cystathionine. A limited double deamination has also been observed. In the absence of catalase, S-(carboxymethyl)homocysteine and S-(beta-carboxyethyl)cysteine have been identified together with ninhydrin-unreactive compounds yielding the above mentioned carboxy compounds upon hydrolysis with HCl. Authentic samples of the monoamino monoketo analogs of cystathionine have been prepared and compared with the enzymatic products. Cyclization of the synthetic products into the ketimine ring is pH-dependent as established by UV spectrum and other assays. Compounds derived from either the oxidation or the reduction of the ketimine have been prepared. It was found that many products of enzymatic and chemical changes of cystathionine and its ketimine described in the present paper are identical with those identified in the urine of cystathioninuric patients. This result indicates the occurrence in humans of secondary metabolic routes of cystathionine centered on the production of cystathionine ketimine, in equilibrium with the open form, which in cystathioninurics is revealed by the lack of cystathionase.     

Evidence for the presence of 7-hydroperoxycholest-5-en-3 beta-ol in oxidized human LDL.

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).     

The autoxidation of arachidonic acid: formation of the proposed SRS-A intermediate.

The air oxidation of 5,8,11,14-eicosatetraenoic [arachidonic] acid and its methyl ester is reported. A mixture of hydroperoxy arachidonic acid products was obtained from the oxidation and subsequent separation of the mixture by high pressure liquid chromatography led to pure hydroperoxides. One of these hydroperoxides, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, is a proposed intermediate in the biosynthesis of slow reacting substance of anaphylaxis.     

Routes of formation and toxic consequences of lipid oxidation products in foods.

Lipid oxidation in foods is initiated by free radical and/or singlet oxygen mechanisms which generate a series of autocatalytic free radical reactions. These autoxidation reactions lead to the breakdown of lipid and to the formation of a wide array of oxidation products. The nature and proportion of these products can vary widely between foods and depend on the composition of the food as well as numerous environmental factors. The toxicological significance of lipid oxidation in foods is complicated by interactions of secondary lipid oxidation products with other food components. These interactions could either form complexes that limit the bioavailability of lipid breakdown products or can lead to the formation of toxic products derived from non-lipid sources. A lack of gross pathological consequences has generally been observed in animals fed oxidized fats. On the other hand, secondary products of lipid autoxidation can be absorbed and may cause an increase in oxidative stress and deleterious changes in lipoprotein and platelet metabolism. The presence of reactive lipid oxidation products in foods needs more systematic research in terms of complexities of food component interactions and the metabolic processing of these compounds.     

PRODUCTS OF THE OXIDATION OF THIOSULFATE BY BACTERIA IN MINERAL MEDIA

Various cultures (previously described), which oxidize thiosulfate in mineral media have been studied in an attempt to determine the products of oxidation. The transformation of sodium thiosulfate by Cultures B, T, and K yields sodium tetrathionate and sodium hydroxide; secondary chemical reactions result in the accumulation of some tri- and pentathionates, sulfate, and elemental sulfur. As a result of the initial reaction, the pH increases; the secondary reactions cause a drop in pH after this initial rise. The primary reaction yields much less energy than the reactions effected by autotrophic bacteria. No significant amounts of assimilated organic carbon were detected in media supporting representatives of these cultures. It is concluded that they are heterotrophic bacteria. Th. novellus oxidizes sodium thiosulfate to sodium sulfate and sulfuric acid; the pH drops progressively with growth and oxidation. Carbon assimilation typical of autotrophic bacteria was detected; the ratio of sulfate-sulfur formed to carbon assimilated was 56:1. It is calculated that 5.1 per cent of the energy yielded by the oxidation of thiosulfate is accounted for in the organic cell substance synthesized from inorganic materials. This organism is a facultative autotroph. The products of oxidation of sodium thiosulfate by Th. thioparus are sodium sulfate, sulfuric acid, and elemental sulfur; the ratio of sulfate sulfur to elemental sulfur is 3 to 2. The pH decreases during growth and oxidation. The elemental sulfur is produced by the primary reaction and is not a product of secondary chemical changes. The bacterium synthesizes organic compounds from mineral substances during growth. The ratio of thiosulfate-sulfur oxidized to carbon assimilated was 125:1, with 4.7 per cent of the energy of oxidation recovered as organic cell substance. This bacterium is a strict autotroph.     

4-Hydroperoxy-2-nonenal is not just an intermediate but a reactive molecule that covalently modifies proteins to generate unique intramolecular oxidation products

α,β-Unsaturated aldehydes generated during lipid peroxidation, such as 4-oxoalkenals and 4-hydroxyalkenals, can give rise to protein degeneration in a variety of pathological states. Although the covalent modification of proteins by these end products has been well studied, the reactivity of unstable intermediates possessing a hydroperoxy group, such as 4-hydroperoxy-2-nonenal (HPNE), with protein has received little attention. We have now established a unique protein modification in which the 4-hydroperoxy group of HPNE is involved in the formation of structurally unusual lysine adducts. In addition, we showed that one of the HPNE-specific lysine adducts constitutes the epitope of a monoclonal antibody raised against the HPNE-modified protein. Upon incubation with bovine serum albumin, HPNE preferentially reacted with the lysine residues. By employing N(α)-benzoylglycyl-lysine, we detected two major products containing one HPNE molecule per peptide. Based on the chemical and spectroscopic evidence, the products were identified to be the N(α)-benzoylglycyl derivatives of N(ε)-4-hydroxynonanoic acid-lysine and N(ε)-4-hydroxy-(2Z)-nonenoyllysine, both of which are suggested to be formed through mechanisms in which the initial HPNE-lysine adducts undergo Baeyer-Villiger-like reactions proceeding through an intramolecular oxidation catalyzed by the hydroperoxy group. On the other hand, using an HPNE-modified protein as the immunogen, we raised a monoclonal antibody against the HPNE-modified protein and identified one of the HPNE-specific lysine adducts, N(ε)-4-hydroxynonanoic acid-lysine, as an intrinsic epitope of the monoclonal antibody. Furthermore, we demonstrated that the HPNE-specific epitopes were produced not only in the oxidized low density lipoprotein in vitro but also in the atherosclerotic lesions. These results indicated that HPNE is not just an intermediate but also a reactive molecule that could covalently modify proteins in biological systems.     

Preferential formation of the hydroperoxide of linoleic acid in choline glycerophospholipids in human erythrocytes membrane during peroxidation with an azo initiator

The formation of phospholipid hydroperoxides was monitored in human red blood cell (RBC) membranes that had been peroxidized with an azo initiator. Peroxidation of RBC membranes caused a profound decrease in the amount of polyunsaturated fatty acids and concomitantly hydroperoxides, as primary products of peroxidation, appeared in the phospholipids. Hydroperoxides were predominantly generated in choline glycerophospholipid (CGP), while the extent of formation of ethanolamine glycerophospholipid (EGP) hydroperoxides was low and their presence was transient. Hydroxy and hydroperoxy moieties in CGP were identified as 9-hydroxy and 13-hydroxy octadecanoic acid, derived from linoleic acid, by gas chromatography-mass spectrometric analysis. No consistent generation of hydroperoxide from arachidonic acid was evident in CGP. The CGP-hydroperoxide accounted for approximately 76% of linoleic acid consumed during peroxidation of RBC membranes. The prominent generation of phospholipid hydroperoxides was observed in the linoleic acid-rich membranes from rabbit RBC, indicating that the level of linoleic acid in phospholipids determins, in part, the extent of formation of phospholipid hydroperoxides. Aldehydic phospholipids, as secondary products of peroxidation, were detected in oxidized membranes. EGP was the most prominent aldehydic phospholipid, while negligible amounts of aldehydic CGP were formed. This study indicates that the process of oxidation of individual phospholipids clearly differs among phospholipids and depends on the structure of each.     

Biophysical and biochemical characterization of active secondary metabolites from Aspergillus allahabadii

Fungal secondary metabolites are a diverse group of natural chemical products with physiological relevance. We aimed to identify bioactive secondary metabolites from Aspergillus allahabadii. We used “activity-guided fractionation” strategy for the isolation of secondary metabolites. Crude extracts showed good antibacterial activity. Two antibacterial secondary metabolites have been isolated from the crude extract. Chemical characterization of these compounds was performed using biophysical techniques (FT-IR, NMR, and mass spectrometry). Structural characterization confirmed these to be pyrone derivatives: 3-hydroxy 2-methyl 4-pyrone and 5-hydroxy-2-(hydroxymethyl)-4H-pyrone. These bioactive pyrone derivatives have been identified as maltol and kojic acid. From our initial observations, we infer that these pyrone derivatives have potent antimicrobial, antioxidant, antidiabetic, and mosquito larvicidal activities and no cytotoxicity. These compounds could have potential therapeutic and biomedical applications, but further mechanistic studies using animal models are very much necessary.     

Oxidation of mannosyl oligosaccharides by hydroxyl radicals as assessed by electrospray mass spectrometry

The hydroxyl radicals are widely implicated in oxidation of carbohydrates during biological and industrial processes being responsible for their structural modifications and causing functional damage. The identification of intermediate oxidation products is hampered by a lack of reliable sensible methods for their detection. In this study, the oxidation of two models of galactomannans (Man₃ and GalMan₂) has been studied in reaction with hydroxyl radical generated by Fenton reaction. The oxidation patterns were assessed using preparative ligand-exchange/size-exclusion chromatography (LEX/SEC) coupled with tandem electrospray mass spectrometry (ESI-MS/MS). This allowed the identification of derived oligosaccharides (OS) containing hexuronic, hexonic, pentonic and erythronic acid residues and neutral OS bearing hydroperoxy, hydrated carbonyl moieties and residues from pyranosyl ring cleavage. The depolymerization products have been also detected upon oxidation of oligomers. This study allowed developing a simple, effective ‘fingerprinting’ protocol for detecting the damage done to mannans by oxidative radicals.     

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.     

Bio-markers of lipid peroxidation in vivo: hydroxyoctadecadienoic acid and hydroxycholesterol

The biological role of lipid peroxidation products has continued to receive a great deal of attention not only for the elucidation of pathological mechanisms but also for their practical application to clinical use as bio-markers. In the last fifty years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. In this decade, F2-isoprostanes from arachidonates and neuroprostanes from docosahexanoates have been proposed as bio-markers. Although these markers are formed by a free radical-mediated oxidation, the yields from the parent lipids are minimal. Compared to these markers, hydroperoxy octadecadienoates (HPODE) from linoleates and oxysterols from cholesterols are yielded by much simpler mechanisms from more abundant parent lipids in vivo. Recently, the method in which both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as total hydroxyoctadecadienoic acid (tHODE) and 7-hydroxycholesterol (t7-OHCh), respectively, was proposed. The concentrations of tHODE and t7-OHCh determined by GC-MS analysis from physiological samples were much higher than that of 8-iso-prostagrandin F(2alpha). In addition to this advantage, hydrogen-donor activity of antioxidants in vivo could be determined by the isomeric-ratio of HODE (9- and 13-(Z,E)-HODE/9- and 13-(E,E)-HODE).     

Occurrence of 9- and 13-keto-octadecadienoic acid in biological membranes oxygenated by the reticulocyte lipoxygenase

Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.     

The reaction of DNA with lipid oxidation products, metals and reducing agents

The interaction of lipid hydroperoxides and secondary oxidation products with DNA was investigated by evaluating the fluorescence formed in the presence of metals and reducing agents. We also investigated the effect of malonaldehyde, because it has been generally considered responsible for the formation of fluorescence with DNA. However, malonaldehyde usually has been estimated by the notoriously unspecific thiobarbituric acid test. At low concentration of oxidation products (1 mM), fluorescence formation required the presence of metals and ascorbic acid. In contrast, a positive thiobarbituric acid reaction was obtained with many lipid oxidation products without metals or ascorbic acid. Monohydroperoxides from autoxidized methyl linoleate and linolenate produced the highest level of fluorescence. Hydroperoxy epidioxides of linolenate and dihydroperoxides of linoleate and linolenate were among the most active secondary products in forming fluorescence with DNA. In contrast, malonaldehyde produced very little fluorescence under our conditions. The thiobarbituric acid values did not correlate with fluorescence formation. This study showed that, in our model reaction system, DNA forms fluorescent products by the breakdown of lipid oxidation products in the presence of metals and ascorbic acid into reactive materials other than malonaldehyde. Therefore, the importance of malonaldehyde in its crosslinking properties with DNA may have been exaggerated in the literature.     

Enzymatic activation of hydrazine derivatives. A spin-trapping study

The oxidative metabolism of hydralazine, isoniazid, iproniazid, and phenylhydrazine has been studied using spin-trapping techniques. The oxidation of these hydrazine derivatives, catalyzed by horseradish peroxidase and prostaglandin synthetase, produces reactive free radical intermediates. Enzymatic activation of hydralazine produce the nitrogen-centered hydralazyl radical (RNHNH); phenylhydrazine formed only the phenyl radical. Iproniazid, on the other hand, formed both the isopropyl radical and a hydroperoxy radical. The formation of the hydroperoxy radical was not inhibited by superoxide dismutase. The horseradish peroxidase-catalyzed oxidation of isoniazid produced two different carbon-centered radicals. The identity of these radicals is not clear; however, they may arise from an acyl (RCO) radical and an alkyl (R) radical.     

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH- substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.