Participation of singlet oxygen in photosensitized oxidation of 1,4-dienoic systems and photooxidation of soybean oil

Sensitized photooxidation of a model 1,4-diene, 4cis, 7cis-undecadiene, was shown to yield 4-hydroperoxy-5trans,7cis-undecadiene and 5-hydroperoxy-3trans,7cis-undecadiene as initial products. Further irradiation (in the presence of the sensitizer) caused the isomerization of 4-hydroperoxy-5trans,7cis-undecadiene to 4-hydroperoxy-5trans,7trans-undecadiene. Oxidation of 4cis,7cis-undecadiene with chemically formed singlet oxygen gave the same initial products as the photosensitized oxidation. 5-Hydroperoxy-3trans,7cis-undecadiene is, however, not formed in the radical autoxidation of the diene. It is concluded that singlet oxygen is the reactive intermediate in the photooxidation. Comparison with this model reaction suggests that the photooxidation of refined soybean oil in propanol also proceeds via singlet oxygen: the photooxidations of both 4,7-undecadiene and soybean oil are inhibited by β-carotene and by triethylamine, but unlike radical chain autoxidation, they are not inhibited by 2,6-di-t-butyl-4-methylphenol. Also soybean oil can act as a sensitizer for the photooxidation of 4,7-undecadiene. Chlorophyl-like sensitizers are probably unimportant in the well refined soybean oil used in this work. The observed photooxidation of the skipped dienoic components of soybean oil, which is probably due to some other, unidentified sensitizer(s), absorbing below 500 nm, can be avoided by using a yellow filter. Presented in part in the symposium “Oxidation Chemistry,” New Chemical Society Meeting, Manchester, April 1972.     

Photo-sensitized oxidation of unsaturated fatty acid methyl esters. The identification of different pathways

The photo-sensitized oxidation of methyl linolenate and methyl oleate was studied using erythrosine and riboflavin as sensitizers. The complex mixtures of hydroperoxides obtained were analyzed for the proportion of conjugated products and, after reduction to the corresponding mixtures of hydroxystearates, for the distribution of positional isomers. By comparing the mixtures with that obtained from autoxidation, it was shown that the riboflavin reaction involved the “Type 1” mechanism of photosensitized oxidation which proceded via the formation of diene-radicals and yielded the same positional isomers of hydroperoxides as autoxidation. Thus, mixtures of the 8, 9, 10, and 11 positional isomers of allylic hydroperoxides were formed from oleate and the 9, 12, 13, and 16 isomers of conjugated diene-hydroperoxides from linolenate oxidation. The erythrosine reaction, on the other hand, proceded via the “Type 2”. mechanism which involved singlet oxygen as the oxygenating species. The mixtures of isomers resulting from oxidation involving singlet oxygen were different from those obtained by autoxidation. Oleate oxidation gave rise to a mixture of only the 9 and 10 positional isomers while the mixture obtained from oxidation of methyl linolenate contained non-conjugated hydroperoxide isomers (with the hydroperoxide group at positions 10 and 15) as well as the conjugated—9, 12, 13, and 16—isomers.     

Mechanistic study of drying of alkyd resins using (Z,Z)- and (E,E)-3,6-nonadiene as model substances

The cobalt-catalysed autoxidative drying of alkyd resins was studied using (Z,Z)-3,6-nonadiene and (E,E)-3,6-nonadiene as model compounds. A large number of reaction products were isolated from the autoxidation mixture using HPLC and preparative size exclusion chromatography and identified with ¹H- and ¹³C-NMR. The identified compounds comprised C₉ hydroperoxides, endoperoxides, epoxides, aldehydes and ketones and some other oxidation products. Their chemical structures pointed to three different types of oxidation processes taking place. Besides the main radical autoxidation reaction, evidence was found for photo-sensitized oxidation involving singlet oxygen. Thirdly, epoxidation occurs via peracids or hydroperoxides formed as intermediates. Because of the large number of possible isomers having very similar physical properties, isolation of dimers required considerable effort. Nevertheless, two dimers were isolated and characterized. Their structures indicate crosslinking to occur by recombination of radicals as termination reaction.     

Autoxidation of fats. I. Preparation and oxidation of alkylbenzene-maleic anhydride adducts

Summary It has been shown that certain alkylbenzenes having a hydrogen atom on the alpha carbon atom react with maleic anhydride to form compounds analogous to the hydroperoxides produced by autoxidation of the alkylbenzenes. The structure of these products has been determined by oxidation with aqueous permanganate and the results have been applied, together with other data, to demonstrate that the reaction proceeds by a free radical chain mechanism involving the abstraction of a hydrogen atom from the alpha carbon atom. In view of the analogy between the products obtained in this investigation and in autoxidation, it is suggested that autoxidation is propagated in a similar manner. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural and Research Administration, U. S. Department of Agriculture.     

Photoassisted oxypolymerization of alkyd resins: Kinetics and mechanisms

A new three-component system for photoassisted oxypolymerization of alkyd resins containing a drier, a photosensitizer and a radical generator was investigated. Polymerization profiles were recorded by real-time infrared spectroscopy for a thin film exposed for 1 h to simulated sunlight radiation. The kinetic results showed that the system follows complex kinetics. Multiple regression analysis was used to model the influence of the drier, the photosensitizer and the radical generator on the final conversion and total polymerization rate during photooxidation. The mechanisms involved were studied through laser spectroscopies. Laser flash photolysis was used to measure the rate constants of reaction between the radicals formed from the photodissociation of the radical generator and the model compounds of alkyd resins, leading to the rapid formation of hydroperoxides. The photosensitizer was expected to produce singlet state molecular oxygen that reacts on the alkyd resin, and time-resolved chemiluminescence technique was used to determine the quenching rate constant of singlet oxygen by model compounds. On the basis of these results, a mechanism for the photoassisted oxypolymerization of alkyd resins is proposed that account for the all the different reaction pathways.     

Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: Cholesterol product analysis as a probe of reaction mechanism

Cholesterol was used as an in situ probe for studying mechanisms of lipid peroxidation in isolated erythrocyte membranes subjected to different prooxidant conditions. The membranes were labeled with [¹⁴C]cholesterol by exchange with prelabeled unilamellar liposomes and photosensitized with hematoporphyrin derivative. Irradiation with a dose of blue light resulted in thiobarbituric acid-detectable lipid peroxidation that was increased markedly by subsequent dark incubation with 0.5–1.0 mM ascorbate (AH⁻). Ascorbate-stimulated lipid peroxidation was inhibited by EDTA, desferrioxamine (DOX) and butylated hydroxytoluene (BHT), suggesting that the process is free radical in nature and catalyzed by membrane-bound iron. Thin layer chromatography and radiometric scanning of extracted lipids from photooxidized membranes revealed that the major oxidation product of cholesterol was the 5α-hydroperoxide (5α-OOH), a singlet oxygen adduct. Post-irradiation treatment with AH⁻/Fe(III) resulted in an almost-total disappearance of 5α-OOH and the preponderance of free radical oxidation products, e.g. 7-ketocholesterol, the epimeric 7α-/7β-hydroperoxides (7α-/7β-OOH) and their respective alcohols (7α-/7β-OH). EDTA, DOX and BHT inhibited the formation of these products, while catalase and superoxide dismutase had no effect. These results are consistent with a mechanism involving 1-electron reduction of photogenerated hydroperoxides to oxyl radical, which trigger bursts of free radical lipid peroxidation. Though generated in this system, partially reduced oxygen species, viz. superoxide, hydrogen peroxide and hydroxyl radical, appear to be relatively unimportant in the autoxidation process. Presented at the symposium “Free Radicals Antioxidants, Skin Cancer and Related Diseases” at the 78th AOCS Annual Meeting in New Orleans, LA, May 1987.     

Antioxidant effects of chlorophyll and pheophytin on the autoxidation of oils in the dark. II. The mechanism of antioxidative action of chlorophyll

To understand the mechanism of the antioxidant effect of chlorophyll on the autoxidation of oils in the dark, antioxidant activities of several derivatives of chlorophyll were compared. Antioxidant activities were observed in chlorophyll derivatives such as protopor-phyrin methyl ester and its magnesium chelated compound. Porphyrin seems to be an essential chemical structure for the antioxidant activity of chlorophyll. Chlorophyll did not decompose the hydroperoxides, but reduced free radicals such as 1,1-diphenyl-2-picrylhydrazyl. Electron spin resonance spectrum of the π-cation radical was recorded during the oxidation of chlorophyll in methyl linoleate solution. These observations suggest that chlorophyll may act as a hydrogen donor to break the chain reaction.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry: II. Methyl linoleate

The gas chromatography-mass spectrometry (GC-MS) approach developed in the preceding paper was applied for qualitative and quantitative investigations of autoxidation products of methyl linoleate. A GC-MS computer summation method was standardized with synthetic 9- and 13-hydroxyoctadecanoate. Equal amounts of 9- and 13-hydroperoxides were found in all samples of linoleate autoxidized at different temperatures and peroxide levels. The results are consistent with the classical free radical mechanism of autoxidation involving a pentadiene intermediate having equivalent sites for oxygen attack at carbon-9 and carbon-13. Minor oxygenated products of autoxidation indicated by GC-MS include keto dienes, epoxyhydroxy monoenes di- and tri-hydroxy monoenes. These hydroxy compounds are presumed to be present in the form of hydroperoxides. The quantitative GC-MS method was found suitable for the analysis of autoxidized mixtures of oleate and linoleate. By this method, it is possible to determine the origin of the hydroperoxides formed in mixtures of these fatty esters. Presented at the AOCS Meeting, Chicago, September 1976.     

Effect of α-, β-, γ-, and δ-Tocotrienol on the Singlet Oxygen Oxidation of Lard

The impacts of four different types of tocotrienol homologues on the singlet oxygen oxidation of lard were evaluated by measuring the headspace oxygen content and the peroxide value. Singlet oxygen oxidation of lard was induced by chlorophyll photosensitization. Samples of 0.100, 0.250, and 0.400 M lard in methylene chloride containing chlorophyll and α‐, β‐, γ‐, or δ‐tocotrienol were prepared and stored under light at 3,000 lux for 4 h. All tocotrienol homologues at 1.20 mM significantly prevented the singlet oxygen oxidation of lard. Chlorophyll under light produced singlet oxygen at 1.09 μmol oxygen/mL headspace/h. A steady state kinetic study showed that tocotrienols reduced the singlet oxygen oxidation of lard by quenching the singlet oxygen. Singlet oxygen reacted with lard at 6.50 × 10⁴M^?1 s^?1. α‐, β‐, γ‐, and δ‐tocotrienol quenched singlet oxygen with the rate of 2.16, 1.99, 2.05, and 0.800 × 10⁷M^?1 s^?1, respectively. Among them, α‐tocotrienol significantly prevented singlet oxygen oxidation of lard.     

A Brief History of Lipid Oxidation

The history of our understanding of fat oxidation is reviewed from the early observations that oxygen was involved to the discovery of the role of singlet oxygen. It includes the debate about relative importance of triglyceride hydrolysis and oxidation; the search for tests that correlated with oxidized flavor; the evidence that oxidation involved the formation of hydroperoxides; the development of tests for hydroperoxides; and the work on the chemistry, mechanism, and kinetics of oxidation by the British Rubber Producers Research Association. The review ends with the effort to isolate and determine the structure of the oxidation products of the common unsaturated fatty acids, their relative rate of oxidation, and the role of singlet oxygen and metals in initiation.     

Untersuchungen über wirkungsmechanismen von lichtschutzmitteln. 1. mitteilung

From mechanistic considerations upon the probability of quenching-processes in the polymer matrix, it was concluded that there exists the possibility of a singlet oxygen mechanism occuring in the photodegradation of polypropylene. Using the sensitized photooxidation of rubrene, it was shown that compounds known to be light stabilizers are able to deactivate, to a greater or lesser extent, singlet oxygen. It is, therefore, not to be excluded that singlet oxygen plays also a role in the photooxidation of polymers. On the other hand, further experiments have shown that these same compounds can also act as radical scavengers or as hydroperoxide-deactivators.     

The free radical oxidation of polyunsaturated lecithins

Two unsymmetric polyunsaturated lecithins were allowed to air oxidize and the primary products of autoxidation were isolated and characterized. 1-Palmitic-2-linoleic-phosphatidylcholine undergoes significant oxidation after 16 hr at room temperature under air. A new phospholipid product may be isolated by reverse phase high pressure liquid chromatography (HPLC) and this HPLC fraction is shown to be made up of lipid hydroperoxides formed by free radical oxidation of the homoconjugated diene of the linoleate component of the lecithin. 1-Stearic-2-arachidonic-phosphatidylcholine undergoes a similar oxidation with the arachidonate polyunsaturated functionality being oxidized. The structure of the oxidation products was established by reduction of hydroperoxide with triphenylphosphine, snake venom hydrolysis of the C-2 ester, and HPLC analysis of the resulting hydroxy fatty acids or their methyl esters.     

Oxidation of all‐cis‐7,10,13,16,19‐docosapentaenoic acid ethyl ester. Hydroperoxide distribution and volatile characterization

The isomeric hydroperoxide distribution and the composition of volatiles generated by oxidation of all‐cis‐7,10,13,16,19‐docosapentaenoic acid ethyl ester (DPA Et) were determined. DPA Et was prepared by using seal blubber oils as raw material and purified by urea complexation and reverse‐phase high‐performance liquid chromatography (HPLC). The DPA Et of over 96% purity thus obtained was dissolved in methanol and subsequently divided into two portions. One portion was added with methylene blue and exposed to a tungsten bulb light at 5 °C for photosensitized oxidation. The other portion was added with 2,2'‐azobis (2,4‐dimethylvaleronitrile) as an azo‐radical initiator and kept in the dark at room temperature for autoxidation. Positional isomers of hydroperoxides generated by autoxidation or photosensitized oxidation of DPA Et were separated by normal‐phase HPLC and detected by a fluorescence detection system as well as UV absorption. The peak components were identified by gas chromatography‐mass spectrometry (GC‐MS). Eight isomeric hydroperoxides, including certain amounts of 7‐, 10‐, 11‐, 13‐, 14‐, 16‐, 17‐, and 20‐hydroperoxy docosapentaenoate, were generated by autoxidation of DPA Et. The photosensitized oxidation of DPA Et yielded not only the above eight hydroperoxide isomers but also two additional isomeric hydroperoxides, 8‐ and 19‐hydroperoxy docosapentaenoate, which are characteristic hydroperoxide isomers generated by singlet oxygen‐mediated oxidation. Volatiles formed by autoxidation of DPA Et at 50 °C were collected and analyzed by solid‐phase micro‐extraction and GC/GC‐MS. A number of aldehydes, ketones, alcohols, acids, furans and hydrocarbons were identified. The formation mechanisms of certain volatiles are discussed.     

Peroxidation of free and esterified fatty acids by horseradish peroxidase

Linoleic and arachidonic acids and unsaturated esterified fatty acids of soybean lecithin liposomes were oxidized by horseradish peroxidase in the presence of hydrogen peroxide. The major products formed in the presence of oxygen were fatty acid hydroperoxides. In the absence of oxygen, other unidentified products were formed. Diene conjugation was about 5 times faster in oxygen than in nitrogen. Malondialdehyde was formed only in the presence of oxygen. Superoxide, singlet oxygen and hydroxyl radical may have been involved in the free fatty acid oxidation system but not in the liposome system. Replacement of hydrogen peroxide with the hydrogen peroxide (and superoxide) generators xanthine oxidase or galactose oxidase caused a more efficient oxidation in the presence of peroxidase than in its absence, suggesting that the in vivo toxicity of hydrogen peroxide and superoxide may be greatly increased in the presence of peroxidase.     

Sulfite-induced lipid peroxidation

Sulfite initiated the peroxidation of linoleic acid and linolenic acid emulsions via a free radical mechanism. Peroxidation of these fatty acids required oxygen and sulfite and occurred with concomitant oxidation of sulfite to sulfate. In reaction mixtures containing linoleic acid, the formation of conjugated diene equaled the formation of hydroperoxide. In reaction mixtures containing linolenic acid emulsions, thiobarbituric acid reactive materials were also formed. Peroxidation was pH-dependent; peroxidation of linoleic acid proceeded between pH 4 and 7, but linolenic acid peroxidation was significant only if pH was below pH 6. The linoleic acid hydroperoxides thus formed were reduced and methylated to methyl hydroxystearate. Analysis of methyl hydroxystearate by gas chromatographymass spectrometry indicated that sulfite-induced peroxidation gave rise to the 9- and 13-hydroperoxy isomers. In addition to the hydroperoxides, sulfite adducts were detected. Hydroquinone, butylated hydroxytoluene and α-tocopherol effectively inhibited both sulfite oxidation and hydroperoxide formation. Conjugated diene formation also was inhibited by 4-thiouridine, suggesting that the reaction is mediated by the sulfite radical. No significant inhibition was observed with the addition of superoxide dismutase, catalase, or the hydroxyl radical scavengers, mannitol ort-butanol. A possible mechanism is presented to account for sulfite-induced peroxidation of linoleic acid.     

Lipid peroxidation in erythrocyte membranes: Cholesterol product analysis in photosensitized and xanthine oxidase-catalyzed reactions

The effects of singlet oxygen- and oxygen radical-induced lipid peroxidation on cell membrane integrity were compared, using the human erythrocyte ghost as a model system. Resealed ghosts underwent lipid peroxidation and lysis (release of trapped glucose-6-P) when irradiated in the presence of uroporphyrin (UP) or when incubated with xanthine (X), xanthine oxidase (XO) and iron. The UP-sensitized process was inhibited by azide but not by phenolic antioxidants, consistent with singlet oxygen (nonradical) involvement. This was confirmed by showing that the predominant photoproduct of membrane cholesterol was the 5α-hydroperoxide. Total hydroperoxide (LOOH) content in UP-photooxidized ghosts increased linearly during the prelytic lag and throughout the period of rapid lysis. Unlike the photoreaction, X/XO/iron-dependent peroxidation and lysis was inhibited by catalase, superoxide dismutase and phenolic antioxidants, indicating O₂ ⁻/H₂O₂ intermediacy and a free radical mechanism. Correspondingly, only radical reactions products of cholesterol were formed, notably the 7α-, 7β-hydroperoxide pair. Membranes lysis had a distinct lag as in photooxidation; however, the LOOH profile was more complex, with an initial lag followed by a sharp increase and then slow decline. X/XO/iron-induced lysis commenced when LOOH levels were 2–3 times higher than in photosensitized lysis, suggesting that the pathways of membrane lesion formation are different in the two systems. In low concentrations, ascorbate exacerbated the damaging effects of photoperoxidation, switching the reaction from primarily singled oxygen- to oxygen radical-dependence, as indicated by cholesterol product analysis.     

Novel hydroperoxides as primary autoxidation products of a model ethoxylated surfactant

Ethoxylated alcohols, widely used as surfactants, are susceptible to oxidation when exposed to air. A complex mixture is formed, in which alkylated aldehydes, alkylated formates, hydroxyaldehydes, and formaldehyde have previously been identified by our group. The compounds identified so fat are all secondary oxidation products, some of which have been shown to be skin sensitizers and irritants. The primary oxidation products from ethoxylated surfactants have been described as peroxides and hydroperoxides, but their structures have not been elucidated more closely. Hydroperoxides are reactive species and can be suspected to be biologically active as skin sensitizers and irritants. In the present study we used a small model compound, diethylenglycol monoethylether (C₂E₂), to facilitate the identification of primary oxidation products formed at autoxidation of ethoxylated surfactants. By performing NMR and HPLC-MS analyses, we found that at least four different hydroperoxides were formed at autoxidation of C₂E₂, one of them dominating in concentration over the others. The hydroperoxide present in the highest concentration was identified as 2-[2-(1-hydroperoxyethoxy)ethoxy]ethanol.     

The kinetics and mechanism of metal-catalyzed autoxidation

The autoxidation of organic compounds, RH, occurs by a radical-catalyzed chain reaction to give hydroperoxides, RO₂H, as primary products. The initial rate is -d[O₂]/dt = k_p[RH] {k_i[Cat]/k_t}^1/2, or in the presence of an inhibitor, (In), k_p[RH](k_i[Cat]/k_I[In]), where k_p is the chain propagation rate; k_i[Cat], the rate of radical catalysis; k_t chain termination rate; k_I[In] rate of inhibitor action. As oxidation proceeds the hydroperoxides break down to give further catalytically active radicals and eventually an autoxidation may reach a maximum rate of k _p ² [RH]²/fk_t, independent of the concentration or nature of the catalyst. Photosensitization, by forming singlet oxygen, can catalyze autoxidation by forming peroxides. Compounds of many transition metals, e.g., Co, Mn, Fe, act as secondary catalysts by promoting the rapid formation of radicals from RO₂H molecules by a one-electron transfer reaction RO-OH + M²⁺→RO· + M³⁺ + OH⁻ and the M³⁺ ions are then reconverted to M²⁺ ions giving further radicals. The overall catalytic activity of a metallic ion is controlled by the slower step of the M²⁺⇌M³⁺ + e redox cycle and depends on the electronic structures of the two ions concerned and on the ligand groups attached to them. These effects are discussed in detail since ligand molecules for transition metal ions can be selected so as either to promote or inhibit autoxidation. Special reference is made to biological catalysts, such as the porphyrins, found in food products. Direct activation of oxygen by metallic complexes rarely seems to occur, but direct oxidation of substrates by metallic compounds is possible. This leads to another redox cycle which is utilized in copper-containing enzymes. One of 28 papers presented at the Symposium, “Metal-Catalyzed Lipid Oxidation,” ISF-AOCS World Congress, Chicago, September 1970.     

Antioxidants and stabilizers LXVII.. Transformation of 4,4′-thiobis(2-methyl-6-tert-butylphenol) under conditions of inhibited autoxidation

The mechanism of the antioxidative effect of thiobisphenols in the inhibition of autoxidation of polyolefins initiated by the decomposition of hydroperoxides was studied by means of a model reaction of 4,4′-thiobis(2-methyl-6-tert-butylphenol) (I) with tert-butylhydroperoxide in the presence CO(II)acetyl acetonate. Products isolated from the reaction mixture prove that in the inhibition of autoxidation, thiobisphenols break as phenols the propagation chains of radical autoxidation, while they decompose as sulphides the hydroperoxides into nonradical products.     

Reaction of oxygen and unsaturated fatty acids

Oxygen reacts readily with unsaturated fatty acids so that every time these compounds are handled there is a danger they will become contaminated with oxidation products. The products formed first are allylic hydroperoxides which are labile molecules that change rapidly to other compounds, some of which are highly flavorous. Sometimes these changes are desirable and may be promoted: frequently they are not and have to be inhibited. Instrumental procedures recently introduced—especially separation by high performance liquid chromatography and identification by¹H and¹³C nuclear magnetic resonance spectroscopy—have led to a renewed interest in this subject. For the nonenzymic processes of autoxidation and photooxygenation we now have a better understanding of the routes leading to the first-formed allylic hydroperoxides and an improved appreciation of the structure of further oxidation products including dihydroperoxides and hydroperoxides which also contain one or more cyclic peroxide units. Direct chemical routes to several of these compounds have also been developed. Oxidation of linoleic acid by plant-derived lipoxygenases gives diene hydroperoxides similar to those produced by autoxidation, except that the former are optically active and the latter racemic. Enzymic oxidation of arachidonic acid and certain related C₂₀ acids in animal systems produces a wide variety of prostaglandins, physiological properties. These compounds have been described as “tomorrow’s drugs”.