Prooxidant effects of inorganic chromium compounds in the autoxidation of methyl linoleate

The prooxidant property of inorganic chromium compounds was determined in methyl linoleate free from natural antioxidants and metals. Prooxidant properties of inorganic chromium compounds appeared in order of sodium chromate > chromium (VI)-oxide > chromium chloride > potassium chromate > chromium (III)-oxide > potassium dichomate. In comparison with the control, additions of chromium compounds induced different amounts of autoxidation products derived from methyl linoleate, such as small amounts of hydroperoxides and conjugated dienes and large amounts of hydroxy groups,α,β,γ,δ-unsaturated carbonyls, isolatedtrans double bonds, polymers, and free radicals. From these analytical data, the catalysis of chromium compounds in the autoxidation of methyl linoleate seemed to be based on their abilities of abstracting a hydrogen from methyl linoleate and decomposing hydroperoxides derived from the autoxidation of methyl linoleate.     

Concurrent oxidation of accumulated hydroperoxides in the autoxidation of methyl linoleate

Summary 1. Kinetic studies showed that concurrent oxidation of preformed hydroperoxides may be expected to take place at all stages of the autoxidation of methyl linoleate. The rate of oxidation relative to the rate of autoxidation of unoxidized ester is determined chiefly by the extent of the accumulation of hydroperoxides. 2. Infrared spectral analysis of hydroperoxides oxidized to various degrees indicated thattrans, trans diene conjugation and isolatedtrans double bonds produced in the autoxidation of methyl linoleate are related to the concurrent oxidation of the accumulated hydroperoxides. 3. The low absorptivity observed for diene conjugation, compared to that which may be expected for the exclusive production ofcis, trans diene conjugated hydroperoxide isomers during the autoxidation of methyl linoleate is attributed to the concurrent oxidation of accumulated hydroperoxides. 4. The effect of antioxidants in giving a well-defined induction period in the oxidation of hydroperoxides isolated from autoxidized methyl linoleate indicated that the oxidation proceeds by a chain reaction. 5. The primary reaction products of the oxidation of hydroperoxides isolated from autoxidized methyl linoleate were found to be polymers formed in a sequence of reaction involving the diene conjugation. 6. Studies on the autoxidation of methylcis-9,trans-11-linoleate showed thatcis, trans isomerization of the conjugated diene took place with the concurrent production of isolatedtrans double bonds and loss of diene conjugation. Hormel Institute publication no. 138. Presented before the American Oil Chemists’ Society, Philadelphia, Pa., Oct. 10–12, 1955. This work was supported by a grant from the Hormel Foundation.     

On the kinetics of the autoxidation of fats. II. Monounsaturated substrates

The autoxidation of methyl oleate and oleic acid shows some differences as compared to the autoxidation of linoleate,e.g., the formation of water at an early stage. Linearization of experimental data on the autoxidation to high oxidation degrees of methyl oleate and other monounsaturated substrates shows that the rate equations previously derived for methyl linoleate in the range of 1–25% oxidation are valid, provided the correct expression for the remaining unreacted substrate is used. With monounsaturated substrates, part of the oxygen is consumed by a secondary oxidation reaction almost from the beginning, and only a certain constant fraction α of the total O₂ consumption is consumed in hydroperoxide formation. The fraction α is different for methyl oleate, oleyl alcohol, oleic acid andcis 9-octadecene, but the rate constant for the hydroperoxide formation is the same for all of them when experimental conditions are the same. The main difference between oleate and linoleate autoxidation is the much faster decomposition of the oleate hydroperoxides relative to their slow formation.     

Kinetic investigation into glucose-, fructose-, and sucrose-activated autoxidation of methyl linoleate emulsion

Autoxidation of methyl linoleate emulsions in aqueous phosphate buffer solutions in the presence of glucose, fructose, and sucrose has been studied by the rate of oxygen uptake. Oxidation rates increased with increasing concentration of sugars in the system. At comparable molar ratios of sugar to methyl linoleate the rate of oxidation in the presence of fructose was greater than with glucose which, in turn, was greater than with sucrose. Oxidation rates increased with pH until a maximum rate was reached at pH 8.00. There was less conjugation and more CO₂ with fructose than with glucose at a comparable level of oxygen uptake and pH value. This suggested concurrent oxidation of methyl linoleate and sugars; fructose is the most readily oxidized of those studied. Sugars seemed to be effective only in combination with the resulting methyl linoleate hydroperoxide. The effect of sugars rests in an activation of the decomposition of the linoleate hydroperoxide, and on the acceleration of the autocatalysis. The activation energy values for the autoxidation of methyl linoleate emulsions in the presence of sucrose, glucose, and fructose are 14.9, 10.6, and 10.6 K. Cal./mol. at pH 5.50; 16.0, 10.8, and 10.4 K. Cal./mol. at pH 7.00; and 14.4, 10.2, and 8.8 K. Cal. at pH 8.00, respectively. Addition of ascorbic acid to the system at zero time or after 2 hrs. increased oxygen absorption. It appeared that oxidized methyl linoleate caused co-oxidation of the ascorbic acid. Additions of nordihydroguaiaretic acid, propyl gallate, and hydroquinone to the system were ineffective in stopping oxidation when they were added after oxidation had commenced. They reduced effectively the rate of oxidation when added at zero time. Presented at the 52nd annual meeting, American Oil Chemists' Society, St. Louis, Mo., May 1–3, 1961. American Meat Institute Foundation Journal Paper No. 215.     

Hydroperoxide formation during autoxidation of conjugated linoleic acid methyl ester

The aim of this study was to investigate whether hydroperoxides are formed in the autoxidation of conjugated linoleic acid (CLA) methyl ester both in the presence and absence of α‐tocopherol. The existence of hydroperoxide protons was confirmed by D₂O exchange and by chemoselective reduction of the hydroperoxide groups into hydroxyl groups using NaBH₄. These experiments were followed by nuclear magnetic resonance (NMR) spectroscopy. The ¹³C and ¹HNMR spectra of a mixture of 9‐hydroper‐oxy‐10‐trans,12‐cis‐octadecadienoic acid methyl ester (9‐OOH) and 13‐hydroperoxy‐9‐cis, 11‐trans‐octadecadienoic acid methyl ester (13‐OOH), which are formed during the autoxidation of methyl linoleate, were studied in detail to allow the comparison between the two linoleate hydroperoxides and the CLA methyl ester hydroperoxides. The ¹³CNMR spectra of samples enriched with one of the two linoleate hydroperoxide isomers were assigned using 2D NMR techniques, namely Correlated Spectroscopy (COSY), gradient Heteronuclear Multiple Bond Correlation (gHMBC), and gradient Heteronuclear Single Quantum Correlation (gHSQC). The ¹³C and ¹H NMR experiments performed in this study show that hydroperoxides are formed during the autoxidation of CLA methyl ester both in the presence and absence of α‐tocopherol and that the major isomers of CLA methyl ester hydroperoxides have a conjugated monohydroperoxydiene structure similar to that in linoleate hydroperoxides.     

A kinetic study of the autoxidation of methyl linoleate and linoleic acid emulsions in the presence of sodium chloride

Autoxidation of linoleic acid and methyl linoleate emulsions in aqueous buffer solutions was studied by the rate of oxygen uptake. The oxidation rates of methyl linoleate emulsions increased with an inerease in the pH of the buffer solution. With linoleic acid, oxidation rates rose until the increase reached its peak at pH 5.50 and then decreased gradually to a minimum at pH 8.00. Oxidation rates of methyl linolente and linoleic acid emulsious decreased with increased concentration of NaCl in the system. The effect of variation of pH of the emulsion in the range investigated was similar to that in emulsions without NaCl. There was no evidence that NaCl accelerated the oxidation rates in the system. The observed inhibitory effect of NaCl may result from the decreased solubility of oxygen in the emulsion with the increased concentration of NaCl. Consequently the availability of oxygen would be a limiting factor in oxidation rates. The activation energy for the monomolecular and bimolecular reactions of methyl linoleate and linoleic acid autoxidation was found to be independent of the pH value and sodium chloride concentration of the system. The energy of activations for the monomolecular and bimolecular reactions of methyl linoleate and of linoleic acid are 22,000, 18,200, 19,600, and 16,400 cal./mol., respectively. Spectrophotometric studies of the autoxidized emulsions of linoleic acid and its methyl ester indicate that the magnitude of the absorption at 2325 Å is the same at different pH values. On the contrary, the secondary products showing absorption at 2775 Å are to some extent dependent on the pH value of the emulsion.     

Study on the oxidative rate and prooxidant activity of free fatty acids

Oleic, linoleic and linolenic acids were autoxidized more rapidly than their corresponding methyl esters. Addition of stearic acid accelerated the rate of autoxidation of methyl linoleate and the decomposition of methyl linoleate hydroperoxides. Therefore, the higher oxidative rate of FFA’s than their methyl esters could be due to the catalytic effect of the carboxyl groups on the formation of free radicals by the decomposition of hydroperoxides. Addition of stearic acid also accelerated the oxidative rate of soybean oil. This result suggests that particular attention should be paid to the FFA content that affects the oxidative stability of oils.     

Peroxide value determination—Comparison of some methods

A comparison between the determination of the peroxide value by the methods of Wheeler and Sully (iodometric titration) and that of Stine, et al. (ferric thiocyanate method) was made. Some oxidized vegetable oils, H₂O₂, t-butyl hydroperoxide, cumene hydroperoxide, methyl oleate hydroperoxides, and methyl linoleate hydroperoxides were used as substrates. One-hundred percent of the methyl linoleate hydroperoxides were recovered by the Wheeler reduction, 85% by the Sully method. The Wheeler method was used to reduce the methyl linoleate hydroperoxides to the corresponding hydroxy acids. In the Sully procedure, the hydroxy acids are only intermediates which are dehydrated to octadecatrienoic acids. One equivalent methyl linoleate hydroperoxide oxidized two equivalents of I⁻ (Wheeler) and four equivalents of Fe²⁺ (Stine, et al.). By way of contrast, H₂O₂ needs only two equivalents I⁻ or Fe²⁺ for reduction. The excess consumption of reduction equivalents in the ferric thiocyanate method probably is caused by secondary reactions of the methyl linoleate hydroperoxide acyl residue.     

Photochemical oxidation of fatty acid esters with and without chlorophyll

1. It has been confirmed that the principal products formed in the oxidation of methyl oleate by oxygen under a variety of conditions are predominantlytrans hydroperoxides. However no inversion of the double bond occurs in unoxidized oleate. Hence the conversion ofcis totrans double bonds and peroxide formation occur together in the same molecules.
2. The autoxidation of methyl linoleate at low temperature yields predominantlycis,trans conjugated hydroperoxides. Autoxidation at 25°C., oxidation catalyzed by visible light, or ultraviolet light and copper soap catalyzed oxidation at temperatures appreciably above 0°C., lead to the formation primarily oftrans,trans conjugated hydroperoxides. The inversion of the second double bond in this case appears to be independent of the peroxide-forming reactions.
3. The photochlorophyll oxidation of methyl linoleate leads to the formation of some unconjugated hydroperoxides, some of which containtrans double bonds.
4. Under all of the conditions employed in the present investigation, the oxidation of methyl oleate and linoleate led primarily to the formation of monomeric peroxides which retained most of the unsaturation of the parent compound.

     

Ion exchange resins and ethyleneimine polymer as antioxidants II: Autoxidation products

The antioxidant effects of ion exchange resins and ethyleneimine polymer on the autoxidation products of methyl linoleate in a heterogeneous reaction system are discussed. Results from analyses of the various autoxidation products from linoleate samples with and without the antioxidants showed that the addi-tion of the antioxidants did not change the original autoxidation mechanism of methyl linoleate. How-ever, the antioxidants did retard the autoxidation in response to their antioxidant activity and, compared with a linoleate control, changed the yields of some autoxidized products such as an increased amount of conjugated diene hydroperoxides in linoleate samples with added ion exchange resins.     

The peroxidizing effect of α-tocopherol on autoxidation of methyl linoleate in bulk phase

In order to understand the effect of α-tocopherol on the autoxidation mechanism of edible oil under storage conditions, methyl linoleate was allowed to autoxidize at 50 C in bulk phase without any radical initiator. The reaction was monitored by determining the production of four isomeric hydroperoxides (13-cis,trans; 13-trans,trans; 9-cis,trans; 9-trans,trans) by high performance liquid chromatographic analysis after reduction. In the absence of α-tocopherol, the rate of autoxidation depended on the sample size, and the duration of the induction period was affected by the initial level of hydroperoxides. However, the distribution of c-t and t-t hydroperoxide isomers remained constant during the propagation period regardless of the sample size. The addition of α-tocopherol at 0.1 and 1.0% caused a linear increase in the amount of hydroperoxides and elevated the distribution of the c-t isomers. The rate of hydroperoxidation appeared to be governed by the initial concentration of α-tocopherol rather than the sample size or the initial hydroperoxide level. This peroxidizing effect of α-tocopherol was suppressed by the presence of ascorbyl palmitate. A mechanism in which chromanoxy radical participates is proposed for the effect of α-tocopherol on lipid autoxidation in bulk phase. It is therefore suggested that α-tocopherol at high concentrations influences the mechanism of autoxidation of edible oil.     

Formation of peroxides in fatty esters. II. Methyl linoleate: application of the polarographic and direct oxygen methods

Summary This study of the prolonged autoxidation of methyllinoleate at 80°C. has included polarographic identification and determination of hydroperoxides, the direct determination of oxygen contents, and catalytic micro-hydrogenation for the determination of unsaturation. The polarographic method has further substantiated the observations of other workers that the principal peroxidic substance formed during the autoxidation of methyl linoleate at 80°C. is a hydroperoxide. The direct oxygen measurements have shown that most of the oxygen absorbed in the initial stages of autoxidation can be accounted for as hydroperoxide. During the latter stages there was a continuous increase of oxygen uptake, half of which can be accounted for as free acid and half as forms other than hydroperoxide, ester, or free acid. By means of the catalytic micro-hydrogenation method it has further been shown that as the autoxidation progresses there is a continuous decrease in unsaturation.     

A possible role for singlet oxygen in the initiation of fatty acid autoxidation

A mechanism for the initiation of autoxidation in fatty acids is proposed which involves singlet state oxygen, formed through a photosensitization reaction, as the reactive intermediate. Both singlet oxygen generated in a radio-frequency gasdischarge, and photosensitization by natural pigments, were shown to catalyze the oxidation of methyl linoleate. The involvement of singlet oxygen was shown by the identification of nonconjugated hydroperoxides as products common to both photooxidation and singlet O₂ oxidation. Nonconjugated hydroperoxides could not be detected among the free radical autoxidation products. Further proof for the above mechanism was gained by showing that compounds known to react strongly with singlet oxygen, inhibited the photooxidation. With the exception of chlorophyll, all sensitizers could be completely inhibited. Although singlet oxygen formation can account for approximately 80% of the observed chlorophyll photooxidation, at least one other mechanism must be involved. It is postulated that proton abstraction by the photoactivated carbonyl group of chorophyll could account for the remaining 20% of the observed photooxidation. The conclusion is drawn that oxygen, excited to its singlet state by a photosensitization process, plays the important role of forming the original hydroperoxides whose presence is necessary before the normal free radical autoxidation process can begin.     

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.     

Distribution of14C after oral administration of (U-14C)labeled methyl linoleate hydroperoxides and their secondary oxidation products in rats

To study the toxicity of low molecular weight (LMW) compounds formed during the autoxidation of oils,¹⁴C-labeled primary monomeric compounds (methyl linoleate hydroperoxides) and secondary oxidation products, i.e., polymer and LMW compounds prepared from autoxidized methyl [U-¹⁴C]linoleate hydroperoxides (MLHPO) were orally administered to rats, and their radioactive distributions in tissues and organs were compared. The polymeric fraction consisted mainly of dimers of MLHPO. For the LMW fraction, 4-hydroxy-2-nonenal, 8-hydroxy methyl octanoate and 10-formyl methyl-9-decenoate were identified as major constituents by gas chromatography-mass spectrometry (GC-MS) after chemical reduction and derivatization. When LMW compounds were administered to rats,¹⁴CO₂ expiration and the excreted radioactivity in urine in 12 hr were significantly higher than those from polymer or MLHPO administration. Maximum¹⁴CO₂ expiration appeared 2–4 hr after the dose of LMW compounds. Radioactivity of the upper part of small intestines six hr after the dose of LMW compounds was higher than the values from administered polymer or MLHPO. The remaining radioactivity in the digestive contents and feces 12 hr after administration of LMW compounds was much lower than the values observed from administered polymer or MLHPO. Among internal organs, the liver contained the highest concentration of radioactivities from polymer, MLHPO and LMW fractions, and an especially higher level of radioactivity was found in liver six hr after the administration of LMW compounds. Six hours after the dose of LMW compounds, a relatively higher level of radioactivity also was detected in kidney, brain, heart and lung. These results show that the LMW compounds from MLHPO autoxidation are more easily absorbed in rat tissues than polymer and MLHPO.     

Isolation and identification of carbonyl compounds formed by autoxidation of ammonium linoleate

1. The isolation and identification of carbonyl compounds formed by autoxidation of pure ammonium linoleate (0.02 M. in water) has been carried out.
2. The autoxidation has been performed according to a method described by Tappel (23). A solution of 0.02 M. ammonium linoleate in 0.1 M. phosphate buffer of pH 6.9, containing 10⁻⁴ M. CuSO₄, was subjected to autoxidation at 37.2°C.
3. The isolation and identification of the D.N.P.-ones of the carbonyl compounds have been carried out, using a method of partition and adsorption chromatography published by van Duin (10, 11).
4. It was demonstrated that the principal compounds formed were n-hexanal, 2-octenal, and 2,4-decadienal.
5. The formation of these aldehydes is fully in line with the theories of autoxidation of unsaturated fatty acids (1, 3, 13) and of the dismutation of the hydroperoxides formed (2).

     

Mechanism of lipoxidase reaction. I. Specificity of hydroperoxidation of linoleic acid

Linoleate hydroperoxides from autoxidation of methyl linoleate and from lipoxidase oxidation of linoleic acid are compared. Data indicate an equal amount of methyl 9- and 13-hydroperoxyoctadecadienoate produced by autoxidation of methyl linoleate, and the exclusive formation of 13-hydroperoxyoctadeca-9,11-dienoic acid from the incubation of lipoxidase with linoleic acid. As a result of these findings, a specific mechanism for the reaction of lipoxidase with linoleic acid is postulated. Presented at the AOCS Meeting, Philadelphia, October 1966. This work was conducted under a Postdoctoral Resident Research Associateship established at the Northern Laboratory by ARS, USDA, in association with the National Academy of Sciences-National Research Council. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Autoxidation of methyl linoleate initiated by the ozonide of allylbenzene

Allylbenzene ozonide (ABO), a model for polyunsaturated fatty acid (PUFA) ozonides, initiates the autoxidation of methyl linoleate (18∶2 ME) at 37°C under 760 torr of oxygen. This process is inhibited by d-α-tocopherol (α-T) and 2,6-di-ert-butyl-4-methylphenol (BHT). The autoxidation was followed by the appearance of conjugated diene (CD), as well as by oxygen-uptake. The rates of autoxidation are proportional to the square root of ABO concentration, implying that the usual free radical autoxidation rate law is obeyed. Activation parameters for the thermal decomposition of ABO were determined under N₂ in the presence of radical scavengers and found to be E_a=28.2 ±0.3 kcal mol⁻¹ and log A=13.6±0.2; k_d (37°C) is calculated to be (5.1±0.3)×10⁻⁷ sec⁻¹. Autoxidation data are also reported for ozonides of 18∶2 ME and methyl oleate (18∶1 ME).     

Photo-initiated peroxidation of lipids in micelles by azaaromatics

The monoazaaromatics, pyridine (1), hexyl nicotinate (2), and quinoline (3) and diazaaromatics pyrimidine (4) and purine (5), readily act as photo-initiators for the peroxidation of methyl linoleate in 0.50 M SDS at 37°C giving free radical chain oxidations of linoleate. Quantitative kinetic runs on the order in substrate, RH, and in the rate of chain initiation, R_i, showed that the classical rate law for autoxidation,-d[O₂]/dt=(k _p/(2 k _t ^1/2))[RH]xR _i ^1/2, is applicable to these photo-initiated oxidations. The oxidizability of methyl linoleate under these conditions is 2.92×10⁻² M^−1/2 s^−1/2. These peroxidations were inhibited by chromanol phenolic antioxidants of the vitamin E class, such as lipid-soluble 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC) and water-soluble 2-carboxy-2,5,7,8-tetramethyl-6-hydroxychroman (Trolox) and derived rate constants for inhibition of peroxidation were k _inh (PMHC)=4.35×10⁴ M⁻¹ s⁻¹ and k _inh (Trolox)=2.81×10⁴ M⁻¹ s⁻¹ during inhibited oxidation of methyl linoleate photo-initiated by 4. The products from photo-initiated peroxidation of methyl linoleate by 1 through 5 were determined by reduction and high-performance liquid chromatography analyses to be the 9-and 13-positional hydroperoxides of the four geometrical isomers: cis-9, trans-11 (6), trans-10, cis-12 (7), trans-9, trans-11 (8), and trans-10, trans-12 (9)-octadecadienoates typical of the free radical chain mechanism of lipid peroxidation. Products from dye-sensitized oxidation by Methylene Blue or Rose Bengal of methyl linoleate gave a product distribution of six hydroperoxides typical of oxidation by singlet oxygen. Thermal or photo-initiated peroxidation of methyl linoleate in SDS gave some selectivity of oxidation at the 13-position of the linoleate chain. The ratio of 13-to 9-oxidation varied in the range 1.23 to 1.14 as the cis/trans to trans/trans ratio of geometric isomers varied from 0.44 to 1.25 during photooxidation of increased amounts of linoleate in SDS. This selectivity is attributed to loss of the pseudo symmetry around the pentadienyl system in the lipid chain in the SDS system during the peroxidation.     

Effect of α-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides

α-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180°C were analyzed by capillary gas chromatography. In the presence of either α-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples. When an isomeric mixture of methyl linoleate hydroperoxides (cis, trans andtrans, trans 9- and 13-hydroperoxides) was decomposed by heat, the presence of α-tocopherol and 1,4-cyclohexadiene caused the relative amounts of pentane and methyl octanoate to decrease and hexanal and methyl 9-oxononanoate to increase. A similar effect of α-tocopherol was observed on the distribution of volatiles formed from a mixture of thetrans,trans 9- and 13-hydroperoxides. This effect of α-tocopherol was, however, insignificant with purecis,trans 13-hydroperoxide of methyl linoleate. The decrease in total volatiles with the hydrogen donor compounds, α-tocopherol and 1,4-cyclohexadiene, indicates a suppression of homolytic β-scission of the hydroperoxides, resulting in a change in relative distribution of volatiles. The increase in hexanal and methyl 9-oxononanoate at the expense of pentane and methyl octanoate in the presence of hydrogen donor compounds supports the presence of a heat-catalyzed heterolytic cleavage (also known as Hock cleavage), which seems to mainly affect thetrans,trans isomers of linoleate hydroperoxides.