Characteristics of Effective and Host Specific Ineffective Strains of Rhizobium japonicum

The formation of dimers in the initial stage of methyl linoleate (ML) autoxidation was demonstrated. The oxidation profile of freshly prepared ML was followed by TLC during autoxidation by aeration at 30°C for 192 hr. After 24 hr of autoxidation, the peroxide value of ML was still 0.6, and two unknown polar spots appeared besides intact ML and methyl linoleate hydroperoxides (MLHPO). These two spots were identified as dimers by successive gel and high performance liquid Chromatographic separations and by molecular weight determination. The ratio of dimers/MLHPO reached a maximum (0.74) after 96 hr of autoxidation. This result indicates that the formation of dimers in the initial stage of autoxidation was slightly less than that of MLHPO. The dimers were linked through ?C?O?O?C? bonds and contained hydroperoxy and/or carbonyl groups and conjugated dienes.     

Further Oxygenated Compounds Produced from Methyl Linoleate Monohydroperoxides at the Process of Autoxidation

The primary stable products, methyl linoleate monohydroperoxides (MLHPO), formed by the autoxidation of methyl linoleate were characterized by gas chromatography-mass spectrometry. MLHPO was converted into methyl hydroxy stearates which consisted of two isomers, methyl 9-hydroxy and methyl 13-hydroxy stearate. Trimethylsilyl ether derivatives of these hydroxy isomers were separated directly by gas chromatography and mass fragmentgraphy. MLHPO was degradated by incubating under aerobic condition at 37°C for a week, and the quantity of MLHPO was determined as hydroxy derivatives. Decrease of MLHPO was almost similar to that of conjugated diene structure, but the peroxide value was not appreciably decreased during the incubation. This fact based on the formation of further oxygenated compounds. After chemical reduction, these compounds were identified as methyl 9,13-hydroxy octadecenoate and methyl 9,12,13- or 9,10,13-trihydroxy octadecenoate, in which oxygen attached to the conjugated diene. The formation mechanisms of these oxygenated compounds are proposed.     

The effect of dietary lipid hydroperoxide on lymphoid tissues in mice

Effects of dietary lipid hydroperoxides on lymphoid tissue were studied in mice. When graded amounts (190, 270 and 310 mg) of methyl linoleate hydroperoxide (MLHPO) were orally administered to male C57BL/6 mice (6 weeks old), necrosis was observed in lymphocytes located among the reticular network in the thymus, and thymus weight was significantly decreased 24 h after the treatment. The spleen weight of mice given MLHPO tended to decrease. Spontaneous chemiluminescence of the thymus was remarkably increased after the dose. Thiobarbituric acid reactants in the liver, thymus and blood were also increased after the dose of MLHPO. At intervals of 3, 6, 12 and 24 h after a dose of 14C-labeled MLHPO, 14C was detected in the blood and liver. Fatty infiltration of the liver was found after the treatment with MLHPO. These findings indicate that oral intake of lipid hydroperoxides causes significant damage to lymphoid tissues of mice.     

Injury of mouse lymphocytes caused by exogenous methyl linoleate hydroperoxides in vitro.

The incubation with methyl linoleate hydroperoxides (MLHPO), a model of lipid peroxides, depressed DNA, RNA and protein syntheses of mouse thymic lymphocytes and increased the amount of thiobarbituric acid-reactive substances in lymphocytes. These phenomena were also found in the splenic lymphoblasts in the DNA synthetic phase (S-phase) obtained by mitogen. Prior culturing with all-rac-alpha-tocopherol increased DNA synthesis in splenic lymphoblasts. Electron microscopically, cytoplasmic micro-organelles of splenic lymphoblasts in the S- and G2-phases were markedly destroyed as compared with nuclei. No discernible changes were observed in not-blastotransformed lymphocytes under these experimental conditions. These findings indicate that thymic lymphocytes and splenic lymphoblasts are affected by exogenous lipid peroxides, and cytoplasmic micro-organelles of splenic lymphoblasts might be markedly damaged by exogenous lipid peroxides as compared to their nuclei.     

Characterization of fluorescent products from reaction of methyl linoleate hydroperoxides with adenine in the presence of Fe2+ and ascorbic acid

The structures of fluorescent products formed in the reaction of methyl linoleate hydroperoxides with adenine, FeSO4 and ascorbic acid were investigated to elucidate the mechanism of interaction. The fluorescent products consisted of at least four major components (I-IV), which could be separated by thin-layer chromatography and high-performance liquid chromatography. Both 2-octenal and 2,4-decadienal, degradation products of methyl linoleate hydroperoxides, reacted with adenine to produce a fluorescent product similar to one of the major compounds (II) formed in the reaction of methyl linoleate hydroperoxides. Spectroscopic data suggest that I and III are the same type of compounds, which have closed ring structures with alpha, beta-unsaturated carbonyl groups between the amino group at the 6-position and the nitrogen at the 1-position of adenine. Component II has a closed ring structure at the same site as I and III, and the presence of an ether linkage was suggested. On the basis of these structures, the involvement of 3-nonenal, methyl 12-oxo-9-dodecenoate and 2-octenal was suggested in the interaction of the methyl linoleate hydroperoxides decomposition products and adenine or DNA in the presence of FeSO4 and ascorbic acid.     

Fluorescence formation from the interaction of DNA with lipid oxidation degradation products

To clarify the mechanism of fluorescence formation between DNA and lipid degradation products in the presence of ferric chloride and ascorbic acid, a number of carbonyl compounds and decomposition products of pure methyl linolenate hydroperoxides were examined. Keto derivatives of methyl ricinoleate, linoleate, and oleate, alkanals and 2-alkenals produced little or no fluorescence with DNA in the presence of ferric chloride-ascorbic acid. 2,4-Alkadienals were more active and 2,4,7-decatrienal was the most active. Mixtures of volatile aldehydes prepared from linolenate hydroperoxide decomposed either thermally or with iron and ascorbate had the same activity as 2,4,7-decatrienal. Higher molecular-weight products from the decomposition of methyl linolenate hydroperoxides showed relatively low activity. beta-Carotene, alpha-tocopherol and other antioxidants effectively reduced the amount of fluorescence formed by linolenate hydroperoxides. The results suggest that, in addition to hydroperoxide decomposition products, singlet oxygen and/or free radical species contribute significantly to the fluorescence formed from the interaction of methyl linolenate hydroperoxides with DNA in the presence of ferric chloride and ascorbic acid.     

Structures of Dimers Produced from Methyl Linoleate during Initial Stage of Autoxidation

A structural investigation on the main fraction of dimers formed during the induction period of autoxidation in methyl linoleate (ML) was carried out. The dimeric fraction (A₂), which was isolated from the autoxidized ML (POV= 18) by various Chromatographic techniques and gave a single spot on TLC, was further separated into four major components (components 1–4) by high performance liquid chromatography (HPLC). The mean molecular weights of these components were found to be 643–655 and component 4 gave the parent peak 652 on an FD-mass spectrum which corresponded to 2 × ML + 4O. The reduced products of each component with stannous chloride were identified in common as methyl 9- or 13-hydroxy octadecadienoate and methyl 9,13-dihydroxy octadecenoate by GC-MS. These results show that all of these dimers contained a peroxide bridge linking between a pair of MLs across C-9 or C-13 on one of the MLs and C-9 or C-13 on the other, with a hydroperoxy group.     

Determination of thiobarbituric acid-reactive substances in oxidized lipids by high-performance liquid chromatography with a postcolumn reaction system

A new high-performance liquid chromatography procedure with a postcolumn reaction system for determination of free malondialdehyde (MDA) and other thiobarbituric acid-reactive substances (TBA-RS) in oxidized lipids in vitro has been developed. Using this procedure, both thermally oxidized methyl linoleate and the degradation products of methyl linoleate hydroperoxides revealed many kinds of lipophilic TBA-RS, but no free MDA was detected on the high-performance liquid chromatography. Similarly, oxidized methyl arachidonate also produced certain kinds of TBA-RS in the lipophilic phase and a small amount of free MDA in the hydrophilic phase. These results indicate that lipophilic TBA-RS produced in oxidized lipids in vitro are major TBA-RS and that the production of free MDA is small, even though the degree of lipid oxidation has previously been estimated as an MDA equivalent measured by the TBA colorimetric test.     

A chemiluminescence study of the decomposition of methyl linoleate hydroperoxides on active substrates

Chemiluminescence (CL) from the decomposition of the hydroperoxides of methyl linoleate (ML) was studied between 70°C and 106°C in an inert atmosphere in the bulk, on neutral, acidic, and basic alumina, and on silica gel. The decomposition of the hydroperoxides in the bulk followed first order kinetics and showed the slowest decomposition rates. The largest signals and the fastest decays occurred on neutral alumina. The rate data indicated bimolecular decomposition of the hydroperoxides adsorbed on adjacent active sites. From sorption data it appeared that the majority of the hydroperoxides were bound by the ester group perpendicular to the surface while the remainder were believed to lie flat, being attached by the hydroperoxide group. The hydroperoxide is susceptible to acid catalysed decomposition on an acidic alumina substrate and this may account for the lower emission intensity observed on this substrate.     

Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine.

Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxides stoichiometrically to yield fluorescent product DPPP oxide, was used as a fluorescent probe for lipid peroxidation in live cells. DPPP was successfully incorporated into U937 cells. Incorporation of DPPP into the cell membrane was confirmed by fluorescence microscopy. Reaction of DPPP with hydroperoxides was examined by monitoring increase in fluorescence intensity of the cell. It was found that lipid-soluble hydroperoxides such as methyl linoleate hydroperoxide preferably react with DPPP, whereas hydrogen peroxide did not react with DPPP located in the membrane. Linear correlation between increase in fluorescence intensity and the amount of methyl linoleate hydroperoxide applied to the cell was observed. DPPP gave little effect on cell proliferation, cell viability or cell morphology for at least 3 d. DPPP oxide, fluorescent product of DPPP, was quite stable in the membrane of living cells for at least 2 d. Fluorescence of DPPP-labeled cells was measured after treating with diethylmaleate (DEM), or 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), or culturing with low serum content. These reagents and culture condition induced dose- and/or time-dependent increase in fluorescence. Addition of vitamin E effectively suppressed increase in fluorescence. When DPPP-labeled cells and DCFH-DA-labeled cells were treated with NO, H(2)O(2), AAPH, and DEM to compare the formation of hydoperoxides in the membrane and cytosol, distinct patterns of peroxide formation were observed. These results indicate that fluorescent probe DPPP is eligible for estimation of lipid peroxidation proceeding in the membrane of live cells, and use of this probe is especially advantageous in long-term peroxidation of the cell.     

Numerical revelation of the kinetic significance of individual steps in the reaction mechanism of methyl linoleate peroxidation inhibited by alpha-tocopherol

A kinetic model was constructed to describe the reactions involved in the oxidation of methyl linoleate (ML) inhibited by alpha-tocopherol (TH). The initial model of the reaction mechanism included 53 individual steps, which were numerically analyzed by the value method based on Hamiltonian systematization of kinetic equations. Good accord was obtained with experimental data at 40 and 50 degrees C. The dominant steps responsible for the antioxidant and pro-oxidant properties of TH in the process of ML peroxidation were revealed. Tocopherol-mediated peroxidation (TMP) and generation of alkoxyl radicals as a result of the reduction of hydroperoxides by TH or the decomposition tocopherol alkyl peroxides are the dominant reactions responsible for the pro-oxidant activities of alpha-tocopherol. The extreme behavior of reaction induction period in relation to TH initial concentration is related to the increase in the ratios of [tocopheroxyl radical]/[peroxyl radical] and the TMP rate/rate of termination by combination of tocopheroxyl and peroxyl radicals.     

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.     

Rat testis lipoxygenase-like enzyme. Characterization of products from linoleic acid.

The linoleate oxidation products of the affinity chromatography-purified lipoxygenase-like enzyme isolated from rat testes microsomes were characterized. Three types of reaction products separated by thin-layer chromatography were generally present: polar byproducts (A and B) and hydroperoxides. The methyl hydroxystearates obtained from the enzymically produced hydroperoxides were analysed by gas-liquid chromatography and showed a ratio of 67% 13-hydroxy isomer to 33% 9-hydroxy isomer. The major polar byproduct was analysed by infrared spectra, nuclear magnetic resonance and mass spectrometry (of the toluene-p-sulphonyl derivative) and its structure was established as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid. The possibility of the existence of a linoleate hydroperoxide isomerase in the affinity-purified preparation is discussed.     

The antioxidative effect of fullerenes during the peroxidation of methyl linoleate in toluene

The antioxidative effect of fullerenes C(60) and C(70) was examined by measuring the inhibition of methyl linoleate (MeL) peroxidation in toluene initiated by 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). The fullerenes retarded the formation of MeL hydroperoxides and lowered the rate of propagation. The reaction rates of fullerenes with AMVN-derived peroxyl radicals were much higher than that of MeL. These results indicate that fullerenes can act as retarders of lipid peroxidation, though their activity is low compared with that of α-tocopherol.     

Structural identification of phosphatidylcholines having an oxidatively shortened linoleate residue generated through its oxygenation with soybean or rabbit reticulocyte lipoxygenase

Phosphatidylcholines (PCs) with platelet-activating factor (PAF)-like biological activities are known to be generated by fragmentation of the sn-2-esterified polyunsaturated fatty acyl group. The reaction is free radical-mediated and triggered by oxidants such as metal ions, oxyhemoglobin, and organic hydroperoxides. In this study, we characterized the PAF-like phospholipids produced on reaction of PC having a linoleate group with lipoxygenase enzymes at low oxygen concentrations. When the oxidized PCs were analyzed by gas chromatography-mass spectrometry, two types of oxidatively fragmented PC were detected. One PC had an sn-2-short chain saturated or unsaturated acyl group (C(8)-C(13)) with an aldehydic terminal; the abundant species were PCs with C(9) and C(13). The other PC had a short chain saturated acyl group (C(6)-C(9)) with a methyl terminal, and the most predominant species was PC with C(8). When the extracts of oxidation products were subjected to catalytic hydrogenation, PCs having saturated acyl groups (C(6)-C(14)) were detected; the most abundant was C(12) species. The less regiospecific formation of PAF-like lipids suggests that they were generated by oxidative fragmentation of PC hydroperoxides formed by non-stereoselective oxygenation of the alkyl radical of esterified linoleate that escaped from the active centers of lipoxygenases. One of the PAF-like PC with an aldehydic terminal was found to be bioactive; it inhibited the production of nitric oxide induced by lipopolysaccharide and interferon-gamma in vascular smooth muscle cells from rat aorta.     

Experimental evidence of the reciprocal oxidation of Bovine Serum Albumin and Linoleate in aqueous solution,initiated by HO free radicals

An investigation of radiation-induced oxidation of aqueous bovine serum albumin (BSA) in the presence of linoleate (LH) at pH 10.5 has been carried out in order to better understand the respective oxidative processes involved in both lipid and protein phases. Solutions containing BSA (15 μmol L⁻¹) and linoleate (15–600 μmol L⁻¹) below the critical micellar concentration (cmc = 2000 μmol L⁻¹), have been irradiated by γ-rays (¹³⁷Cs) at radiation doses ranging from 10 to 400 Gy (dose rate 9.5 Gy min⁻¹). It can be noticed that, in the absence of BSA, the main hydroperoxides formed from HO^•-induced linoleate oxidation below the cmc, do not exhibit a conjugated dienic structure. This was also verified in the presence of BSA. Selected chemical markers of oxidation have been monitored: non-conjugated dienic hydroperoxides and conjugated dienes (without hydroperoxide function) for linoleate oxidation, and carbonyl groups for BSA oxidation. We have shown that for the lowest linoleate concentration (15 μmol L⁻¹) in the presence of BSA (15 μmol L⁻¹), the formation of conjugated dienes was not observed, meaning that LH was not exposed to HO^• radicals attack. However, non-conjugated dienic lipid hydroperoxides were simultaneously detected, indicating that LH was secondarily oxidised by BSA oxidised species. Moreover, the oxidation of linoleate was found to be enhanced by the presence of BSA. For the highest linoleate concentration (600 μmol L⁻¹), the expected protection of BSA by LH was not observed, even if LH monomers were responsible for the total scavenging of HO^• radicals. In this latter case, the formation of non-conjugated dienic lipid hydroperoxides was lower than expected. Those results showed that BSA was not oxidised by the direct action of HO^• radicals but was undergoing a secondary oxidation by non-dienic lipid hydroperoxides and/or lipid radical intermediates, coming from the HO^•-induced linoleate oxidation.     

The Antioxidative Effect of Fullerenes during the Peroxidation of Methyl Linoleate in Toluene

The antioxidative effect of fullerenes C₆₀ and C₇₀ was examined by measuring the inhibition of methyl linoleate (MeL) peroxidation in toluene initiated by 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN). The fullerenes retarded the formation of MeL hydroperoxides and lowered the rate of propagation. The reaction rates of fullerenes with AMVN-derived peroxyl radicals were much higher than that of MeL. These results indicate that fullerenes can act as retarders of lipid peroxidation, though their activity is low compared with that of α-tocopherol.     

Limitations of the hemoglobin method for detecting lipid hydroperoxides

The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides, trilinoleoylglycerol hydroperoxides and phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were different. Some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. Front these results, the Hb method needs to be carefully applied to biological materials that contain mixtures of different typos of lipid classes.     

Methyl linoleate ozonide as a substrate for rat glutathione S-transferases: reaction pathway and isoenzyme selectivity

The 9,10-mono-ozonide of methyl linoleate was shown to be a substrate for rat hepatic cytosolic, rat lung cytosolic and rat hepatic microsomal glutathione S-transferases (GST). The activities of lung cytosol and liver microsomes with methyl linoleate ozonide (MLO) were found to be high relative to the activity demonstrated by liver cytosol, as compared with their respective activities towards 1-chloro-2,4-dinitrobenzene (CDNB). Only a slight catalytic activity towards the ozonide was noticed for rat lung microsomes. Isoenzyme 2-2 exhibited the highest specific activity (208 nmol/min/mg) when isoenzymes 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 7-7 were compared. This isoenzyme accounts for approx. 25% of cytosolic GST protein in rat lung, while in rat liver it represents approx. 9%. This may partly explain the high activity towards the ozonide noticed for rat lung cytosol. No stable conjugates were formed as products of the reaction of MLO with glutathione; although two glutathione-conjugates were noticed on TLC, they were only formed as intermediate compounds. Coupling of an aldehyde dehydrogenase assay or a glutathione reductase assay to the GST-catalyzed conjugation, demonstrated that oxidized glutathione and aldehydes are formed as the major products in the reaction. To further confirm the formation of aldehydes, the products of the GST-catalyzed reaction were incubated with 2,4-dinitrophenylhydrazine, which resulted in hydrazone formation. In conclusion, the activity of the GST towards the ozonide of methyl linoleate is similar to their peroxidase activity with lipid hydroperoxides as substrates.     

Fluorescence formation and heme degradation at different stages of lipid peroxidation

Secondary oxidative products of autoxidized methyl linoleate were divided into three groups (SP-I, SP-II and SP-III), which were then compared as to their abilities to form fluorescent substances and to degrade heme. SP-III showed a marked ability to produce two fluorescent substances exhibiting an excitation maximum at 350-360 nm and an emission maximum at 410-430 nm, while SP-I showed a more strongly degradative effect on heme than SP-III. The heme degradation was observed in parallel with the changes of TBA value in an early stage of lipid peroxidation and the fluorescence formation markedly increased according to the decrease of TBA value in a later stage. The results suggested that there are different reactive substances which bring about fluorescence formation and heme degradation and that they are produced at different stages of lipid peroxidation.