Junji Terao

Antioxidant activity of β-carotene-related carotenoids in solution

The effect of the antioxidant activity of β-carotene and related carotenoids on the free radical-oxidation of methyl linoleate in solution was examined by measuring the production of methyl linoleate hydroperoxides. Canthaxanthin and astaxanthin which possess oxo groups at the 4 and 4′-positions in the β-inonone ring retarded the hydroperoxide formation more efficiently than β-carotene and zeaxanthin which possess no oxo groups. The rates of autocatalytic oxidation of canthaxanthin and astaxanthin were also slower than those of β-carotene and zeaxanthin. These results suggest that canthaxanthin and astaxanthin are more effective antioxidants than β-carotene by stabilizing the trapped radicals.

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.

Preparation of hydroperoxy and hydroxy derivatives of rat liver phosphatidylcholine and phosphatidylethanolamine

A convenient method for the preparation of hydroperoxy and hydroxy derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is described. PC and PE obtained from rat liver were oxidized with singlet oxygen by using methylene blue as the photosensitizer, and their hydroperoxides were isolated with the aid of reverse phase liquid chromatography. The hydroxy derivatives were obtained by reducing the hydroperoxides with sodium borohydride. The results of gas chromatography mass spectrometry revealed that hydroxy fatty acid components of the hydroxy derivatives were derived from isomeric hydroperoxides of oleic acid, linoleic acid, arachidonic acid and docosahexanoic acid. Normal phase high performance liquid chromatography did not separate the hydroperoxy and hydroxy derivatives from the respective unoxidized phospholipids, although unoxidized PC and PE were separated from each other. However, the hydroperoxy and hydroxy derivatives could be distinguished from unoxidized phospholipid species on reversed phase thin layer chromatography.

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

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

High-performance liquid chromatographic determination of phospholipid peroxidation products of rat liver after carbon tetrachloride administration

A method to detect and determine phospholipid peroxidation products in a biological system was developed using reversed-phase high performance liquid chromatography and normal-phase HPLC. Reversed-phase HPLC could separate phosphatidylcholine (PC) hydroperoxides and phosphatidylethanolamine (PE) hydroperoxides of rat liver from the respective phospholipids. A linear relationship was observed between these hydroperoxides and their peak areas on the chromatogram. In the experiment with rats administered CCl4, reversed-phase HPLC gave prominent, large peaks attributable to the peroxidation of phospholipids, and the peroxide level of the liver phospholipids was tentatively determined. Normal-phase HPLC analysis confirmed that both PC and PE in the liver phospholipids were peroxidized after CCl4 treatment. Neither the thiobarbituric acid value of the liver homogenate nor the fatty acid composition of the liver phospholipid fraction showed any significant difference between CCl4-treated and control rats. It is concluded that normal-phase HPLC and reversed-phase HPLC can complement each other to serve as a direct and sensitive method for the determination of lipid peroxide levels in a biological source. However, it was difficult to distinguish phospholipid hydroperoxides from their hydroxy derivatives.

Electrochemical detection of phospholipid hydroperoxides in reverse-phase high performance liquid chromatography

Hydroperoxy derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) can be separated from their respective phospholipids by reverse-phase high performance liquid chromatography (HPLC). However, ultraviolet absorption due to conjugated diene cannot detect the hydroperoxy group. In this work, an electrochemical (EC) detector was first applied to the analysis of hydroperoxy phospholipids. Both the PC and PE hydroperoxides from rat liver were reduced quantitatively by the glassy carbon electrode at −300 mV vs Ag/AgCl. Since neither the hydroxy derivatives nor unoxidized phospholipids showed any response, it would seem this technique can be used to distinguish phospholipid hydroperoxides from their hydroxy derivatives. Thus, the reverse phase HPLC-EC detection method is proposed for the specific analysis of hydroperoxy phospholipids in biological tissues.

Singlet Oxygen-Initiated Photooxidation of Unsaturated Fatty Acid Esters and Inhibitory Effects of Tocopherols and β-Carotene

It has been suggested that photosensitized oxidation initiates oxidative deterioration of vegetable oils (1–3). Chlorophyll-like pigments present in oils seem to act as sensitizers by absorbing visible light to produce hydroperoxides in unsaturated fatty acids. This reaction has been categorized into two classes, Type I and Type II, as mentioned by Foote (4). Type I reaction involves the production of free radicals by interaction of the excited sensitizer with a substrate. In the Type II process, an excited sensitizer produces singlet oxygen by transferring excitation from the sensitizer to oxygen. This active oxygen molecule reacts with olefinic double bonds to produce hydroperoxides by a concerted ene type mechanism (5).

Thiobarbituric acid reaction of methyl arachidonate monohydroperoxide isomers

Methyl ester of monohydroperoxy eicosatetraenoic acid (MeHPETE) was prepared from methylene blue sensitized photooxidation products of methyl arachidonate. The thiobarbituric acid (TBA) value of MeHPETE was increased by adding ferrous sulfate to the reaction mixture. A linear relationship existed betweent he TBA value and the concentration of MeHPETE when ferrous sulfate was added. By using high performance liquid chromatography, MeHPETE was separated into 5 fractions whose isomeric compositions were determined by gas chromatography-mass spectrometry. The results of the TBA test for each fraction suggest that all of the MeHPETE isomers are positive to the TBA test. It is concluded that each isomer of HPETE formed by peroxidation of arachidonic acid in a biological system can yield TBA-reacting materials during the test reaction.

Structural analysis of hydroperoxides formed by oxidation of phosphatidylcholine with singlet oxygen

Soybean phosphatidylcholine (PC) and dilinoleoyl PC (di-18∶2 PC) were oxidized with singlet molecular oxygen using methylene blue as the photosensitizer. The oxidation products, PC monohydroperoxides (PC-MHP) and PC dihydroperoxides (PC-DHP), were isolated by reverse phase liquid chromatography, and their structures were analyzed by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). Signals for the hydroperoxy proton appeared downfield in NMR spectra of PC-MHP and PC-DHP. Soybean PC-MHP and di-18∶2 PC-MHP were converted to trimethylsilyl (TMS) derivatives of hydrogenated diglycerides when treated with phospholipase C and hydrogenated. Thetert-butyldimethylsilyl (TBDMS) derivatives of hydrogenated diglycerides were also prepared from di-18∶2 PC-MHP. Fragmentation of the TMS and TBDMS derivatives was obtained in electron impact mass spectra. The isomeric composition of hydroperoxylinoleate component in di-18∶2 PC-MHP was determined by methanolysis of the hydrogenated diglyceride and mass chromatographic analysis of the resulting isomeric hydroxy octadecanoates.

Hydroperoxides formed by ferrous ion-catalyzed oxidation of methyl linolenate

An emulsion of methyl linolenate was allowed to oxidize with a catalyst of ferrous sulfate and ascorbic acid. Three oxidation products were isolated, and their hydrogenated derivatives were characterized as the isomeric mixture of methyl monohydroxyoctadecanoate (monoOH), methyl 9,16-dihydroxyoctadecanoate (diOH), and the isomeric mixture of methyl trihydroxyoctadecanoate (triOH). The monoOH isomers and diOH apparently were derived from methyl monohydroperoxyoctadecatrienoate (monoHPO) and methyl dihydroperoxyoctadecatrienoate (diHPO), respectively. Two triOH isomers (the 9,10,12- and 13,15,16-isomers) were thought to be derived from the products containing cyclic peroxide-hydroperoxide structure. 9,16-diHPO was produced by the incubation of monoHPO with ferrous sulfate and ascorbic acid. Moreover, the experiment using18O2 demonstrated that mono-HPO yielded 9,16-diHPO by reacting with oxygen molecule. 9,10,13- and/or 9,12,13- and 12,13,16- and/or 12,15,16-triOH isomers were also detected in the hydrogenated derivatives of oxidation products from monoHPO.