Setsuro Matsushita

Coloring conditions of thiobarbituric acid test for detecting lipid hydroperoxides

The coloring reaction of the thiobarbituric acid test for hydroperoxides was completely inhibited by the addition of EDTA. Therefore, it was necessary to add a metal salt to the reaction mixture to complete the reaction and also to add an antioxidant to prevent autoxidation when unoxidized unsaturated fatty acids co-exist. The optimal pH of the reaction was found at 3.6 using glycine-hydrochloric acid buffer.

Thiobarbituric acid test for detecting lipid peroxides

The thiobarbituric acid (TBA) test has been used in the field of medical science in recent years to detect lipid peroxides. In this case, it is necessary for hydroperoxides to be decomposed to secondary products during the reaction. When purified methyl linoleate and methyl linolenate monohydro-peroxides were used as the sample for the TBA test, they did not decompose entirely to secondary products, but did so completely when an iron catalyst (ferrous sulfate) was added. However, the iron catalyst also accelerated the autoxidation of coexisting unsaturated fatty acids. Therefore, the addition of antioxidants was required. Fifteen min of heating was sufficient to complete the reaction. With additions of catalyst and antioxidant to the TBA test, it may be possible to make useful distinctions between hydroperoxides and secondary products of lipid oxidation.

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.

A colorimetric microdetermination of peroxide values utilizing aluminum chloride as the catalyst

A colorimetric microassay is described for the determination of lipid hydroperoxides. Hydroperoxides are reacted with potassium iodide in the presence of an acid catalyst and liberated iodine is measured. Aluminum chloride, an alcohol-soluble Lewis acid, is used as catalyst. Liberated iodine is measured colorimetrically at 560 nm after addition of starch in 0.01 N hydrochloric acid. The range of the measurement was 0.05–0.5 μmol of hydroperoxides.

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.

Chemical reactivity of the nucleic acid bases. I. Antioxidative ability of the nucleic acids and their related substances on the oxidation of unsaturated fatty acids

Abstract The nucleic acids und their related substances exhibit antioxidative effects on the oxidation of linoleic acid by air. Especially adenine, guanosine, xanthine, hypoxanthine, uric acid, uracil, orotic acid, and ribonucleic acid were as effective antioxidants as α-tocopherol, nordihydroguaiaretic acid, and butylhydroxyanisole. Nucleic acids and their related substances may be used as antioxidants for lipids.

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).