Takashi Kaneda

Autoxidation of ethyl eicosapentaenoate and docosahexaenoate

The extent of oxidation of ethyl esters of eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) was compared quantitatively with that of ethyl linoleate (Lo) and ethyl linolenate (Ln) by oxygen uptake and formation of conjugated diene, hydroperoxide and secondary oxidation products. EPA and DHA esters were oxidized rapidly even at 5 C in the dark after an induction period of 3–4 days, while the induction periods of Ln and Lo esters were 20 days and more than 60 days, respectively. Oxygen uptake of EPA and DHA esters after the induction period was 5.2 and 8.5 times faster than that of ethyl Ln, respectively. Hydroperoxides of EPA and DHA esters are much less stable than those of ethyl Lo. The peroxide value is not necessarily a good indication of oxidation in these polyenoic acids because a considerable amount of secondary products is formed at the early stage of oxidation. Polymers were found to be major secondary products in these polyenoic esters.

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.

Antioxidant effects of chlorophyll and pheophytin on the autoxidation of oils in the dark. I: Comparison of the inhibitory effects

The effects of chlorophyll and pheophytin on the autoxidation of oils in the dark were investigated by oven tests. The results indicated that both chlorophyll and pheophytin show antioxidant activity when methyl linoleate is used as substrate. Furthermore, chlorophyll retarded the oxidative deterioration of triglycerides in rapeseed and soybean oils at 30 C. Among the four chlorophyll derivatives (chlorophylls a and b and pheophytins a and b), chlorophyll a showed the strongest antioxidant activity. The antioxidant effects of chlorophyll and pheophytin depended on the storage temperature and the kinds of oil used as substrate.

Prooxident activities of chlorophylls and their decomposition products on the photooxidation of methyl linoleate

Although chlorophylls in oils affect oxidative stabilities, little has been known about the prooxidant activity of the chlorophylls or their decomposition products. To evaluate the prooxidant activities of chlorophyll derivatives, chlorophyll (CHL) A and B, pheophytin (PHY) A and B and pheophorbide (PHO) A and B were added to methyl linoleate (ML). The sample oils were then subjected to photooxidation at O C and the peroxide value and absorbance at 234 nm were measured. Chlorophylls catalyzed oxidation of methyl linoleate at concentrations greater than 2.2×10−9mol/g ML, and CHL B showed a stronger prooxidant activity, ca. twice that of CHL A under the same light intensity. PHY and PHO exhibited stronger prooxidant activities than CHL, the prooxidant activity of PHO being the strongest among them. Moreover, photosensitive activity was found in PHY as well as in CHL. These results suggest that particular attention should be paid to the decomposition products of CHL that affect the quality of vegetable oils.

Residual amounts of chlorophylls and pheophytins in refined edible oils

The quantities of chlorophyll (CHL) A and B, and pheophytin (PHY) A and B in 10 kinds of refined edible oils were estimated by the fluorometric method. The results revealed that CHL and PHY were present in commercial edible oils. PHY A showed the highest content at ca. 67% in total pigments. Compositional ratios of CHL and PHY were similar in different kinds of plant oils. Through the analysis of rapessed oils at every refining step, we determined that PHY is not formed during oil refining. In the autoxidation of soybean oils to which various amounts of CHL mixtures had been added, the peroxide value of tested oils increased in proportion to the total chlorophyll content. In addition, the compositional changes of the 4 components during autoxidation were investigated.

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.

Development of an ultra-high sensitive photon counting system and its application to biomedical measurements

Abstract The sensitivity of the photon counting method for the detection of extra-weak optical intensity was improved on the introduction of the enhanced single photoelectron counting technique incorporated with a selected photomultiplier operated under optimum conditions. With this ultra-high sensitive photon counting system it has been possible to measure the difference between the luminescence of the blood of healthy and diseased human subjects (both have extremely weak values).

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.

Dimers formed in oxygenated methyl linoleate hydroperoxides

The formation of dimers was demonstrated in methyl linoleate hydroperoxides decomposed by bubbling with dry air at 30 C. The dimer fraction isolated by gel permeation chromatography was further fractionated by successive silicic acid column and high performance liquid chromatography. Major components were analyzed after derivatizations by gas chromatography-mass spectrometry with several ionization methods, i.e., electron impact, chemical ionization and field desorption.After aeration for 90 min, 1.4% of the hydroperoxides were decomposed. However, almost all of the secondary products were dimers, while polar monomeric and low molecular products were negligible. After aeration for 390 min, both polar monomeric (5.0%) and low molecular (0.8%) compounds formed, but dimers and polymers (18.1%) were still the major products. These results show the importance of polymerization in the aerobic breakdown of hydroperoxides.The dimers isolated from hydroperoxides aerated 90 min could be separated into two fractions according to their polarities. The dimers identified usually were composed of octadecadienoate and oxygenated octadecenoate moieties crosslinked through either ether or peroxy linkages across C-9 or C-13 positions. The oxygen-containing functional groups found in the dimers include hydroperoxy, hydroxy and oxo groups. The polar dimers had two of these groups per molecule, while the less polar dimers had one. The main constituents of dimers were linked through peroxy bridges and found to be similar to the dimers previously identified in autoxidized methyl linoleate. These dimers are suggested as important intermediates in linoleate oxidation and as precursors of flavor deterioration.

Characterization of toxic compound in thermally oxidized oil

In preliminary experiments, soybean oil was heated at 275 C for 12 hr in the presence of N2 or air. Feeding studies with rats showed that the oil heated in the presence of air, oxidatively polymerized oil, retarded weight gain more than that heated with N2, thermally polymerized oil. Oxidatively polymerized oil was fractionated with silicic acid column chromatography, and the fraction eluted with ether (fraction III) proved to be most toxic to mice. For chemical studies combined with bioassays, additional oxidatively polymerized oil was prepared by aeration at 185 C for 90 hr, and the product was fractionated by silicic acid column chromatography. The material originally eluted with ether (fraction III) was eluted in stages, and fraction IIIc proved to be most toxic in a mouse bioassay. IR, UV and NMR analyses did not indicate the presence of C−O−O−C or C−O−C linkages or aldehyde groups or aromatic compounds. Fraction IIIc was converted to its methyl esters and molecularly distilled to yield two fractions: one containing unpolymerized fatty acid esters and one giving evidence of more functional groups per molecule and of dimeric material. This “dimeric” fraction was more toxic to mice. IR analysis of this fraction revealed carbonyl groups, and NMR showed several functional groups on alkyl chains. Treatment with sodium borohydride and with hydroiodic acid revealed no C−O−O−C and no C−O−C linkages. Mass spectrography showed peaks at mass 586 and 293; in the reduced fraction a peak also occurred at mass 143, suggestive of cleavage of some of the chains. It is tentatively concluded that a highly toxic material formed in oil heated in air is a dimer of triglyceride molecules.