Light-Induced Lipid Peroxidation in Isolated Chloroplasts and Role of α-Tocopherol

Lipid peroxidation in isolated chloroplasts illuminated by visible light and the role of α-tocopherol in chloroplasts were studied. The TBA reactants and fluorescent products derived from lipid peroxidation were formed by illumination. Peroxidation was inhibited by free radical scavengers and ¹O₂ quenchers. Hydroxy methyl octadecanoates, which were the reduced and hydrogenated products of lipid hydroperoxides, were detected. Among them, 10-and 15-hydroxy methyl octadecanoates were generated from ¹O₂ oxidation. On the other hand, lipid hydroperoxides did not accumulate in this peroxidation process. The amount of α-tocopherol in the chloroplasts decreased with lipid peroxidation, and α-tocopheryl quinone was produced. The results indicate that α-tocopherol acts as a free radical scavenger for photo-oxidation of chloroplasts.     

Reaction Products from Various Tocopherols with Trimethylamine Oxide and Their Antioxidative Activities

The structures of many reaction products obtained when various tocopherols (Toc’s) and trimethylamine oxide (TMAO) were treated in liquid paraffin under a nitrogen stream at 180°C, were determined and their antioxidative activities were investigated.

The reaction products (Toc dimers) isolated were as follows: α-tocopheryl ethane from α-Toc; 5-(γ-tocopheryloxy)-γ-Toc, 5-(γ-tocopheryl)-γ-Toc (two kinds) and α-tocopheryl ethane from γ-Toc; 5-(δ-tocopheryloxy)-δ-Toc from δ-Toc.

The two 5-(γ-tocopheryl)-γ-Toc’s are atropisomers of each other (TLC (Rf): 0.75, 0.45—benzene) and isomerization occurred within 20 min when they were treated under nitrogen at 180°C.

All Toc dimers, in particular 5-(γ-tocopheryloxy)-γ-Toc, have antioxidative activities and excellent synergism with TMAO in inhibiting the oxidation of lard kept in the dark at 60°C.     

High-performance liquid chromatography and gas chromatography-mass spectrometry determination of specific lipid peroxidation products in vivo

Peroxidation of membrane lipids has been implicated in the toxicity of reactive oxygen intermediates and of several hepatotoxins, but the specific products of this peroxidation in vivo have not been chemically identified. A method for the isolation, identification, and quantitation of specific lipid hydroperoxy and hydroxy acids formed in vivo has been developed. Hydroxylated derivatives of linoleic, arachidonic, and docosahexaenoic acids formed in mouse liver phosphatidylcholines following carbon tetrachloride administration were isolated by high-pressure liquid chromatography and identified as the trimethylsilyl ether methyl ester derivatives by gas chromatography-mass spectrometry. This methodology should be important for the investigation of the role of lipid peroxidation in a variety of normal physiologic and pathologic processes.     

Decomposition products of Dimers Arising from Secondary Oxidation of Methyl Linoleate Hydroperoxides

Dimers formed in aerated methyl linoleate hydroperoxides were decomposed in liquid paraffin by bubbling with dry air at 30°C for 24 hr to identify the decomposition products. The aerated dimers were fractionated according to their molecular weights by gel permeation chromatography. Identification of the monomeric (25.6%) and low molecular fission products (10.8%) by gas chromatography-mass spectrometry showed the major monomers as methyl hydroxy-octadecadienoate, methyl hydroxy (or hydroperoxy)-epoxy-octadecenoate, methyl dihydroxy (or hydroperoxy)-octadecenoate, methyl trihydroxy (or hydroperoxy)-octadecenoate; and the major fission products as methyl 8-hydroxy-octanoate, 4-hydroxy (or hydroperoxy)-nonanal or -2-nonenal, methyl 12-oxo-9-hydroxy (or hydroperoxy)-dodecanoate or -10-dodecenoate, and methyl 11-oxo-9-undecenoate.

The monomeric products were presumed to be derived from alkoxy radicals generated by the cleavage of peroxy linkages in the dimers, whereas the low molecular products were suggested to be raised by the direct carbon-carbon scission of oxygenated ester moieties on both sides of the peroxy bonds.     

Autoxidation and Photosensitized Oxidation of Methyl Eicosapentaenoate: Secondary Oxidation Products

Methyl eicosapentaenoate (methyl 5,8,11,14,17-eicosapentaenoate) was subjected to autoxidation and methylene blue-sensitized photooxidation. The secondary oxidation products were separated, and characterized by gas chromatography-mass spectrometry. The autoxidation products included hydroperoxy endoperoxide isomers, prostaglandin-like hydroperoxy bicyclic endoperoxide isomers and 5,18-dihydroperoxide. The photosensitized oxidation products included only dihydroperoxide isomers as the secondary products.     

ω-Hydroxylation of α-tocopheryl quinone reveals a dual function for cytochrome P450-4F2 in vitamin E metabolism

α-Tocopherol (α-TOH) is the primary lipophilic radical trapping antioxidant in human tissues. Oxidative catabolism of α-tocopherol (αTOH) is initiated by ω-hydroxylation of the terminal carbon (C-13) of the isoprenoid sidechain followed by oxidative transformations that sequentially truncate the chain to yield the 2,5,7,8-tetramethyl(3′carboxyethyl)-6-hydroxychroman (α-CEHC). After conjugation to glucuronic acid, 3′-carboxyethyl-6-hydroxychroman glucuronide is excreted in urine. We report here that the same enzyme that accomplishes this task, the cytochrome P450 monooxygenase CYP-4F2, can also ω-hydroxylate the terminal carbon of α-tocopheryl quinone. A standard sample of ω-OH-α-tocopheryl quinone (ω-OH-α-TQ) was synthesized as a mixture of stereoisomers by allylic oxidation of α-tocotrienol using SeO₂ followed by double-bond reduction and oxidation to the quinone. After incubating human liver microsomes or insect cell microsomes expressing only recombinant human CYP-4F2, cytochrome b5, and NADPH P450 reductase with d₆-α-tocopheryl quinone (d₆-αTQ), we showed that the ω-hydroxylated (13-OH) d₆-α-TQ was produced. We further identified the production of the terminal carboxylic acid d₆-13-COOH-αTQ. The ramifications of this discovery to the understanding of tocopherol utilization and metabolism, including the quantitative importance of the αTQ-ω-hydroxylase pathway in humans, are discussed.     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Mass‐spectrometric characterization of phospholipids and their primary peroxidation products in rat cortical neurons during staurosporine‐induced apoptosis

The molecular diversity of phospholipids is essential for their structural and signaling functions in cell membranes. In the current work, we present, the results of mass spectrometric characterization of individual molecular species in major classes of phospholipids – phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), sphingomyelin (CerPCho), and cardiolipin (Ptd₂Gro) – and their oxidation products during apoptosis induced in neurons by staurosporine (STS). The diversity of molecular species of phospholipids in rat cortical neurons followed the order Ptd₂Gro > PtdEtn >> PtdCho >> PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd₂Gro >> PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd₂Gro, was represented in cortical neurons by the greatest variety of both total and peroxidizable molecular species. Quantitative fluorescence HPLC analysis employed to assess the oxidation of different classes of phospholipids in neuronal cells during intrinsic apoptosis induced by STS revealed that three anionic phospholipids – Ptd₂Gro >> PtdSer > PtdIns – underwent robust oxidation. No significant oxidation in the most dominant phospholipid classes – PtdCho and PtdEtn – was detected. MS‐studies revealed the presence of hydroxy‐, hydroperoxy‐ as well as hydroxy‐/hydroperoxy‐species of Ptd₂Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd₂Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H₂O₂, confirmed that molecular identities of the products formed were similar to the ones generated during STS‐induced neuronal apoptosis. The temporal sequence of biomarkers of STS‐induced apoptosis and phospholipid peroxidation combined with recently demonstrated redox catalytic properties of cyt c realized through its interactions with Ptd₂Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd₂Gro and PtdSer peroxidation products. These oxidation products participate in mitochondrial membrane permeability transition and in PtdSer externalization leading to recognition and uptake of apoptotic cells by professional phagocytes.     

Adaptations to oxidative stress induced by vitamin E deficiency in rat liver

Vitamin E deficiency in rats led to a sequence of antioxidant defense adaptations in the liver. After three weeks, α-tocopherol concentration was 5% of control, but ascorbate and ubiquinol concentrations were 2- to 3-fold greater than control. During the early phase of adaptation no differences in markers of lipid peroxidation were observed, but the activities of both cytochrome b5 reductase and glucose-6-phosphate dehydrogenase were significantly greater in deficient livers. By nine weeks, accumulation of lipid peroxidation end products began to occur along with declining concentrations of ascorbate, and higher NQO1 activities. At twelve weeks, rat growth ceased, and both lipid peroxidation products and cytosolic calcium-independent phospholipase A2 reached maximum concentrations. Thus, in growing rats the changes progressed from increases in both ubiquinol and quinone reductases through accumulation of lipid peroxidation products and loss of endogenous antioxidants to finally induction of lipid metabolizing enzymes and cessation of rat growth.     

Quenching Effect of Tocopherols on the Methyl Linoleate Photooxidation and Their Oxidation Products

The quenching effect of α-, γ- and δ-tocopherols on the methylene blue sensitized photo- oxidation of methyl linoleate was investigated, and the ¹O₂. quenching ability of tocopherols was determined. The ¹O₂ quenching rate constants for α-, γ- and δ-tocopherols in ethanol were estimated to be 2.6 × 10⁸ m^?1 sec^?1, 1.8 × 10⁸ m^?1 sec^?1 and 1.0 × 10⁸ m^?1 sec^?1, respectively. And the rate constants for the chemical reaction between each tocopherol and ¹O₂ were 6.6 × 10⁶ m^?1 sec^?1, 2.6 × 10⁶ m^?1 sec^?1 and 0.7 × 10⁶ m^?1 sec^?1 for α-, γ- and δ-tocopherols, respectively. The results show that α-tocopherol is the most effective compound toward ¹O₂ among the three tocopherols. The photooxidation of each tocopherol produced two peroxides which, after chemical reduction, were identified to be tocopherol hydroquinone by gas chromatography-mass spectrometry analysis. The photooxidation mechanism of these tocopherols was assumed to be different from that of autoxidation.     

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k₁ = 0.003 hr^?1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k₁ = 0.145 and 0.162 hr^?1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A₂ may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley-Liss, Inc.     

Fragility of Erythrocytes from Chicks and Rats Fed Dilauryl Succinate and Related Compounds

Susceptibility to hydrogen peroxide of the erythrocytes from chicks and rats fed dilauryl succinate and related compounds with and without supplementation of dl-α-tocopheryl acetate was determined.

Dilauryl succinate, lauryl alcohol, n-decyI alcohol, myristyl alcohol, and lauraldehyde were confirmed to make the erythrocytes from the chicks fragile. Supplemented dl-α-tocopheryl acetate of 200 mg per kg of diet completely prevented the hemolysis induced by these compounds. Dilauryl succinate also makes the rat’s erythrocytes fragile and supplemented dl-α-tocopheryl acetate prevented the hemolysis of the rats, but ethoxyquin was not. The symptoms of encephalomalacia in the chick is preceded by increased hemolysis value of the erythrocytes, and this hemolysis value dropped after the appearance of encephalomalacia.     

Quinone methides—and not dehydrodopamine derivatives—as reactive intermediates of β-sclerotization in the puparia of flesh fly Sarcophaga bullata

Cuticular phenoloxidase(s) from Sarcophaga bullata larvae oxidized a variety of o-diphenolic compounds. While catechol, 3,4-dihydroxybenzoic acid, dopa, dopamine, and norepinephrine were converted to their corresponding quinone derivatives, other catechols such as 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 3,4-dihydroxyphenyl glycol, 3,4-dihy-droxymandelic acid, and N-acetyldopamine were oxidized to their side-chain oxygenated products. In addition, the enzyme-catalyzed oxidation of the latter group of compounds accompanied the formation of colorless catecholcuticle adducts consistent with the operation of β-sclerotization. Radioactive trapping experiments failed to support the participation of 1,2-dehydro-N-acetyldopamine as a freely formed intermediate during phenoloxidase-mediated oxidation of N-acetyldopamine. When specifically tritiated substrates were provided, cuticular enzyme selectively removed tritium from [7-³H]N-acetyldopamine and not from either [8-³H] or [ring-³H]N-acetyldopamine during the initial phase of oxidation. The above results are consistent with the generation and subsequent reactions of quinone methides as the initial products of enzyme-catalyzed N-acetyldopamine oxidation and confirm our hypothesis that quinone methides and not 1,2-dehydro-N-acetyldopamine are the reactive intermediate of β-sclerotization of sarcophagid cuticle. Quinone methide sclerotization resolves a number of conflicting observations made by previous workers in this field.     

Characteristics of Chlorophyll Fluorescence and Membrane-Lipid Peroxidation During Senescence of Flag Leaf in Different Cultivars of Rice

With japonica rice 98-08, indica hybrids Shanyou 63, Gangyou 881, and X07S/Zihui 100, and sub-species hybrid Peiai 64S/9311 as materials, chlorophyll (Chl) content, Chl a fluorescence parameters, and membrane lipid peroxidation in flag leaf were measured at late developmental stages under natural conditions. F_v/F_m, q_P, _PS2, and electron transport rate gradually decreased while q_N increased conversely. Excessive photon energy led to the accumulation of active oxygen (O₂ ^–), H₂O, malonyldialdehyde, and products of membrane lipid peroxidation, and resulted in reduced Chl content and early ageing subsequent to the photooxidation during flag leaf senescence. There was obvious diversification of these parameters among rice cultivars. In comparison with japonica cv. 98-08 (tolerant to photooxidation), F_v/F_m decreased in indica cv. Shanyou 63 (susceptible to photooxidation) with greater accumulation of active oxygen and a sharp drop in Chl content, which resulted in yellowish early ageing, and affected the filling and setting of rice grains. The mechanism for premature ageing in indica rice was related to irradiance and temperature at filling stages. On a sunny day at above 25 °C, the reaction centre of photosystem 2 (PS2) exhibited a dynamic change on reversible inactivation. Under the intense irradiance at noon, PS2 function in indica rice exhibited obvious down-regulation and photoinhibition. Under intense irradiance with lowered temperatures, PS2 resulted in photo-damage and early ageing, related to the degradation of PS2-D1 protein and the inhibition of endogenous protection systems such as the xanthophyll cycle and enzymes scavenging active oxygen. Hence for high-yield breeding, based on a good plant-type and utilising heterosis and tolerance of photooxidation, the selection of japonica rice or a sterile line with the japonica genotype as female is a strategy worthy of consideration.     

Vitamin E Activity of 1-Thio-α-Tocophero as Measured by the Rat Curative Myopathy Bioassay

The bioactivity of the acetate of the all-racemic, 1-thio analog of a-tocopherol (all-rac-]-thio-α-tocopheryl acetate) has been determined by measuring its ability to decrease plasma levels of pyruvate kinase in vitamin E deficient rats using the curative myopathy bioassay. The thio analog is only 0.22 times as active as RRR-α-tocopheryl acetate and is therefore approximately 0.33 times as active as all-rac-α-tocopheryl acetate, since the latter has been shown to be 1.47 times less active than RRR-α-tocopheryl acetate in the same bioassay (H. Weiser, M. Vecchi and M. Schlachter, Internal. J. Vit. Nutr. Res. 55 149-158 (1985)). The 0.33:1.0 ratio is similar to the ratio of 0.41:1.0 measured for the in vitro antioxidant activities of the corresponding free phenols. This finding lends further support to our view that the vitamin E activity in the curative myopathy bioassay of close structural analogs of α-tocopherol is determined primarily by the in vitro antioxidant activity of the analog relative to α-tocopherol, consistent with the belief that vitamin E functions primarily as a general purpose, lipid-soluble antioxidant in mammals.     

An alpha-tocopherol binding protein in rat liver cytoplasm.

Gel filtration and sucrose density gradient analysis of rat liver high speed supernatant revealed the presence of a protein capable of binding [³H] α-tocopherol. The protein sedimented with an S value of 3.0. Gel filtration yielded an estimated molecular weight of 31,000. Specificity for α-tocopherol was demonstrated by competition for binding of [³H]α-tocopherol with unlabeled α-tocopherol, but not with α-tocopheryl quinone or α-tocopheryl acetate. Pronase digestion completely abolished binding.     

Coupling of cytochrome and quinone turnovers in the photocycle of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides.

A minimal kinetic model of the photocycle, including both quinone (Q-6) reduction at the secondary quinone-binding site and (mammalian) cytochrome c oxidation at the cytochrome docking site of isolated reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides, was elaborated and tested by cytochrome photooxidation under strong continuous illumination. The typical rate of photochemical excitation by a laser diode at 810 nm was 2.200 s-1, and the rates of stationary turnover of the reaction center (one-half of that of cytochrome photooxidation) were 600 +/- 70 s-1 at pH 6 and 400 +/- 50 s-1 at pH 8. The rate of turnover showed strong pH dependence, indicating the contribution of different rate-limiting processes. The kinetic limitation of the photocycle was attributed to the turnover of the cytochrome c binding site (pH < 6), light intensity and quinone/quinol exchange (6 < pH < 8), and proton-coupled second electron transfer in the quinone acceptor complex (pH > 8). The analysis of the double-reciprocal plot of the rate of turnover versus light intensity has proved useful in determining the light-independent (maximum) turnover rate of the reaction center (445 +/- 50 s-1 at pH 7.8).     

Interconversion between Rotenone and Dalpanol

Photosensitized oxidation of trioleoylglycerol (TO), trilinoleoylglycerol (TL), trilinolenoylglycerol (TLn) and vegetable oil triacylglycerols (triglycerides, TG) was carried out in isopropanol using methylene blue as a photosensitizer. Isomeric compositions of hydroperoxy fatty acid components of the oxidized TG were determined by hydrogenation, methanolysis and mass chromatographic analysis of the resulting methyl hydroxy octadecanoate. TO gave 9- and 10-isomers; TL, 9-, 10-, 12- and 13-isomers; and TLn, 9-, 10-, 12-, 13-, 15- and 16-isomers. It was concluded that each unsaturated fatty acid component of vegetable oil TG yields isomeric hydroperoxides during photosensitized oxidation in a manner similar to the corresponding unsaturated fatty acid methyl ester. TL monohydroperoxides were isolated from the photooxidized TL and hydrolyzed by pancreatic lipase. The hydrolysis products consisted of dilinoleoylglycerol, monolinoleoylglycerol, linoleic acid and their respective hydroperoxides. Formation of a hydroperoxy fatty acid component was observed during photoirradiation of vegetable oils in the bulk phase without methylene blue. The isomeric compositions of the resulting methyl hydroxy octadecanoate support the idea that singlet oxygen is responsible for the formation of hydroperoxides in the initial stage of photooxidation.     

Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinephrine by cuticular phenoloxidase from Sarcophaga bullata

The mechanism of oxidation of two related sclerotizing precursors—N-acetyldopamine and N-acetylnorepinephrine—by the cuticular phenoloxidase from Sarcophaga bullata was studied and compared with mushroom tyrosinase-mediated oxidation. While the fungal enzyme readily generated the quinone products from both of these catecholamine derivatives, sarcophagid enzyme converted N-acetyldopamine to a quinone methide derivative, which was subsequently bound to the cuticle with the regeneration of o-dihydroxy phenolic function as outlined in an earlier publication [Sugumaran: Arch Insect Biochem Physiol, 8, 73 (1988)]. However, it converted N-acetylnorepinephrine to its quinone and not to the quinone methide derivative. Proteolytic digests of N-acetyldopamine-treated cuticle liberated peptides that had covalently bound catechols, while N-acetylnorepinephrine-treated cuticle did not release such peptides. Acid hydrolysis of N-acetyldopamine-treated cuticle, but not N-acetylnorepinephrine-treated cuticle liberated 2-hydroxy-3′,4′-dihydroxyacetophenone and arterenone. These results further confirm the unique conversion of N-acetyldopamine to its corresponding quinone methide derivative and N-acetylnorepinephrine to its quinone derivative by the cuticular phen-oloxidase. Significance of this differential mechanism of oxidation for sclerotization of insect cuticle is discussed.     

Bio-markers of lipid peroxidation in vivo: hydroxyoctadecadienoic acid and hydroxycholesterol

The biological role of lipid peroxidation products has continued to receive a great deal of attention not only for the elucidation of pathological mechanisms but also for their practical application to clinical use as bio-markers. In the last fifty years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. In this decade, F2-isoprostanes from arachidonates and neuroprostanes from docosahexanoates have been proposed as bio-markers. Although these markers are formed by a free radical-mediated oxidation, the yields from the parent lipids are minimal. Compared to these markers, hydroperoxy octadecadienoates (HPODE) from linoleates and oxysterols from cholesterols are yielded by much simpler mechanisms from more abundant parent lipids in vivo. Recently, the method in which both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as total hydroxyoctadecadienoic acid (tHODE) and 7-hydroxycholesterol (t7-OHCh), respectively, was proposed. The concentrations of tHODE and t7-OHCh determined by GC-MS analysis from physiological samples were much higher than that of 8-iso-prostagrandin F(2alpha). In addition to this advantage, hydrogen-donor activity of antioxidants in vivo could be determined by the isomeric-ratio of HODE (9- and 13-(Z,E)-HODE/9- and 13-(E,E)-HODE).