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.

Preferential hydrolysis of monohydroperoxides of linoleoyl and linolenoyl triacylglycerol by pancreatic lipase.

Four triacylglycerols (TGs) containing palmitoyl and linoleoyl or linolenoyl groups in known positions were synthesized and pancreatic lipase hydrolysis of their monohydroperoxides was investigated. TG monohydroperoxides did not deactivate the lipase and were hydrolyzed at almost the same degrees as their original TGs. In the hydrolysis of unoxidized TGs, pancreatic lipase showed almost the same reactivity on palmitoyl, linoleoyl and linolenoyl groups at the 1(3)-positions. However, this enzyme had fatty acid specificity for TG monohydroperoxides and the molar concentration of hydroperoxy linoleic or linolenic acid liberated from 1(3)-positions of TG monohydroperoxides were 1.6-2.4-times higher than that of the unoxidized fatty acid from the corresponding 3(1)-positions. The susceptibility of hydroperoxy acyl components of TG monohydroperoxides to pancreatic lipase hydrolysis is explained by its molecular structure and hydrophilic property.

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.

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.

Effects of a carotene-deficient diet on measures of oxidative susceptibility and superoxide dismutase activity in adult women.

The effect of consuming a low carotene diet (approximately 60 micrograms carotene/day) on oxidative susceptibility and superoxide dismutase (SOD) activity in women living in a metabolic research unit was evaluated. The diet had sufficient vitamins A, E, and C. The women ate the diet supplemented with 1500 micrograms/day beta-carotene for 4 days (baseline), then the unsupplemented diet for 68 days (depletion), followed by the diet supplemented with > 15,000 micrograms/day carotene for 28 days (repletion). Production of hexanal, pentanal, and pentane by copper-oxidized plasma low density lipoproteins from carotene-depleted women was greater than their production of these compounds when repleted with carotene. Erythrocyte SOD activity was depressed in carotene-depleted women; it recovered with repletion. Thiobarbituric acid reactive substances in plasma of carotene-depleted women were elevated and diminished with repletion. Dietary carotene seems to be needed, not only as a precursor of vitamin A, but also to inhibit oxidative damage and decrease oxidation susceptibility.

Reduced oxidative susceptibility of LDL from patients participating in an intensive atherosclerosis treatment program.

The goal of this investigation was to determine whether participation in an atherosclerosis treatment program would reduce the oxidative susceptibility of LDL from patients with coronary artery disease. The treatment program included intensive exercise therapy, stress management, and consumption of a diet containing 10% fat. The size and antioxidant and lipid contents of LDL particles from 25 patients were analyzed at baseline and after 3 mo of therapy. The susceptibility of LDL to copper-mediated oxidation was measured by a conjugated diene assay and headspace gas chromatography (HSGC). Atherosclerosis treatment significantly reduced plasma total cholesterol and apolipoprotein B concentrations and the molar ratio of LDL cholesterol ester to apolipoprotein B (P < 0.01). The LDL content of alpha-tocopherol and beta-carotene was increased (27% and 17%, respectively, P < 0.04) and the molar ratio of LDL cholesterol ester the sum of LDL alpha-tocopherol and LDL beta-carotene decreased from 159 at baseline to 122 at 3 mo (P < 0.01). The lag phase of LDL conjugated diene formation increased 24%, whereas the maximum rate of oxidation slowed 29% (P < 0.01). As assessed by HSGC, copper-catalyzed formation of volatile lipid oxidation products was reduced 15% (P < 0.007); the reduction in volatiles was correlated with an increase in the alpha-tocopherol content of LDL (r=-0.48, P < 0.01). The principal determinants of reduced LDL oxidative susceptibility were the particle contents of alpha-tocopherol and beta-carotene. To our knowledge, this is the first report to document a reduction in LDL oxidation in coronary artery disease patients undergoing atherosclerosis-reversal therapy.

Secondary products of lipid oxidation.

In the last decade, a multitude of secondary products have been identified from the radical and photosensitized oxidations of polyunsaturated lipids. These secondary products consist of oxygenated monomeric materials including epoxy-hydroperoxides, oxo-hydroperoxides, hydroperoxy epidioxides, dihydroperoxides, hydroperoxy bis-epidioxides, and hydroperoxy bicycloendoperoxides. More recently, higher molecular weight dimeric compounds have been identified from autoxidized methyl linoleate and linolenate. Decomposition of these oxidation products form a wide range of carbonyl compounds, hydrocarbons, furans, and other materials that contribute to the flavor deterioration of foods and that are implicated in biological oxidation. The interaction of some of these degradation products with DNA may be involved in cell-damaging reactions.

Cyclic fatty esters: synthesis, characterization, and lipolysis of isomeric triglycerides of 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoic acid.

Triglycerides of a model cyclic fatty acid (CFA) 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoic acid (Ia) were synthesized for biological and toxicity evaluations. The monoacid triglyceride II (CyCyCy) was interesterified with triolein (OOO) to obtain mixtures of diacid triglycerides: III (OOCy), IV (OCyO), V (OCyCy), and VI (CyOCy). The interesterification mixtures were separated by preparative HPLC into two 'critical pairs' of isomeric triglycerides. Triglycerides III-VI were synthesized and a 13C-NMR method was developed to estimate 'critical pairs'. CFA-triglycerides were characterized by IR, NMR, HPLC and capillary GLC, and their relative rates of hydrolysis by lipase were compared. Although tricyclin (II) was completely resistant to lipolysis, triglycerides III and VI hydrolyzed significantly slower than triolein, and the 'critical pairs' hydrolyzed as readily as triolein. Therefore, partial CFA-triglycerides formed in heat-abused fats can undergo lipolysis and likely be absorbed like normal dietary fats.

Low-density lipoprotein antioxidant activity of phenolic compounds and polyphenol oxidase activity in selected clingstone peach cultivars.

The antioxidant potential of eight clingstone peach cultivars was investigated by determining phenolic compounds and inhibition of low-density lipoprotein (LDL) oxidation. Cultivars low in polyphenol oxidase (PPO) were also selected to minimize enzymatic browning. Inhibition of LDL oxidation varied from 17.0 to 37.1% in peach flesh extract, from 15.2 to 49.8% in whole peach extract, and from 18.2 to 48.1% in peel extract. Total phenols were 432.8-768.1 mg/kg in flesh extract, 483.3-803.0 mg/kg in whole extract, and 910.9-1922.9 mg/kg in peel extract. The correlation coefficient between relative LDL antioxidant activity and concentration of total phenols was 0.76. Peel PPO activity was higher than flesh activity in most cultivars. The lowest PPO and specific activities were found in the Walgant cultivar, followed by Kakamas and 18-8-23. These three cultivars combine the desirable characteristics of strong antioxidant activity, low PPO activity, and lower susceptibility to browning reactions.

Antioxidant activity of selected Spanish wines in corn oil emulsions.

Wines contain phenolic compounds that may be useful for preventing lipid oxidation as dietary antioxidants. This study was aimed at evaluating the antioxidant activity in corn oil emulsions of seventeen selected Spanish wines and two California wines. The inhibition of hydroperoxide formation at 10 microM gallic acid equivalents (GAE) varied from 8.4% to 40.2% with the red wines, from 20.9% to 45.8% with the rosé wines, and from 6.5% to 47.0% with the white wines. The inhibition of hydroperoxide formation at 20 microM GAE varied from 11.9% to 34.1% with the red wines, from 0.1% to 34. 5% with the rosé wines, and from 3.3% to 37.2% with the white wines. The inhibition of hexanal formation at 10 microM GAE varied from 23. 6% to 64.4% with the red wines, from 42.7% to 68.5% with the rosé wines, and from 28.4% to 68.8% with the white wines. The inhibition of hexanal formation at 20 microM GAE varied from 33.0% to 46.3% with the red wines, from 11.3% to 66.5% with the rosé wines, and from -16.7% to +21.0% with the white wines. The antioxidant effect decreased with increasing concentration. This antioxidant activity was related to the five main groups of phenolic compounds identified in wines by HPLC. The relative antioxidant activity correlated positively with the total phenol content of wines (by the Folin-Ciocalteu method and by HPLC), benzoic acids, anthocyanins, flavan-3-ols, and flavonols, for the inhibition of hydroperoxides and hexanal at 10 and 20 microM GAE.

Activities of antioxidants are affected by colloidal properties of oil-in-water and water-in-oil emulsions and bulk oils.

The activity of alpha-tocopherol, Trolox, propyl gallate, gallic acid, methyl carnosoate, and carnosic acid was studied in two oil-in-water (o/w) emulsions, in two water-in-oil (w/o) emulsions, and in bulk oil with and without added emulsifiers. All antioxidants had either moderate or higher activity in bulk oil than in the emulsions. In most emulsions, the most polar antioxidants, propyl gallate and gallic acid, exhibited either prooxidant activity or no antioxidant activity. Methyl carnosoate was the most active antioxidant in w/o emulsions but was less active than Trolox in o/w emulsions. alpha-Tocopherol was less active in bulk oil than in emulsions, but its activity in bulk oil was markedly enhanced by the addition of o/w emulsifiers. Partitioning of antioxidants, hydrogen bonding, interphase transport, surface accessibility, and interaction of emulsifier with antioxidants are considered to be important parameters that determine antioxidant activity in lipid-containing systems.

Lactoferrin in infant formulas: effect on oxidation.

Lactoferrin is an iron transport protein present in human milk at an average concentration of 1.4 mg/mL. Commercially modified infant formulas based on cow's milk contain much lower amounts of lactoferrin (0.1 mg/mL lactoferrin) and soy based formulas have none. In addition to its role in iron transport, lactoferrin has bacteriostatic and bactericidal activities. Infant formulas are supplemented with relatively large amounts of iron (up to 12 mg/L). The effect of various concentrations of added lactoferrin and supplemental iron on lipid oxidation was tested in two different infant formulas. The extent of oxidation in the formulas as a function of time was determined by formation of hydroperoxides, production of hexanal, and fluorescence. On the basis of all three of these determinations, lactoferrin acted as an antioxidant in the absence and presence of different concentrations of supplemented iron. Lactoferrin inhibited oxidation in a concentration-dependent manner even at concentrations beyond its capacity to bind iron at its two high affinity binding sites. Lactoferrin can be used, therefore, as a dual purpose additive in infant formulas and similar food products for its antioxidant and its antimicrobial properties.

Comparison of natural polyphenol antioxidants from extra virgin olive oil with synthetic antioxidants in tuna lipids during thermal oxidation.

Polyphenols extracted from extra virgin olive oil (EVOO) were tested for their ability to inhibit lipid oxidation of canned tuna. Hydroperoxide formation during oxidation was monitored by measurement of peroxide value and decomposition of hydroperoxides by static headspace gas chromatographic analysis of volatiles. In tuna oxidized at 40 and 100 degrees C, 400 ppm of the EVOO polyphenols was an effective antioxidant as compared with 100 ppm of a 1:1 mixture of the synthetic antioxidants butylated hydroxytoluene and butylated hydroxyanisole. However, at concentrations <100 ppm, the EVOO phenolic compounds promoted hydroperoxide formation and decomposition. The EVOO polyphenols were effective antioxidants when added to heated tuna muscle in the presence of either brine or refined olive oil. The oxidation rate in tuna muscle packed in brine was higher than that of tuna packed in refined olive oil. The EVOO polyphenols had higher antioxidant activity in the brine samples than in the refined olive oil. The higher antioxidant activity of EVOO polyphenols in tuna packed in brine may be explained by their greater affinity toward the more polar interface between water and the fish oil system.

Effect of lactoferrin on oxidative stability of corn oil emulsions and liposomes.

Interest in using lactoferrin in foods for its antimicrobial activity inspired the present study of its antioxidant activity. Natural bovine lactoferrin inhibited oxidation in buffered corn oil emulsions and lecithin liposome systems at pH 6.6 and 50 degrees C. The antioxidant activity increased with lactoferrin concentration in both phosphate- and Tris-buffered emulsions, but not in both buffered liposome systems. A mixture of 1 microM lactoferrin and 0.5 microM ferrous ions was a significantly better antioxidant than 1 microM lactoferrin alone in Tris-buffered emulsions and in phosphate-buffered liposomes. Lactoferrin was a prooxidant at 1 microM in phosphate-buffered liposomes and at 15 and 20 microM in Tris-buffered liposomes. Copper was a stronger prooxidant than iron in both buffered emulsions. Lactoferrin decreased the prooxidant effect of iron, but not of copper, in emulsions. The antioxidant or prooxidant activities of lactoferrin depended on the lipid system, buffer, its concentration, the presence of metal ions, and oxidation time.

Spanish sparkling wines (Cavas) as inhibitors of in vitro human low-density lipoprotein oxidation.

Forty-seven dealcoholized sparkling wines (cava) from the Penedès area in Spain were tested for their antioxidant activity in a low-density lipoprotein system. The effect of different quality-related parameters, such as harvest year or grape variety, was investigated. Twenty-two phenolic compounds were separated by high-performance liquid chromatography and identified by comparing their retention time and their ultraviolet spectra with those of pure standards. When tested at the same total phenol concentration, the antioxidant activity of these white sparkling wines was found to be similar to that reported for red wines. This activity was positively correlated with the total phenolic content, trans-caffeic acid, coumaric acid, protocatechuic acid, and quercetin 3-glucuronide. The wines made of the classic cava wine coupage had superior antioxidant activity compared to those of other cultivars.

Mutagenicity tests of lipid oxidation products in Salmonella typhimurium: monohydroperoxides and secondary oxidation products of methyl linoleate and methyl linolenate.

Nine hydroperoxy and hydroperoxy-epidioxy oxidation products derived from either autoxidation (AO) or photosensitized oxidation (PO) of methyl linoleate (MLo) or methyl linolenate (MLn) were tested for mutagenic activity by the Salmonella typhimurium his+ reversion assay using strains TA100, TA98, TA102, TA97 and TA1537. All nine oxidation products, monohydroperoxides from AO-MLn (I) or from PO-MLn (II), dihydroperoxides from PO-MLo (III), AO-MLn (IV) or PO-MLn (V), hydroperoxy epidioxides from PO-MLo (VI), AO-MLn (VII) or PO-MLn (VIII) and hydroperoxy bis-epidioxides from PO-MLn (IX), were weakly mutagenic in strains TA97 and/or TA100. The hydroperoxy epidioxides (VI-IX) exhibited significantly greater activity in strain TA97 than did the monohydroperoxides (I, II) or the dihydroperoxides (III-V). In strain TA100, all of the oxidation products tested exhibited similar activity. No major differences between products derived from autoxidized and photooxidized MLn (I v. II, IV v. V, VII v. VIII) were obtained. Rat-liver S-9 reduced the toxicity of all oxidation products to the tester strains. The greatest mutant yields were usually obtained in the presence of S-9, but mutagenic potency was sometimes greater without S-9. The structural feature common to all of the mutagenic oxidation products was the presence of a hydroperoxy group, suggesting that this characteristic is responsible for the observed mutagenicity, either directly or through a common degradative pathway to reactive products of lower molecular weight.

Headspace gas chromatography of volatile lipid peroxidation products from human red blood cell membranes.

An improved headspace capillary gas chromatographic (GC) method was developed to measure the oxidative susceptibility of human red blood cell (RBC) membranes. This method analyzed volatile peroxidation products of both n-6 (hexanal and pentane) and n-3 (propanal) polyunsaturated fatty acids. Oxidative susceptibility tests were standardized by incubating in a sealed 10-mL headspace bottle 0.25 or 1 mL of human RBC membrane in 40 mM phosphate buffer for 1 hr at 37 degrees C with a mixture of Fe++, ascorbic acid and H2O2. Sodium dodecyl sulfate increased significantly the amount of hexanal measured by headspace GC. By this standard headspace method, in one series of red blood cell membranes (RBCM) samples a four-fold variation in oxidative susceptibility was observed in RBCM from blood freshly drawn from six healthy subjects. In another series of RBCM samples a sixteen-fold variation in oxidative susceptibility was noted in frozen RBCM from blood freshly drawn from five healthy subjects. Correlation between hexanal formation and polyunsaturated fatty acids (PUFA) depletion provided good evidence that under these standard conditions hexanal is exclusively derived from the oxidation of arachidonic acid. Hydroperoxides of arachidonic acid are more readily formed and decomposed than those of linoleic acid in the presence of Fe++, ascorbic acid and H2O2 to produce hexanal as the main product that can be readily analyzed by headspace GC. This method may provide a useful tool to study susceptibility toward lipid peroxidative damage in human RBC membranes.

Cyclic fatty esters: Synthesis and characterization of methyl ω-(6-alkyl-3-cyclohexenyl) alkenoates.

Diunsaturated C18 cyclic fatty acid methyl esters of known structure and configuration were synthesized as model derivatives of cyclic fatty acids formed in heat-abused vegetable oils for characterization and further biological evaluation. The Wittig reaction was used to prepare 5 pure methyl esters: (a) 12-(3-cyclohexenyl)-11-dodecenoate, (b) 11-(6-methyl-3-cyclohexenyl)-10-undecenoate, (c) 10-(6-ethyl-3-cyclohexenyl)-9-decenoate, (d) 9-(6-propyl-3-cyclohexenyl)-8-nonenoate and (e) 8-(6-butyl-3-cyclohexenyl)-7-octenoate. Diels-Alder cycloaddition reactions between 1,3-butadiene and appropriate (E)-2-alkenals produced 3-cyclohexenal intermediates. The appropriate methyl ω-bromoesters and their triphenylphosphonium bromides were made and converted to their respective ylids with NaOCH3 in DMF. The appropriate 3-cyclohexenals and phospho-ylids were reacted, and the desired cyclic ester products were isolated in crude yields of 30-83% as liquids and fractionally distilled. The crude cyclic esters were purified either by preparative TLC or by saponification-esterification. Double bonds in purified cyclic esters weretrans-isomerized and hydrogenated. Each derivative was characterized by IR,(1)H-NMR,(13)C-NMR, capillary GLC and GC-MS. On the basis of these analyses, no positional isomers were detected, Z-unsaturated isomers were produced in better than 90% purity, and the alkyl and ester ring substituents were predominantlytrans to each other.

Effect of alpha-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides.

alpha-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180 degrees C were analyzed by capillary gas chromatography. In the presence of either alpha-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples. When an isomeric mixture of methyl linoleate hydroperoxides (cis, trans, and trans,trans 9- and 13-hydroperoxides) was decomposed by heat, the presence of alpha-tocopherol and 1,4-cyclohexadiene caused the relative amounts of pentane and methyl octanoate to decrease and hexanal and methyl 19-oxononanoate to increase. A similar effect of alpha-tocopherol was observed on the distribution of volatiles formed from a mixture of the trans,trans 9- and 13-hydroperoxides. This effect of alpha-tocopherol was, however, insignificant with pure cis,trans 13-hydroperoxide of methyl linoleate. The decrease in total volatiles with the hydrogen donor compounds, alpha-tocopherol and 1,4-cyclohexadiene, indicates a suppression of homolytic beta-scission of the hydroperoxides, resulting in a change in relative distribution of volatiles. The increase in hexanal and methyl 9-oxononanoate at the expense of pentane and methyl octanoate in the presence of hydrogen donor compounds supports the presence of a heat-catalyzed heterolytic cleavage (also known as Hock cleavage), which seems to mainly affect the trans,trans isomers of linoleate hydroperoxides.

Cyclic fatty esters: hydroperoxides from autoxidation of methyl 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoate.

Autoxidation of the cyclic fatty acid ester, methyl 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoate (I) was investigated to characterize the hydroperoxide isomers formed and to provide basic information on their chemistry, detection and effect on the quality of polyunsaturated cooking oils. Oxidation at 60 C with 1% hydroperoxide initiator produced a monohydroperoxide fraction containing five positional isomers (7-, 11-, 12-, 13- and 14-OOH), resolved by high performance liquid chromatography, as their allylic hydroxy esters. Their structures were established by 1H- and 13C-NMR spectroscopy and by capillary gas chromatography-mass spectrometry (GC-MS) as trimethylsilyl ether derivatives. Two additional isomers (8- and 9-OOH) were detected by GC-MS in only trace (less than 1%) quantities. Capillary GC resolved some geometric and stereoisomers, as well as positional isomers. Compared to photosensitized oxidation, two additional positional isomers (11- and 14-OOH) were produced by autoxidation. More stereoisomers were formed, and oxidation of the ring double bond was favored 8:1 over that of the side chain. This selectivity may be attributed to greater steric hindrance for oxygen attack at the side-chain double bond. A free radical mechanism is proposed to explain the greater isomeric complexity of the hydroperoxide products compared to photosensitized oxidation.