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.     

Yellow Reaction Products from Tocopherols and Trimethylamine Oxide

Characteristic yellow substances obtained when tocopherols were treated with trimethylamine oxide under a nitrogen stream at 180°C were purified by means of silicagel column chromatography and preparative TLC. Their structures were determined by spectroscopic studies.

Treatment of α-tocopherol with trimethylamine oxide gave 7-formyl-β-tocopherol, 5-formyl-γ-tocopherol-3-en and 5-formyl-γ-tocopherol. Yellow reaction products from γ- and (δ-tocopherols with trimethylamine oxide were 5-formyl-γ-tocopherol and 5-formyl-δ-tocopherol, respectively.     

Solvent influence on model oxidations of alpha-tocopherol

(+/-)-alpha-Tocopherol has been oxidised with t-butyl hydroperoxide in chloroform in order to simulate in vivo oxidations due to lipid hydroperoxides. t-Butyl hydroperoxide proved to be a weak oxidant and failed to oxidise alpha-tocopherol in 3 h at 60 degrees C. Inclusion of a small amount of ethanol in the reaction mixture brought about immediate oxidation and the formation of a new product, 5-ethoxymethyl-7,8-dimethyltocol in addition to the spiro dimer and spiro trimer of alpha-tocopherol, alpha-tocopherylquinone and 5-formyl-7,8-dimethyltocol. Formation of 5-ethoxymethyl-7,8-dimethyltocol increased with increasing concentrations of ethanol, up to a maximum of 59% at 20% ethanol. Further increase in ethanol concentration brought about a decrease in the oxidation of alpha-tocopherol and in the formation of 5-ethoxymethyl-7,8-dimethyltocol. Oxidation of the tocopherol model compound 2,2,5,7,8-pentamethyl-6-hydroxychroman under similar conditions produced the analogous product, 5-ethoxymethyl-2,2,7,8-tetramethyl-6-hydroxychroman together with 5-formyl-2,2,7,8-tetramethyl-6-hydroxychroman and 2-(3'-hydroxy-3'-methylbutyl)-3,5,6-trimethylbenzo-1,4-quinone.     

Microbial Oxidation of α-Methylstyrene and β-Methylstyrene

An unidentified bacterial strain S107B1, isolated from soil by use of isopropylbenzene as a carbon source, was shown to bring about oxidation of α-methylstyrene and β-methylstyrene,

One of the oxidation products produced from α-methylstyrene was identified as the new compound, (—)-cis-23-dihydroxy-1-isopropenyl-6-cyclohexene.

The same strain S107B1 also oxidized β-methylstyrene and produced 3-phenylpropionaldehyde and benzoic acid.

From these results, the existence of reductive step for the aerobic degradation of these aromatic hydrocarbons by this strain was made clear. The initial attack on these aromatic hydrocarbons and a cyclohexenediol compound formed from α-methylstyrene were discussed.     

Oral a-tocopherol Supplementation Inhibits Lipid Oxidation in Established Human Atherosclerotic Lesions

Background: Much experimental evidence suggests that lipid oxidation is important in atherogenesis and in epidemiological studies dietary antioxidants appear protective against cardiovascular events. However, most large clinical trials failed to demonstrate benefit of oral antioxidant vitamin supplementation in high-risk subjects. This paradox questions whether ingestion of antioxidant vitamins significantly affects lipid oxidation within established atherosclerotic lesions. Methods and results: This placebo-controlled, double blind study of 104 carotid endarterectomy patients determined the effects of short-term α-tocopherol supplementation (500 IU/day) on lipid oxidation in plasma and advanced atherosclerotic lesions. In the 53 patients who received α-tocopherol there was a significant increase in plasma α-tocopherol concentrations (from 32.66±13.11 at baseline to 38.31±13.87 (mean±SD) μmol/l, p<0.01), a 40% increase (compared with placebo patients) in circulating LDL-associated α-tocopherol (p<0.0001), and their LDL was less susceptible to ex vivo oxidation than that of the placebo group (lag phase 115.3±28.2 and 104.4±15.7 min respectively, p<0.02). Although the mean cholesterol-standardised α-tocopherol concentration within lesions did not increase, α-tocopherol concentrations in lesions correlated significantly with those in plasma, suggesting that plasma α-tocopherol levels can influence lesion levels. There was a significant inverse correlation in lesions between cholesterol-standardised levels of α-tocopherol and 7β-hydroxycholesterol, a free radical oxidation product of cholesterol. Conclusions: These results suggest that within plasma and lesions α-tocopherol can act as an antioxidant. They may also explain why studies using <500 IU α-tocopherol/day failed to demonstrate benefit of antioxidant therapy. Better understanding of the pharmacodynamics of oral antioxidants is required to guide future clinical trials.     

γ-Tocopherol biokinetics and transformation in humans

Background: The uptake and biotransformation of γ-tocopherol (γ-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of γ-T biology in healthy volunteers and their response to γ-T supplementation.

Methods: A single bolus of 100 mg of deuterium labeled γ-T acetate (d²-γ-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d² and d⁰-γ-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-γ-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of γ-T supplementation on α-T concentrations in plasma and α-T and γ-T metabolite formation were also assessed by HPLC or GC-MS analysis.

Results: At baseline, mean plasma α-T concentration was approximately 15 times higher than γ-T (28.3 vs. 1.9 µmol/l). In contrast, plasma γ-CEHC concentration (0.191 µmol/l) was 12 fold greater than α-CEHC (0.016 µmol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 µmol, respectively) suggesting that the clearance of α-CEHC from plasma was more than 40 times that of γ-CEHC. After d²-γ-TAC administration, the d² forms of γ-T and γ-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d⁰ species were hardly affected. Mean total concentrations of γ-T and γ-CEHC in plasma and urine peaked, respectively, between 0–9, 6–12 and 9–24 h post-supplementation with increases over baseline levels of 6–14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d²-γ-T equivalents was approximately 7 mg. Baseline levels of γ-T correlated positively with the post-supplementation rise of (d⁰ + d²) – γ – T and γ-CEHC levels in plasma, but correlated negatively with urinary levels of (d⁰ + d²)-γ-CEHC. Supplementation with 100 mg γ-TAC had minimal influence on plasma concentrations of α-T and α-T-related metabolite formation and excretion.

Conclusions: Ingestion of 100mg of γ-TAC transiently increases plasma concentrations of γ-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of γ-T nor the concentration and metabolism of α-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios α-T to γ-T and γ-CEHC to α-CEHC.     

α-Tocopherol supplementation does not affect monocyte endothelial adhesion or C-reactive protein levels but reduces soluble vascular adhesion molecule-1 in the plasma of healthy subjects

Abstract

Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (α-tocopherol). We have tested the hypothesis that α-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 μM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received α-tocopherol supplements (400 IU RRR-α-tocopherol/day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM-1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-κB in isolated resting monocytes, nor any effect of α-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and α-tocopherol concentration. In conclusion, α-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration.     

Antioxidant Effect of Tocopherols on Autoxidation of Milk Fat

Antioxidant activity of d-α-, dl-β-, d-γ- and d-δ-tocopherol was investigated with fatty acid methylester of milk fat from which unsaponifiable matter had been removed. Autoxidation was carried out at 50°C and its degree was indicated by peroxide value, α- or β-Tocopherol was more effective at lower concentrations (0.003 and 0.01%) than at higher concentrations (0.05, 0.1 and 0.5%). The antioxidant activity of γ- and δ-tocopherol was increased with the increase of tocopherol concentration within the range of 0.001 to 0.5%. The order of antioxidant activity of these tocopherols, which was compared in terms of the time to reach 30 meq of peroxide value, varied with the concentration; γ > β > δ > α at 0.001%, α > γ > β > δ at 0.003%, γ > δ > β > α at 0.01%, and δ > γ > β > α at the concentrations more than 0.05%. α-Tocopherol at the concentration of 0.003%, which corresponded to the concentration in original milk fat, was more effective than other tocopherols at the same concentration and α-tocopherol at other concentrations. Synergism due to the combination of β-, γ-, or δ-tocopherol with 0.003% of α-tocopherol was not observed.     

Occurrence of γ-(Guanylureido)butyric Acid in a Red Alga,Gymnogongrus flabelliformis

From a red alga, Gymnogongrus flabelliformis, a new guanidino compound of an empirical formula, C₆H₁₂O₃N₄ was isolated. The decomposition products identified were carbon dioxide, guanidine and γ-aminobutyric acid on hydrolysis at 120°C, succinic acid and guanylurea on oxidation with potassium permanganate, and γ-aminobutyric and γ-ureidobutyric acids on hydrolysis with barium hydroxide, respectively. These results led the authors to postulate that the substance is 1-amidino-3-(3-carboxypropyl)urea or γ-(guanylureido)butyric acid.     

Photosynthetic apparatus of chilling-sensitive plants IX. The involvement of α-tocopherol in the electron transport chain and the anti-oxidizing system in chloroplasts of tomato leaves

1. The role of tocopherols in tomato chloroplasts from fresh, cold and dark-stored as well as stored and illuminated leaves was studied.2. The cold and dark storage of leaves results in a loss of chloroplast α- and γ-tocopherols of about 30–40% accompanied by an increase in chloroplast δ-tocopherol of about 40%. On illumination of stored leaves, an elevation of α- and γ-tocopherol level to about 110 and 95% of the control, respectively, occurs, whilst δ-tocopherol content is not affected.3. Experiments performed with 2,2-diphenyl-1-picrylhydrazyl-treated chloroplasts show that only about 70% of total α-tocopherol is functionally active in the electron transport of Photosystem II between the diphenyl-carbazide (DPC) donation site and the inhibition site of DBMIB.4. A small amount of α-tocopherol quinone (about 10% of α-tocopherol content) is found in chloroplasts from fresh, fresh and illuminated as well as cold and dark-stored tomato leaves, whereas the illumination of the latter increases the chloroplast α-tocopherol quinone content 3-fold. Moreover, following the illumination of chloroplasts from cold and dark-stored as well as stored and illuminated leaves, the oxidation of exogenous α-tocopherol to α-tocopherol quinone is 2-fold faster then in chloroplasts from fresh leaves.5. The primary product (‘α-tocopheroxide’) formed during the α-tocopherol oxidation by illuminated chloroplasts was identified as 8a-hydroxy-α-tocopheron.6. Exogenous α-tocopherol inhibits the lipid photoperoxidation by about 40–50% in chloroplasts from all three kinds of tomato leaf.7. The results seem to suggest that chloroplast α-tocopherol is involved in both electron transport of PS II and antioxidizing system of chloroplasts.     

PCR-verified microarray analysis and functional in vitro studies indicate a role of α-tocopherol in vesicular transport

Global gene expression profiles of livers from mice, fed diets differing in α-tocopherol content, were compared using DNA microarray technology. Three hundred and eighty nine genes were found to significantly differ in their expression level by a factor of 2 or higher between the high and the low α-tocopherol group. Functional clustering using the EASE software identified 121 genes involved in transport processes. Twenty-one thereof were involved in (synaptic) vesicular trafficking. Up-regulation of syntaxin 1C (Stx1c), vesicle-associated membrane protein 1 (Vamp1), N-ethylmaleimide-sensitive factor (Nsf) and syntaxin binding protein 1 (Stxbp1, Munc18-1) was verified by real time PCR. At a functional level, α-tocopherol increased the secretory response in RBL and PC12 cells. Although here detected in liver, the α-tocopherol-responsive pathways are also relevant to neurotransmission. A role of α-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe α-tocopherol deficiency.     

α-Tocopherol injections in rats up-regulate hepatic ABC transporters, but not cytochrome P450 enzymes

The role of hepatic xenobiotic regulatory mechanisms in modulating hepatic α-tocopherol concentrations during excess vitamin E administration remains unclear. We hypothesized that increased hepatic α-tocopherol would cause a marked xenobiotic response. Thus, we assessed cytochrome P450 oxidation systems (phase I), conjugation systems (phase II), and transporters (phase III) after daily α-tocopherol injections (100mg/kg body wt) for up to 9days in rats. α-Tocopherol injections increased hepatic α-tocopherol concentrations nearly 20-fold, along with a 10-fold increase in the hepatic α-tocopherol metabolites α-CEHC and α-CMBHC. Expression of phase I (CYP3A2, CYP3A1, CYP2B2) and phase II (SULT2A1) proteins and/or mRNAs was variably affected by α-tocopherol injections; however, expression of phase III transporter genes was consistently changed by α-tocopherol. Two liver efflux transporter genes, ABCB1b and ABCG2, were up-regulated after α-tocopherol injections, whereas OATP, a liver influx transporter, was down-regulated. Thus, an overload of hepatic α-tocopherol increases its own metabolism and increases expression of genes of transporters that are postulated to lead to increased excretion of both vitamin E and its metabolites.     

GmTMT2a from soybean elevates the α-tocopherol content in corn and Arabidopsis

Tocochromanol, or vitamin E, plays a crucial role in human and animal nutrition and is synthesized only by photosynthetic organisms. γ-Tocopherol methyltransferase (γ-TMT), one of the key enzymes in the tocopherol biosynthetic pathway in plants, converts γ, δ-tocopherols into α-, β-tocopherols. Tocopherol content was investigated in 15 soybean cultivars and GmTMT2 was isolated from five varieties based on tocopherol content. GmTMT2a was expressed in E. coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. Overexpression of GmTMT2a enhanced α-tocopherol content 4–6-fold in transgenic Arabidopsis, and α-tocopherol content increased 3–4.5-fold in transgenic maize seed, which correlated with the accumulation of GmTMT2a. Transgenic corn that is α-tocopherol-rich may be beneficial for animal health and growth.     

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.     

Electron nuclear double resonance studies of radicals produced by the PbO2 oxidation of α-tocopherol and its model compound in solution

The electron nuclear double resonance (ENDOR) spectra of chromanoxyl radicals obtained by the PbO₂ oxidation of α-tocopherol and its model compound were observed in t-butylbenzene, and the proton hyperfine coupling constants were correctly determined. Each of the two β- and γ-methylene protons in the chromanoxyl ring shows an equivalent hyperfine splitting, suggesting that the heterocyclic ring attached to the aromatic ring are coplanar with the plane of the aromatic system. A comparison of the hyperfine couplings in α-tocopheroxyl radical and its model shows that the introduction of a long-isoprenoid-chain in the α-tocopherol in place of a methyl group in the model compound has very little effect on the unpaired spin distribution or molecular structure of the chromanoxyl skeleton. The results of McLachlan molecular orbital (MO) calculations were found to be in satisfactory agreement with the ‘experimental’ spin densities evaluated from the hyperfine coupling constants.     

Antioxidant activity of 3-methyl-1-phenyl-2-pyrazolin-5-one

Summary

The antioxidant activity of an anti-ischemic agent, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), was examined. The pKa value of MCI-186 is 7.0 and the rate of oxidation of MCI-186 initiated with an azo compound increased with increasing pH, suggesting that the anionic form of MCI-186 is much more reactive than the non-ionic form. The major products were 3-methyl-1-phenyl-2-pyrazolin-4,5-dione (4,5-dione) and 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB). Hydrolysis of 4,5-dione gave OPB. The minor intermediate product was 4-hydroxy-4-(3-methyl-1-phenyl-1H-pyrazolin-5-on-4-yl)-3-methyl-1-phenyl-1H-pyrazolin-5-one (BPOH). The nucleophilic attack of the anionic form of MCI-186 to 4,5-dione is likely to give BPOH. MCI-186 (50 μM) inhibited the aerobic oxidation at 37°C of 5.2 mM unilamellar soybean phosphatidylcholine (PC) liposomal membranes, initiated with a water-soluble initiator, as efficientlyas did ascorbate (100 μM). MCI-186 (50 μM) also inhibited the oxidation of the same PC liposomal membranes, this time initiated with a lipid-soluble initiator, almost as efficiently as did α-tocopherol (2 μM). Furthermore, the combination of MCI-186 with ascorbate or α-tocopherol showed almost complete inhibition of PC oxidation induced by both initiators. These data suggest that MCI-186 may work as a good antioxidant in cellular systems as well as in cell-free systems.     

Inhibition of Mammalian 15-Lipoxygenase-Dependent Lipid Peroxidation in Low-Density Lipoprotein by Quercetin and Quercetin Monoglucosides

Lipoxygenase is suggested to be involved in the early event of atherosclerosis by inducing plasma low-density lipoprotein (LDL) oxidation in the subendothelial space of the arterial wall. Since flavonoids such as quercetin are recognized as lipoxygenase inhibitors and they occur mainly in the glycoside form, we assessed the effect of quercetin and its glycosides (quercetin 3-O-β-glucopyranoside, Q3G; quercetin 4′-O-β-glucopyranoside, Q4′G; quercetin 7-O-β-glucopyranoside, Q7G) on rabbit reticulocyte 15-lipoxygenase (15-Lox)-induced human LDL lipid peroxidation and compared it with the inhibition obtained by ascorbic acid and α-tocopherol, the main water-soluble and lipid-soluble antioxidants in blood plasma, respectively. Quercetin inhibited the formation of cholesteryl ester hydroperoxides (CE-OOH) and endogenous α-tocopherol consumption effectively throughout the incubation period of 6 h. Ascorbic acid exhibited an effective inhibition only in the initial stage and LDL preloaded with fivefold α-tocopherol did not affect the formation of CE-OOH compared with the native LDL. CE-OOH formation was inhibited by both quercetin and quercetin monoglucosides in a concentration-dependent manner. Quercetin, Q3G, and Q7G exhibited a higher inhibitory effect than Q4′G (IC₅₀: 0.3–0.5 μM for quercetin, Q3G, and Q7G and 1.2 μM for Q4′G). While endogenous α-tocopherol was completely depleted after 2 h of LDL oxidation, quercetin, Q7G, and Q3G prevented the consumption of α-tocopherol. Quercetin and its monoglucosides were also exhausted during the LDL oxidation. These results indicate that quercetin glycosides as well as its aglycone are capable of inhibiting lipoxygenase-induced LDL oxidation more efficiently than ascorbic acid and α-tocopherol.     

Oxidative stress,growth factor production and budding in potato tubers during cold storage

In order to verify the role played by oxidation in the budding of potato tubers (Solanum tuberosum L. cv. Kennebec), the physiological events occurring below bud at 4°C have been studied for a period of 6 months. The low temperature storage induced an increase in the degree of unsaturation and a decrease in the ratio of saturated/unsaturated fatty acids of membrane polar lipids with a subsequent increase of lipid hydroperoxides (LOOH). Cold stress increased both enzymatic antioxidative activities (superoxide dismutase, SOD, E.C.1.15.1.1; catalase, CAT, E.C. 1.11.1.6), and α-tocopherol levels thus protecting membrane's polyunsaturated lipids. Between 0 and 15 days of storage SOD/CAT ratio, α-tocopherol, LOOH levels and the degree of lipid unsaturation showed strong variations. After 30 to 120/150 days the antioxidative system seemed to reach a homeostasis different from that of time 0, accompanied by a constant increase of indole-3-acetic acid (IAA) after 60 days. The antioxidative system, after 150 days, lost its efficiency while LOOH levels were maintained higher than time 0 and IAA concentration was sufficient to allow sprouting.     

Effect of α-tocopherol tissue levels on beef quality

To evaluate meat quality of beef with different α-tocopherol tissue levels, 55 feedlot steers were fed a barley-based finisher diet with four vitamin E supplementation levels (0, 350, 700 and 1400 IU DL-α-tocopheryl acetate/animal per day) for 120 days. Although the increase in oxidation levels overtime was much smaller (P < 0.001) in the high-medium and high groups, α-tocopherol tissue levels did not affect (P > 0.05) pH, proximate analysis, drip and cooking losses, and shear force of steaks. No effect of α-tocopherol tissue levels was found in retail evaluation of steaks after a short ageing time of 6 days, but with 21 days of ageing, a delay in formation of metmyoglobin (P = 0.008) was observed in steaks with higher tissue levels of α-tocopherol. Similar results were found for ground beef (25% fat) prepared from 6-day aged meat. Thus, higher α-tocopherol tissue levels protect ground beef and long-aged steaks from discolouration and lipid oxidation.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.