Vitamin E: action, metabolism and perspectives

Natural vitamin E includes four tocopherols and four tocotrienols. RRR-alpha-tocopherol is the most abundant form in nature and has the highest biological activity. Although vitamin E is the main lipid-soluble antioxidant in the body, not all its properties can be assigned to this action. As antioxidant, vitamin E acts in cell membranes where prevents the propagation of free radical reactions, although it has been also shown to have pro-oxidant activity. Non-radical oxidation products are formed by the reaction between alpha-tocopheryl radical and other free radicals, which are conjugated to glucuronic acid and excreted through the bile or urine. Vitamin E is transported in plasma lipoproteins. After its intestinal absorption vitamin E is packaged into chylomicrons, which along the lymphatic pathway are secreted into the systemic circulation. By the action of lipoprotein lipase (LPL), part of the tocopherols transported in chylomicrons are taken up by extrahepatic tissues, and the remnant chylomicrons transport the remaining tocopherols to the liver. Here, by the action of the "alpha-tocopherol transfer protein", a major proportion of alpha-tocopherol is incorporated into nascent very low density lipoproteins (VLDL), whereas the excess of alpha-tocopherol plus the other forms of vitamin E are excreted in bile. Once secreted into the circulation, VLDL are converted into IDL and LDL by the action of LPL, and the excess of surface components, including alpha-tocopherol, are transferred to HDL. Besides the LPL action, the delivery of alpha-tocopherol to tissues takes place by the uptake of lipoproteins by different tissues throughout their corresponding receptors. Although we have already a substantial information on the action, effects and metabolism of vitamin E, there are still several questions open. The most intriguing is its interaction with other antioxidants that may explain how foods containing small amounts of vitamin E provide greater benefits than larger doses of vitamin E alone.     

Vitamin E: function and metabolism.

Although vitamin E has been known as an essential nutrient for reproduction since 1922, we are far from understanding the mechanisms of its physiological functions. Vitamin E is the term for a group of tocopherols and tocotrienols, of which alpha-tocopherol has the highest biological activity. Due to the potent antioxidant properties of tocopherols, the impact of alpha-tocopherol in the prevention of chronic diseases believed to be associated with oxidative stress has often been studied, and beneficial effects have been demonstrated. Recent observations that the alpha-tocopherol transfer protein in the liver specifically sorts out RRR-alpha-tocopherol from all incoming tocopherols for incorporation into plasma lipoproteins, and that alpha-tocopherol has signaling functions in vascular smooth muscle cells that cannot be exerted by other forms of tocopherol with similar antioxidative properties, have raised interest in the roles of vitamin E beyond its antioxidative function. Also, gamma-tocopherol might have functions apart from being an antioxidant. It is a nucleophile able to trap electrophilic mutagens in lipophilic compartments and generates a metabolite that facilitates natriuresis. The metabolism of vitamin E is equally unclear. Excess alpha-tocopherol is converted into alpha-CEHC and excreted in the urine. Other tocopherols, like gamma- and delta-tocopherol, are almost quantitatively degraded and excreted in the urine as the corresponding CEHCs. All rac alpha-tocopherol compared to RRR-alpha-tocopherol is preferentially degraded to alpha-CEHC. Thus, there must be a specific, molecular role of RRR-alpha-tocopherol that is regulated by a system that sorts, distributes, and degrades the different forms of vitamin E, but has not yet been identified. In this article we try to summarize current knowledge on the function of vitamin E, with emphasis on its antioxidant vs. other properties, the preference of the organism for RRR-alpha-tocopherol, and its metabolism to CEHCs.     

Vitamin E, antioxidant and nothing more

All of the naturally occurring vitamin E forms, as well as those of synthetic all-rac-alpha-tocopherol, have relatively similar antioxidant properties, so why does the body prefer alpha-tocopherol as its unique form of vitamin E? We propose the hypothesis that all of the observations concerning the in vivo mechanism of action of alpha-tocopherol result from its role as a potent lipid-soluble antioxidant. The purpose of this review then is to describe the evidence for alpha-tocopherol's in vivo function and to make the claim that alpha-tocopherol's major vitamin function, if not only function, is that of a peroxyl radical scavenger. The importance of this function is to maintain the integrity of long-chain polyunsaturated fatty acids in the membranes of cells and thus maintain their bioactivity. That is to say that these bioactive lipids are important signaling molecules and that changes in their amounts, or in their loss due to oxidation, are the key cellular events that are responded to by cells. The various signaling pathways that have been described by others to be under alpha-tocopherol regulation appear rather to be dependent on the oxidative stress of the cell or tissue under question. Moreover, it seems unlikely that these pathways are specifically under the control of alpha-tocopherol given that various antioxidants other than alpha-tocopherol and various oxidative stressors can manipulate their responses. Thus, virtually all of the variation and scope of vitamin E's biological activity can be seen and understood in the light of protection of polyunsaturated fatty acids and the membrane qualities (fluidity, phase separation, and lipid domains) that polyunsaturated fatty acids bring about.     

Flavonoids can replace alpha-tocopherol as an antioxidant

Endogenous antioxidants such as the lipid-soluble vitamin E protect the cell membranes from oxidative damage. Glutathione seems to be able to regenerate alpha-tocopherol via a so-called free radical reductase. The transient protection by reduced glutathione (GSH) against lipid peroxidation in control liver microsomes is not observed in microsomes deficient in alpha-tocopherol. Introduction of antioxidant flavonoids, such as 7-monohydroxyethylrutoside, fisetin or naringenin, into the deficient microsomes restored the GSH-dependent protection, suggesting that flavonoids can take over the role of alpha-tocopherol as a chain-breaking antioxidant in liver microsomal membranes.     

Is vitamin E the only lipid-soluble,chain-breaking antioxidant in human blood plasma and erythrocyte membranes?

The concentration of lipid-soluble, chain-breaking antioxidants in human plasma and in erythrocyte ghosts have been determined for the first time by an inhibited-autoxidation method. The results are very similar to the concentrations of vitamin E measured for the same blood components by the HPLC method. It is concluded that vitamin E, which is largely present as alpha-tocopherol, is the only significant lipid-soluble, chain-breaking type of antioxidant present in human blood. The concentration of vitamin E in the plasma lipids divided by the concentration of vitamin E in the ghost membrane lipids is approximately a constant despite the large differences in vitamin E-intake and in plasma lipid concentrations in different individuals. Vitamin E/lipid ratios for plasma and ghosts were larger for subjects taking a supplement of alpha-tocopherol acetate of 100 IU per week, compared to nonsupplemented subjects (based on data from a limited number of subjects). A larger supplement of 2800 IU per week did not significantly increase the vitamin E/lipid ratios.     

Lipoxygenase-catalysed degradation of carotenoids from tomato in the presence of antioxidant vitamins

Carotenoid extract from ripe tomato fruit was subjected to a lipoxygenase-catalysed co-oxidation in the presence of vitamin C and vitamin E at different concentrations. Relative retention (%) of major carotenoids by the experimental mixture was used as an index of their degradation and interaction with the antioxidants. Oxidation-prevention activity of each antioxidant against pigment co-oxidation as impacted by their molar concentration was studied. beta-Carotene was found to be the most sensitive pigment, followed by lycoxanthin and lycopene. Ascorbic acid when added in the range of 0-1.8 mM interacted with the different carotenoids by different modes. Evidence was given on regeneration, by ascorbic acid, of lycopene during the course of co-oxidation. The concentration required for alpha-tocopherol acetate to exhibit antioxidative effect was 10 times higher than that of ascorbic acid. beta-Carotene was prevented, by alpha-tocopherol acetate, faster than lycoxanthin and lycopene. The latter carotenoids differed substantially in their interaction with the lipophilic antioxidant at only the lowest concentration (0.3 mM). It was established that under the given conditions there is no synergism between vitamin C and vitamin E that improves their oxidation prevention against co-oxidation of carotenoids. Moreover, the combined use of antioxidants caused more oxidative degradation of beta-carotene.     

A method to measure antioxidant activity in organic media: application to lipophilic vitamins

The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS(.+)) can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase in an organic medium. The ABTS radical is easily generated in acidified ethanol medium in about 100 s with a stability of 1.7 x 10(-3) (-deltaabs/min) monitored at 730 nm. Other organic solvents, such as methanol or acetone, have lower radical generation times but the radical is less stable. The addition of Trolox or a lipophilic antioxidant such as alpha-tocopherol or beta-carotene produces a decrease in absorbance that can be used to estimate antioxidant capacity. Using a spectrophotometric end-point method and microplate-reader equipment, we have developed a method that estimates the antioxidant activity of different lipophilic vitamins. The use of Trolox as an antioxidant standard led to a limit of detection of 0.08 nmoles and limit of quantitation of 0.28 nmoles, while similar values were obtained for alpha-tocopherol and beta-carotene. The relative antioxidant activity values obtained by different antioxidants showed that alpha-tocopherol has a similar antioxidant potential to Trolox and that beta-carotene has 2.6 times the antioxidant potential of Trolox. In our opinion, this method can be useful for estimating the antioxidant activity in lipophilic samples and as a complement to other methods that measure antioxidant activity in aqueous media.     

Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma

Oxidative stress and the role of antioxidants are currently one of the most important subjects in the field of life science. In the present study, we assessed the oxidation of plasma lipids induced by free radicals and its inhibition by antioxidants with a fluorescence probe BODIPY. Vitamin E and C-depleted plasma was used to evaluate the inherent action of several antioxidants. BODIPY reacted with free radicals in plasma to emit fluorescence (ex. 510 nm, em. 520 nm), which was suppressed by the antioxidants in a concentration-dependent manner. However, the suppression of fluorescence emission by antioxidants did not always correlate quantitatively with the suppression of lipid peroxidation. For example, alpha-tocopherol suppressed BODIPY fluorescence but enhanced the peroxidation of plasma lipids in the absence of ascorbic acid. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without a phytyl side chain, almost completely suppressed both fluorescence emission and lipid peroxidation in the plasma. These results show that BODIPY can be used as a convenient probe for radical scavenging, but that care should be taken for the evaluation of antioxidant capacity.     

Interaction of ascorbate and alpha-tocopherol enhances antioxidant reserve of erythrocytes during anemia in visceral leishmaniasis

Visceral leishmaniasis (V.L.) is associated with enhanced lipid peroxidation along with impaired function of antioxidant defense system in erythrocytes. The effect of chronic treatment with ascorbate and alpha-tocopherol was studied on erythrocytes in hamsters infected with Leishmania donovani. Combination treatment with both antioxidants proved to be a potential suppressor of lipid hydroperoxide formation as well as hypotonic osmotic lysis during the leishmanial infection. Positive correlations between the depleted levels of erythrocyte ascorbate, GSH and alpha-tocopherol exhibit proportionate alterations in the nonenzymatic antioxidant levels at different stages of infection. Indirect measurement of transmembrane electron transfer as ferricyanide reduction suggests an active participation of endogenous contents of ascorbate and alpha-tocopherol in the protection against oxidative damage of membrane lipids. Cooperative behavior of both antioxidants in the ferricyanide reducing capacity was further evinced by resealing the ghosts in presence of exogenous ascorbate and alpha-tocopherol. Furthermore, intravesicular ascorbate serves in the defense of extravesicular ferricyanide induced oxidation of endogenous alpha-tocopherol. The results suggest an interacting role of ascorbate and alpha-tocopherol in maintaining the antioxidant reserve of erythrocytes during anemia in V.L.     

Topically applied vitamin E prevents massive cutaneous inflammatory and oxidative stress responses induced by double application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice

Vitamin E (alpha-tocopherol) is a promising chemopreventive and pharmacologically safe agent, which can be exploited or tested against skin cancer. It is an established antioxidant with an ability to ameliorate the UV-induced skin damage and chemically induced inflammation in lungs. However, there are some conflicting reports about its role as a modulator of chemically induced promotion. We evaluated its efficacy in preventing the inflammatory and oxidative stress responses in a double 12-O-tetradecanoylphorbol-13-acetate (TPA) application tumor skin promotion protocol. Double application of TPA was undertaken to produce massive inflammatory and oxidative stress responses. Topical TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion. Vitamin E application 30 min prior to TPA treatment (10 nmol) inhibited induction of hydrogen peroxide, myeloperoxidase (MPO) activity, xanthine oxidase (XO) activity and lipid peroxidation (LPO). Vitamin E also positively modulated altered antioxidants of mouse skin. Histological examination also revealed marked improvement. These results confirm the efficacy of vitamin E against early inflammatory and oxidative stress responses, which are hallmark of tumor promotion and provide rational basis for chemopreventive action of vitamin E in skin cancer.     

Vitamin E: underestimated as an antioxidant

Some 80 years after its discovery, vitamin E has experienced a renaissance which is as surprising as it is trivial. Although vitamin E is essential for reproduction, in rats at least, and deficiency causes neurological disorders in humans, the main interest in the last decades has concentrated on its antioxidant functions. This focus has highly underestimated the biological importance of vitamin E, which by far exceeds the need for acting as a radical scavenger. Only recently has it become clear that vitamin E can regulate cellular signaling and gene expression. Out of the eight different tocols included in the term vitamin E, alpha-tocopherol often exerts specific functions, which is also reflected in its selective recognition by proteins such as the alpha-tocopherol transfer protein and alpha-tocopherol-associated proteins. Vitamin E forms other than alpha-tocopherol are very actively metabolised, which explains their low biopotency. In vivo, metabolism may also attenuate the novel functions of gamma-tocopherol and tocotrienols observed in vitro. On the other hand, metabolites derived from individual forms of vitamin E have been shown to exert effects by themselves. This article focuses on the metabolism and novel functions of vitamin E with special emphasis on differential biological activities of individual vitamin E forms.     

Vitamin E and its function in membranes

Vitamin E is a fat-soluble vitamin. It is comprised of a family of hydrocarbon compounds characterised by a chromanol ring with a phytol side chain referred to as tocopherols and tocotrienols. Tocopherols possess a saturated phytol side chain whereas the side chain of tocotrienols have three unsaturated residues. Isomers of these compounds are distinguished by the number and arrangement of methyl substituents attached to the chromanol ring. The predominant isomer found in the body is alpha-tocopherol, which has three methyl groups in addition to the hydroxyl group attached to the benzene ring. The diet of animals is comprised of different proportions of tocopherol isomers and specific alpha-tocopherol-binding proteins are responsible for retention of this isomer in the cells and tissues of the body. Because of the lipophilic properties of the vitamin it partitions into lipid storage organelles and cell membranes. It is, therefore, widely distributed in throughout the body. Subcellular distribution of alpha-tocopherol is not uniform with lysosomes being particularly enriched in the vitamin compared to other subcellular membranes. Vitamin E is believed to be involved in a variety of physiological and biochemical functions. The molecular mechanism of these functions is believed to be mediated by either the antioxidant action of the vitamin or by its action as a membrane stabiliser. alpha-Tocopherol is an efficient scavenger of lipid peroxyl radicals and, hence, it is able to break peroxyl chain propagation reactions. The unpaired electron of the tocopheroxyl radical thus formed tends to be delocalised rendering the radical more stable. The radical form may be converted back to alpha-tocopherol in redox cycle reactions involving coenzyme Q. The regeneration of alpha-tocopherol from its tocopheroxyloxyl radical greatly enhances the turnover efficiency of alpha-tocopherol in its role as a lipid antioxidant. Vitamin E forms complexes with the lysophospholipids and free fatty acids liberated by the action of membrane lipid hydrolysis. Both these products form 1:1 stoichiometric complexes with vitamin E and as a consequence the overall balance of hydrophobic:hydrophillic affinity within the membrane is restored. In this way, vitamin E is thought to negate the detergent-like properties of the hydrolytic products that would otherwise disrupt membrane stability. The location and arrangement of vitamin E in biological membranes is presently unknown. There is, however, a considerable body of information available from studies of model membrane systems consisting of phospholipids dispersed in aqueous systems. From such studies using a variety of biophysical methods, it has been shown that alpha-tocopherol intercalates into phospholipid bilayers with the long axis of the molecule oriented parallel to the lipid hydrocarbon chains. The molecule is able to rotate about its long axis and diffuse laterally within fluid lipid bilayers. The vitamin does not distribute randomly throughout phospholipid bilayers but forms complexes of defined stoichiometry which coexist with bilayers of pure phospholipid. alpha-Tocopherol preferentially forms complexes with phosphatidylethanolamines rather than phosphatidylcholines, and such complexes more readily form nonlamellar structures. The fact that alpha-tocopherol does not distribute randomly throughout bilayers of phospholipid and tends to form nonbilayer complexes with phosphatidylethanolamines would be expected to reduce the efficiency of the vitamin in its action as a lipid antioxidant and to destabilise rather than stabilise membranes. The apparent disparity between putative functions of vitamin E in biological membranes and the behaviour in model membranes will need to be reconciled.     

Antioxidant alpha-tocopherol ameliorates glycemic control of GK rats, a model of type 2 diabetes

We have shown recently that oxidative stress by chronic hyperglycemia damages the pancreatic beta-cells of GK rats, a model of non-obese type 2 diabetes, which may worsen diabetic condition and suggested the administration of antioxidants as a supportive therapy. To determine if natural antioxidant alpha-tocopherol (vitamin E) has beneficial effects on the glycemic control of type 2 diabetes, GK rats were fed a diet containing 0, 20 or 500 mg/kg diet alpha-tocopherol. Intraperitoneal glucose tolerance test revealed a significant increment of insulin secretion at 30 min and a significant decrement of blood glucose levels at 30 and 120 min after glucose loading in the GK rats fed with high alpha-tocopherol diet. The levels of glycated hemoglobin A1c, an indicator of glycemic control, were also reduced. Vitamin E supplementation clearly ameliorated diabetic control of GK rats, suggesting the importance of not only dietary supplementation of natural antioxidants but also other antioxidative intervention as a supportive therapy of type 2 diabetic patients.     

Cell death caused by selenium deficiency and protective effect of antioxidants

Selenium is an essential trace element and it is well known that selenium is necessary for cell culture. However, the mechanism underlying the role of selenium in cellular proliferation and survival is still unknown. The present study using Jurkat cells showed that selenium deficiency in a serum-free medium decreased the selenium-dependent enzyme activity (glutathione peroxidases and thioredoxin reductase) within cells and cell viability. To understand the mechanism of this effect of selenium, we examined the effect of other antioxidants, which act by different mechanisms. Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely blocked selenium deficiency-induced cell death, although alpha-tocopherol (biologically the most active form of vitamin E) could not preserve selenium-dependent enzyme activity. Other antioxidants, such as different isoforms and derivatives of vitamin E, BO-653 and deferoxamine mesylate, also exerted an inhibitory effect. However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, displayed no such effect. Dichlorodihydrofluorescein (DCF) assay revealed that cellular reactive oxygen species (ROS) increased before cell death, and sodium selenite and alpha-tocopherol inhibited ROS increase in a dose-dependent manner. The generation of lipid hydroperoxides was observed by fluorescence probe diphenyl-1-pyrenylphosphine (DPPP) and HPLC chemiluminescence only in selenium-deficient cells. These results suggest that the ROS, especially lipid hydroperoxides, are involved in the cell death caused by selenium deficiency and that selenium and vitamin E cooperate in the defense against oxidative stress upon cells by detoxifying and inhibiting the formation of lipid hydroperoxides.     

Dietary antioxidants as inhibitors of the heme-induced peroxidation of linoleic acid: mechanism of action and synergism

In this work, a quantitative kinetic model for investigating the heme-induced peroxidation of linoleic acid and its inhibition by two important dietary antioxidants, quercetin and alpha-tocopherol, is developed. The main conclusions of this work are: (1) The time dependence of the lipid hydroperoxide concentration is critically dependent on the rate constant for lipid hydroperoxide cleavage, initial fraction of lipid hydroperoxides among the pool of conjugated dienes, and rate of heme degradation. (2) The lipophilic antioxidant alpha-tocopherol acts as a chain-breaking antioxidant that quickly reduces 1-2 eq of lipid peroxyl radicals (inhibition of propagation), whereas the more hydrophilic antioxidant quercetin is only marginally chain-breaking but capable of reducing 4-5 eq of iron-oxo initiator (inhibition of initiation). (3) Based on comparisons between experimental peroxidation curves and simulated curves assuming additivity, it can be concluded that combinations of alpha-tocopherol and quercetin are generally synergistic. The kinetic analysis and HPLC titrations of the antioxidants both suggest that synergism mainly arises from a capacity of alpha-tocopherol to regenerate some quercetin oxidation products still endowed with a reducing activity.     

Recycling and antioxidant activity of tocopherol homologs of differing hydrocarbon chain lengths in liver microsomes

Tocopherols (vitamin E) function as inhibitors of lipid peroxidation in biomembranes by donating a hydrogen atom to the chain propagating lipid radicals, thus giving rise to chromanoxyl radicals of the antioxidant. We have shown that alpha-tocopherol homologs differing in the lengths of their hydrocarbon side chains (alpha-Cn) manifest strikingly different antioxidant potencies in membranes. The antioxidant activity of tocopherol homologs during (Fe2+ + ascorbate)- or (Fe2+ + NADPH)-induced lipid peroxidation in rat liver microsomes increased in the order alpha-tocopherol (alpha-C16) less than alpha-C11 less than alpha-C6 less than alpha-C1. Chromanoxyl radicals generated from alpha-tocopherol and its more polar homologs by an enzymatic oxidation system (lipoxygenase + linolenic acid) can be recycled in rat liver microsomes by NAD-PH-dependent electron transport or by ascorbate. The efficiency of recycling increased in the same order: alpha-tocopherol (alpha-C16) less than alpha-C11 less than alpha-C6 less than alpha-C1. Thus the high efficiency of regeneration of short-chain homologs of vitamin E may account for their high antioxidant potency.     

Effect of phytyl side chain of vitamin E on its antioxidant activity

Inhibition of the oxidation of methyl linoleate and soybean phosphatidylcholine in homogeneous solution and in aqueous dispersion by four chain-breaking antioxidants, vitamin E (alpha-tocopherol), 2,2,5,7,8-pentamethyl-6-chromanol, 2,6-di-tert-butyl-4-methylphenol, and stearyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, was studied to examine the effect of the phytyl side chain of vitamin E on its antioxidant activity. These four antioxidants exerted similar antioxidative activities. They were also effective as antioxidants in protecting the oxidation of soybean phosphatidylcholine liposomes in water dispersion. However, when they were incorporated into dimyristoyl phosphatidylcholine liposomes, only 2,2,5,7,8-pentamethyl-6-chromanol and 2,6-di-tert-butyl-4-methylphenol could suppress the oxidation of soybean phosphatidylcholine liposomes dispersed in the same aqueous system. It was concluded that the antioxidative properties of vitamin E and its model without the phytyl side chain are quite similar within micelles and liposomes as well as in homogeneous solution but that the phytyl side chain enhances the retainment of vitamin E in liposomes and suppresses the transfer of vitamin E between liposomal membranes.     

Inhibition of the metmyoglobin-induced peroxidation of linoleic acid by dietary antioxidants: Action in the aqueous vs. lipid phase

The gastric digestion of food containing oxidizable lipids and iron catalysts for peroxide decomposition such as (met)myoglobin from muscle meat can be accompanied by an extensive formation of potentially toxic lipid hydroperoxides. An early protective action by dietary antioxidants in the gastro-intestinal tract is plausible, especially for poorly bioavailable antioxidants such as polyphenols. Hence, the ability of antioxidants to inhibit lipid peroxidation initiated by dietary iron in mildly acidic emulsions is a valuable and general model. In this work, the ability of some ubiquitous dietary antioxidants representative of the main antioxidant classes (alpha-tocopherol, the flavonol quercetin, beta-carotene) to inhibit the metmyoglobin-induced peroxidation of linoleic acid is investigated by UV-visible spectroscopy and HPLC in mildly acidic emulsions. The phenolic antioxidants quercetin and alpha-tocopherol come up as the most efficient peroxidation inhibitors. Inhibition by quercetin essentially proceeds in the aqueous phase via a fast reduction of an unidentified activated iron species (with a partially degraded heme) produced by reaction of metmyoglobin with the lipid hydroperoxides. This reaction is faster by, at least, a factor 40 than the reduction of ferrylmyoglobin (independently prepared by reacting metmyoglobin with hydrogen peroxide) by quercetin. By contrast, alpha-tocopherol mainly acts in the lipid phase by reducing the propagating lipid peroxyl radicals. The poorer inhibition afforded by beta-carotene may be related to both its slower reaction with the lipid peroxyl radicals and its competitive degradation by autoxidation and/or photo-oxidation.