Erythrocyte Membrane Fatty Acid Composition of Smokers and Non-Smokers: Effects of Vitamin E Supplementation

Erythrocyte membrane fatty acid composition of smokers and non-smokers: effects of vitamin E supplementation

KM Brown, PC Morrice and GG Duthie

Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, Scotland, UK

Objective: Adequate dietary intakes of vitamin E and essential polyunsaturated fatty acids are important to maintain cell membrane integrity, and de®ciencies have been associated with smoking related cardiovascular disease. Suf®cient vitamin E is required to prevent free radical mediated peroxidation of membrane lipids. Consequently, smokers may have a greater requirement for this antioxidant. To investigate, we assessed the concurrent in¯uences of smoking, vitamin E supplementation and red blood cell (RBC) PUFA composition on the susceptibility of the cells to lipid peroxidation in adult males. Design and subjects: Thirty male smokers and thirty male non-smokers were randomly ascribed to daily 280 mg vitamin E or placebo supplements for 10 weeks. RBC were analysed at weeks 0 and 10 for fatty acid methyl esters, vitamin E, and their susceptibility to in vitro H2O2 induced lipid peroxidation. Results: Concentrations of essential fatty acids (EFA) in RBC were lower in smokers than in non-smokers. Supplementation with vitamin E increased levels of RBC EFA in smokers to match those of non-smokers. Furthermore, the ratio of vitamin E to PUFA in RBC from smokers and non-smokers was inversely correlated with their susceptibility to peroxidation. Conclusions: An adequate vitamin E to PUFA ratio is required to protect cell membranes from oxidative damage. The signi®cant correlation between susceptibility to peroxidation and the PUFA content of RBC before supplementation suggests an inadequate intake of vitamin E in relation to PUFA intake. Moreover, the requirement for vitamin E appears to be greater in smokers than in non-smokers. Sponsorship: BASF Germany, Tobacco Products Research Trust, Scottish Of®ce Agriculture, Environment, and Fisheries Department. Descriptors: Vitamin E; polyunsaturated fatty acids; lipid peroxidation; smoking

Introduction free radical load incurred by smokers arises from the cigarette smoke, each puff containing an estimated 1014 Cell membrane stability is essential to normal cell function, free radicals in the tar phase and 1015 in the gas phase and is in¯uenced by many factors including dietary intakes (Church & Pryor, 1985), and from increased phagocytic of fat and antioxidant nutrients. The structure and function activity of alveolar macrophages and neutrophilic granulo- of membranes largely depends upon the presence of dif- cytes in peripheral blood. These are associated with the ferent classes of phospholipids and fatty acids, and it is here respiratory burst reaction (RBR), which generates OÁ7 and that vitamin E is believed to exert its fundamental bio- 2 hypochlorous acid via NADPH-oxidase and myeloperox- logical action as an antioxidant (Tappel, 1962; Burton idase systems respectively (Clausen, 1991). & Ingold, 1981; Burton et al, 1983). Lipid peroxidation, A common index of oxidative modi®cation of important initiated when a reactive radical species abstracts a hydro- biological molecules in smokers is an increase in the gen atom from relatively weak C±H methylene bonds of concentration of products of peroxidation in blood polyunsaturated fatty acids, is delayed or prevented by (Duthie, 1992; Brown et al, 1994; Mezzetti et al, 1995). antioxidant defences. The inhibition of lipid peroxidation For example, plasma concentrations of F -isoprostanes in by vitamin E is believed to be important in reducing the 2 smokers are double those in non-smokers, indicative of risk of many diseases associated with human growth increased free radical mediated peroxidation of arachidonic development and aging (Dargel, 1992; Gutteridge, 1993; acid (independent of cyclooxygenase mediated oxidation) Karp & Robertson, 1986). The antioxidant activity of (Morrow et al, 1995). Moreover, the generation of mal- vitamin E also counteracts the oxidative burden imposed ondialdehyde (MDA), an end product of lipid peroxidation, by cigarette smoke (Duthie et al, 1991; Hoshino et al, 1990; in vivo, may be mediated by H O , particularly in RBC Brown et al, 1994; Metzzetti et al, 1995; Brown et al, 2 2 where H O is generated from the dismutation of super- 1997). Smoking provides a further in¯uence over mem- 2 2 oxide produced by the autooxidation of haemoglobin (Giu- brane stability and integrity (Niki et al, 1993; Tsuchiya et livi, 1994). al, 1993), which may be compounded by the lower anti- To assess the effects of smoking, vitamin E and RBC oxidant status of smokers (Duthie, 1993). The sustained PUFA composition on susceptibility of the cells to lipid peroxidation in vitro, we examined the effect of vitamin E supplementation in smokers and non-smokers on vitamin E Correspondence: Dr KM Brown. Received 2 August 1997; revised 24 October 1997; accepted 25 October and PUFA concentrations in RBC, and their susceptibility 1997 to peroxidation. Erythrocyte membrane fatty acid composition KM Brown et al 146 Subjects and methods Statistical analysis The effects of smoking and vitamin E were examined using The supplementation protocol included four treatment two-way analysis of variance, and estimated as the groups to form a 262 factorial design ie. smokers vs unweighted average of the effects within the smoking and non-smokers and placebo vs vitamin E supplementation. non-smoking groups. The effect was tested for signi®cance Thirty males who had smoked for 10 y (>15 cigarettes by t-test. Simple linear regression analysis was used to smoked/d) and thirty males who had never smoked were describe the relationship between independent and depen- each given daily for 10 weeks, a capsule containing either a dent variables. supplement 280 mg dl-a-tocopherol acetate (all-rac-a- tocopherol acetate) or visually identical hydrogenated Results coconut oil of negligible vitamin E content (< 5 mg). Blood samples were collected into heparin treated evacu- Age and body mass index were the same in all four groups ated tubes from each fasted subject at the start and end (smokers placebo, S P: smokers vitamin E, S E: of treatment. After centrifugation of blood (15006g, non-smokers placebo, NS P: non-smokers vitamin 4C, 15 min), the plasma was removed and the red cells E, NS E) (Table 1). Erythrocyte vitamin E concentrations were washed three times in iso-osmotic phosphate buffered did not signi®cantly differ between smokers and non- saline (pH 7.4) and resuspended to the original volume. The smokers at week 0 but increased equally (P < 0.001) in cells were then aliquoted and snap frozen in liquid nitrogen both groups given vitamin E but remained unchanged in the prior to storage at 770C. smokers and non-smokers receiving the placebo (Table 2). Lipid extractions from erythrocytes were undertaken The susceptibility of RBC to H2O2 induced in vitro lipid using the Modi®ed Folch Method (Hamilton et al, 1992), peroxidation was signi®cantly greater in smokers than in with L-a-phosphatidylcholine, dihepta-decanol (0.2 ml) as non-smokers, and signi®cantly decreased in both groups the internal standard. After weighing, the lipid was re- supplemented with vitamin E (Table 2). dissolved in 2 ml of chloroform. Samples were protected The predominant fatty acids extracted from RBC of all from light and stored at 720C in the presence of butylated subjects were palmitic, stearic and behenic acid (Table 3). hydroxytoluene to prevent autooxidation. This method of The percentage of extractable PUFA methyl esters from lipid extraction allows a 95±99% extraction of lipids. Prior RBC was lower in smokers than in non-smokers (Table 3). to gas liquid chromatographic separation, methyl ester This was mainly attributable to lower extractions of the n-6 derivatives of free fatty acids were prepared and extracted series of a-linoleic (P < 0.001), g-linolenic (P < 0.001), into hexane containing 0.02% BHT. GLC separation of arachidonic (P < 0.01), and docosatriaenoic (P < 0.02) methyl esters was undertaken on a Hewlett Packard series acids, and of the n-3 series a-linolenic (P < 0.02), docosa- II gas chromatograph, using a 50 m60.25 mm fused silica triaenoic (P < 0.01), docosapentaenoic (P < 0.001), and capillary column coated with a SIL 88, 0.25 m thick polar docosahexaenoic (DHA) (P < 0.001) acids. Following sup- liquid phase (Chrompack UK Ltd., Millharbour London). plementation with vitamin E, the proportions of a-linoleic, Vitamin E concentrations in RBC was determined by a arachidonic, docosatriaenoic, adrenic, a-linolenic, docosa- ¯uorimetric technique, sensitive in the range of 0.10±50 mg triaenoic and DHA fatty acids in smokers increased to those tocopherol/ml, with a recovery of between 91±100% in non-smokers (Table 3). There were also differences (Taylor et al, 1976). Lipid peroxidation was estimated between the smokers and non-smokers before and after indirectly as a derivative of malondialdehyde (MDA), supplementation in the RBC vitamin E to PUFA ratios generated by the reactivity of MDA with thiobarbituric (Table 2). These were lower in non-smokers than in acid (TBA). RBC susceptibility to peroxidation was deter- smokers (P < 0.01), re¯ecting the lower PUFA concentra- mined on 100 ml fresh washed, resuspended red cells tions in RBC of smokers, and increased following vitamin diluted with 900 ml PBS containing 4 mM sodium azide, E supplementation in all subjects (P < 0.02). to prevent the destruction of added H2O2 by catalase. The The susceptibility of RBC from supplemented non- cells were incubated with 1.5% (w/v) H2O2) for 1 h at 37 C smokers to H2O2 induced lipid peroxidation was related after which the reaction was stopped by the addition of to their concentrations of methyl esters of eicosapentaenoic 1 ml of 20% (w/v) trichloroacetic acid. The suspensions acid (P < 0.0001, r 0.887) (Figure 1) and docosatriaenoic were centrifuged at 24006g for 5 min, the supernatant acid (P < 0.001, r 0.800) (Figure 2). A similar but non- removed and frozen slowly with 2% butylated hydroxyto- signi®cant relationship occurred in the smokers. There was luene to 770C prior to ®nal analysis of thiobarbituric acid however no such correlation in RBC of subjects given reactive substances (TBARS) using ¯uorescence detection vitamin E. Furthermore, the ratio of vitamin E to PUFA adapted from the method of Yagi (Yagi et al, 1987). Plasma concentrations in RBC was inversely correlated with RBC ascorbic acid concentration was measured by reverse phase susceptibility to peroxidation in smokers (P < 0.001, hplc using an ion-pairing reagent with UV detection on r 70.810), and non-smokers (P < 0.01, r 70.681) samples stored in 10% (w/v) metaphosphoric acid (Ross, (Figure 3). 1994). a Assessment of habitual food intake using a food intake Table 1 Anthropometric characteristics and indices of smoking habits questionnaire was used as an estimation of nutrient intake. S P S E NS P NS E The dietary questionnaire was developed initially for the Caerphilly and Speedwell collaborative heart disease stu- Age years 48 Æ 0.2 46 Æ 0.4 47 Æ 0.6 45 Æ 0.2 Body mass indexb 23 Æ 0.6 24 Æ 0.8 26 Æ 1 28 Æ 1.2 dies (Caerphilly et al, 1984, 1985) and The Diet and Cigarettes smoked/d 19 Æ 0.4 20 Æ 0.4 0 0 Reinfarction trial (Burr et al, 1989), and has been modi®ed Cotinine (mg/L) 256 Æ 22 279 Æ 23 < 6 < 6 and improved on the basis of these studies. Nutritional a analysis of the questionnaire was undertaken using Tinuviel Mean Æ SE; S P, smokers given placebo; S E, smokers given 280mg vitamin E; NS P, non-smokers given placebo; NS E, non-smokers Software DIETQ Nutritional Analysis Suite, Version 2; given 280mg vitamin E. Stoke Gifford Bristol. b In kg/m2. Erythrocyte membrane fatty acid composition KM Brown et al 147 Table 2 Effect of supplementation with vitamin E or placebo for 10 weeks on erythrocyte vitamin E concentrations, susceptibility of erythrocytes to H2O2-induced lipid peroxidation, erythrocyte vitamin E:PUFA ratio and plasma ascorbate concentration of smokers and non-smokersa

S P S E NS P NS E

RBC vitamin E (mmol/gHb) Week 0 16.8 Æ 3.5 19.1 Æ 4.2 15.7 Æ 4.4 20.0 Æ 4.1 Week 10 16.9 Æ 4.1 30.5 Æ 1.02 18.4 Æ 6.3 30.6 Æ 3.92 TBARS (nmol/gHb) Week 0 436 Æ 423 438 Æ 294 308 Æ 44 321 Æ 32 Week 10 447 Æ 47 321 Æ 485 355 Æ 34 168 Æ 282 RBC vitamin E : PUFA (mmol/L : mg/100mg) Week 0 7.3 Æ 0.583 6.5 Æ 0.763 5.1 Æ 0.57 4.7 Æ 0.44 Week 10 6.0 Æ 0.86 10.2 Æ 1.42 5.7 Æ 0.67 5.7 Æ 0.565 Plasma ascorbate mmol/L Week 0 27 Æ 43 25 Æ 76 35 Æ 3 39 Æ 9 Week 10 26 Æ 53 21 Æ 56 32 Æ 4 32 Æ 5

a Mean Æ s.e.m.; S P, smokers given placebo; S E, smokers given 280mg vitamin E; NS P, non- smokers given placebo; NS E, non-smokers given 280mg vitamin E; PUFA, polyunsaturated fatty acid; Hb, haemoglobin; TBARS, thiobarbituric reactive substances; RBC, red blood cells. 2,5 Signi®cance of vitamin E effect: 2P < 0.001, 5 P < 0.02. 3,4,6 Signi®cance of smoking effect: 3 P < 0.01, 4 P < 0.02, 6P < 0.001.

Table 3 Predominant fatty acids in red blood cells of smokers and non-smokers presented as % composition of total lipid extracta

Non-smokers Non-smokers Smoker Smoker Fatty acid Week 0 Week 10 Week 0 Week 10

Myristic C14:0 2.7 Æ 0.1 2.7 Æ 0.3 3.5 Æ 0.13 2.6 Æ 0.3 Palmitic C16:0 26.4 Æ 2.7 26.3 Æ 3.1 29.9 Æ 0.4 26.4 Æ 2.1 Stearic C18:0 20.8 Æ 2.9 19.3 Æ 3.4 23.7 Æ 0.10 17.1 Æ 1.9 Behenic C22:0 7.5 Æ 0.2 6.7 Æ 0.4 11.2 Æ 0.053 8.0 Æ 2.0 Oleic C18:1 n-9 3.8 Æ 0.10 3.7 Æ 1.0 3.4 Æ 0.12 3.4 Æ 0.2 Elaidic C18:1 n-9 0.8 Æ 0.01 1.0 Æ 0.02 2.0 Æ 0.012 1.8 Æ 0.1 Gondoic C20:1 n-9 2.1 Æ 0.10 2.4 Æ 0.9 2.3 Æ 0.13 2.2 Æ 0.1 a-linoleic C18:2 n-6 3.5 Æ 0.07 4.4 Æ 0.9 1.7 Æ 0.022 4.1 Æ 0.95 g-linolenic C18:3 n-6 2.1 Æ 0.1 4.1 Æ 0.7 0.1 Æ 0.012 0.1 Æ 0.01 Arachidonic C20:4 5.5 Æ 0.9 5.2 Æ 1.2 4.1 Æ 0.63 7.9 Æ 1.15 Docosatriaenoic C22:3 n-6 2.1 Æ 0.4 2.0 Æ 0.1 1.7 Æ 0.14 2.2 Æ 0.17 Adrenic C22:4 n-6 1.6 Æ 0.01 1.8 Æ 0.2 1.4 Æ 0.1 1.8 Æ 0.96 a-linolenic C18:3 n-3 7.8 Æ 0.9 6.6 Æ 0.8 6.9 Æ 0.44 9.9 Æ 0.95 EPA C20:5 n-3 2.3 Æ 0.2 2.4 Æ 0.1 2.2 Æ 0.01 2.6 Æ 0.3 Docosatriaenoic C22:3 n-3 5.4 Æ 0.13 5.4 Æ 0.2 3.6 Æ 0.23 7.1 Æ 2.05 DPA C22:5 n-3 2.1 Æ 0.1 2.3 Æ 0.2 0.9 Æ 0.12 0.8 Æ 0.1 DHA C22:6 n-3 3.4 Æ 0.3 3.8 Æ 0.2 1.5 Æ 0.022 2.2 Æ 0.16

a Mean Æ s.e.m. 2,3,4 Signi®cance of smoking effect 2P < 0.001, 3P < 0.01, 4P < 0.02. 5,6,7 Signi®cance of vitamin E effect: 5P < 0.001, 6P < 0.01, 7 P < 0.02.

The differences between smokers and non-smokers can Discussion not be attributed to dietary intake since it was similar in all subjects. Estimated energy intake was lower in smokers The aerobic environment of mammalian cells renders their than non-smokers, although not signi®cantly, and the % membrane lipids and nucleic acids highly susceptible to energy supplied by protein, fat and carbohydrate did not peroxidation. Polyunsaturated fatty acids (PUFA:H) are differ between the groups (Table 4). The greatest contribu- highly susceptible to autooxidation because of their relation of energy from fat was from saturated sources, and the tively weak C±H methylene bonds which readily undergo least from polyunsaturated sources. Cholesterol intake was free-radical-mediated hydrogen abstraction. similar in smokers and non-smokers, but non-smokers The primary products of lipid peroxidation are lipid consumed signi®cantly more EPA than smokers. Dietary hydroperoxides, and the number formed is dependent on vitamin E intake ranged between 3±11 mg/d and was the the number of double bonds (n) present on a PUFA, and same in smokers and non-smokers. Although plasma ascor- equals 2n-2 (Esterbauer, 1993). Thus linoleic acid (18:2) bate was lower in smokers than non-smokers (Table 2), yields 2 lipid hydroperoxides; linolenic (18:3) 4; arachido- mean dietary intake was not signi®cantly different, and in nic (20:4) 6; eicosapentaenoic acid (20:5) 8; and docosa- the range of 27±104 mg/d in non-smokers and 16±84 mg/d hexaenoic (20:6) 10. The breakdown of these lipid in smokers (Table 4). hydroperoxides produces many highly cytotoxic aldehydes, Erythrocyte membrane fatty acid composition KM Brown et al 148

Figure 1 Correlation between RBC H2O2 induced in vitro lipid peroxidation and RBC methyl esters of n-3 series eicosapentaenoic acid in non-smokers before supplementation.

Figure 2 Correlation between RBC H2O2 induced in vitro lipid peroxidation and RBC methyl esters of n-6 series docosatriaenoic acid in non-smokers before supplementation.

in particular 4-hydroxynonenal (4-HNE), 4-hydroxyhexe- oxygen centered radicals generated from nitric oxide in the nal (4-HHE), and MDA (Esterbauer et al, 1991). The lower gas phase of cigarette smoke may have initiated erythrocyte concentrations of methyl esters of the n-3 and n-6 fatty membrane lipid peroxidation in vivo generating lipid acids containing 3 or more methylene interrupted double hydroperoxides and their breakdown products, including bonds in RBC from smokers compared with non-smokers MDA. may be indicative of increased MDA generation. Most As a caution against increased lipid peroxidation, the peroxidation in biomembranes involves C20:4 n-6 and panel on Dietary Reference Values for Food Energy and C22:6 n-3 which results in decreases in their concentrations Nutrients for the UK (RHSS 41, 1991), recommend that in the membrane (Slater et al, 1987). This was clearly PUFAs should not provide more than 10% of total energy evident in the RBC of smokers, and was not a re¯ection of intake. Regardless of the total amount of PUFA consumed, differences in dietary intake. It is unclear therefore, why intake of vitamin E must be suf®cient to prevent peroxida- RBC of smokers were more susceptible to in vitro perox- tion of PUFA in circulating lipoproteins and cell mem- idation, where MDA is generated from PUFA and lipid branes. Although plasma tocopherol to cholesterol ratios in hydroperoxides during the assay. This observation may be both smokers and non-smokers were within the normal UK attributable to hydrogen peroxide already present in the population range (Brown et al, 1994), the correlations sample generated from a quinone radical system identi®ed between susceptibility to peroxidation and PUFA concen- in the tar phase of cigarette smoke (Pryor & Stone, 1993). trations in RBC of non-smokers suggest that a vitamin E A semiquinone radical generated from this system is cap- intake estimated to be in the range of 3±11 mg/d is not able of reducing oxygen to superoxide which then dismu- suf®cient to prevent H2O2 induced lipid peroxidation of red tates to form hydrogen peroxide. Furthermore, carbon and cells in vitro. The poor correlation in the smokers probably Erythrocyte membrane fatty acid composition KM Brown et al 149 increased the concentration of EPA in LDL and RBC. The EPA enriched LDL was highly susceptible to oxidation. Vitamin E supplementation of 400 mg daily for four weeks ameliorated both the effects of smoking and ®sh oil supplementation. Although estimation of EPA intake from the dietary questionnaire in our study suggests that smokers consume less than non-smokers, this is not re¯ected in RBC EPA concentrations which are similar in all subjects. Furthermore, the concentration in RBC from smokers increased following vitamin E supplementation suggesting that their initial lower levels may be attributable to increased peroxidation of EPA. PUFA of the n-3 and n-6 series are derived from a- linoleic (C18:2 n-6) and a-linolenic (C18:3 n-3). Most Figure 3 Correlation between RBC H2O2 induced in vitro lipid perox- mammals, including humans, accumulate C18:2 n-6 and idation and RBC vitamin E:PUFA ratio following 10 weeks supplementa- its desaturation and elongation products, g-linolenic, ara- tion in smokers (d) and non-smokers (m). chidonic, and adrenic acids. This is re¯ected in the fatty acid pro®les of RBC of the population in the present study. Table 4 Daily macronutrient intake data from food frequency The desaturation of C18:2 n-6 to g-linolenic is rate limiting, questionnaire for smokers and non-smokers as is the desaturation and elongation of C18:3 n-3 to EPA and DHA, again re¯ected in the RBC. The lower concen- Nutrient Non-smokers Smokers trations of n-3 and n-6 EFA in the RBC of smokers may Energy MJ 9.6 Æ 1.6 8.5 Æ 1.3 re¯ect EFA pro®les of other biomembranes rendering the Protein g 86 Æ 4.4 85 Æ 4.5 smoker at greater risk of chronic in¯ammatory disease. Carbohydrate g 286 Æ 9.1 253 Æ 6.5 Fat g 87 Æ 5.6 76 Æ 4.7 % energy total fat 33 Æ 2.3 33 Æ 2.4 Conclusions % energy SFA 14 Æ 1.7 12 Æ 1.6 % energy MUFA 11 Æ 1.4 11 Æ 1.2 Supplementation of smokers with vitamin E markedly % energy PUFA 5 Æ 1.3 6 Æ 1.4 decreased in vitro erythrocyte lipid peroxidation as % energy protein 15 Æ 1.5 17 Æ 1.7 % energy carbohydrate 58 Æ 8.4 47 Æ 2.5 assessed by the generation of TBARS. The limitations of Cholesterol mg 352 Æ 12.3 347 Æ 11.5 the TBARS assay as a marker of peroxidation is well EPA mg 183 Æ 30 147 Æ 302 recognised, but the inhibitory effect of vitamin E on the Vitamin E 6 Æ 1.4 5 Æ 1.7 generation of TBARS suggests that it is a valid marker of in Vitamin C 63 Æ 5.2 57 Æ 4.6 vitro induced lipid peroxidation. The protective effect of 1 Mean Æ s.e.m. SFA, saturated fatty acids; EPA, eicosapentaenoic acid. increasing vitamin E concentration may be indicative that 2 Signi®cance of smoking effect 2P < 0.02. the dietary vitamin E intake, even of the non-smokers, was sub-optimum. This is consistent with the report that 95% of a randomly selected population from the same locality as re¯ects an increase in peroxidation of RBC membrane used in the present study consumed less than the US PUFA. Furthermore, the strong inverse relationship Recommended Dietary Allowance for this antioxidant between RBC susceptibility to peroxidation and vitamin (Duthie et al, 1993; RDA, 1989). A further complication E:PUFA ratio following 10 weeks vitamin E supplementa- in establishing optimum intake of vitamin E may be tion suggests that higher concentrations of vitamin E are attributable to the signi®cant differences in plasma ascor- required to protect membrane PUFA against in vitro bate between smokers and non-smokers. Since dietary peroxidation. The close association between vitamin E vitamin C intake was similar in all subjects, the low and PUFA was observed in a study on 40 non-smoking plasma ascorbate concentrations in smokers probably subjects fed a high ®sh oil diet for 10 weeks (Padmanab- re¯ects an increased ascorbate turnover (Brown et al, ham et al, 1993). The ingestion of 15 g ®sh oil forti®ed 1997). Kinetic studies demonstrate an intracellular inter- with 15 mg vitamin E, decreased a-tocopherol concentra- action between vitamin C and vitamin E whereby the tions in plasma, platelets and red blood cells and increased membrane-bound oxidized vitamin E is regenerated non- plasma MDA. Continued supplementation for another eight enzymically by ascorbate at the membrane cytosol interface weeks with ®sh oil forti®ed with 200 mg vitamin E (Chan, 1993). reversed the rise in plasma MDA, and restored plasma The dependence of an individual's vitamin E require- and cell tocopherol concentrations, suggesting that increas- ment on dietary PUFA makes it dif®cult to provide recom- ing PUFA intake without suf®cient vitamin E enhances mendations on adequate intake. It is reported that the 2.5 susceptibility to peroxidation. In a cross over dietary and 97.5 centile intakes of n-6 PUFA by men are 5.1 and intervention study with 26 subjects providing diets with 29 g/d (Gregory et al, 1990). No recommended nutrient 34% energy from PUFA or MUFA, in vitro Cu(II) induced intake (RNI) is made for vitamin E in respect of high PUFA LDL oxidation generated signi®cantly more TBARS during intake in the UK. 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