Biology of vitamin E

Ciba Foundation symposium 101

1983

Pitman

London

Biology of vitamin E

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Biology of vitamin E

Ciba Foundation symposium 101

1983

Pitman

London

0 Ciba Foundation 1983

ISBN 0 272 79748 0

Published in November 1983 by Pitman Books Ltd., 128 Long Acre, London WC2E 9AN, UK Distributed in North America by CIBA Pharmaceutical Company (Medical Education Division), Post Office Box 12832, Newark, NJ 07101, USA

Suggested series entry for library catalogues Ciba Foundation symposia

Ciba Foundation symposium 101 ix + 260 pages, 40 figures, 38 tables

British Library Cataloguing in publication data Biology of vitamin. E.-(Ciba Foundation symposium;

101) 1. Vitamin E-Congresses I. Porter, Ruth 111. Series 574.19’26 Q801.T6

11. Whelan, Julie

Printed in Great Britain at The Pitman Press, Bath

Contents

Symposium on Biology of Vitamin E, held at the Ciba Foundation, London, 8-10 March 1983

The subject for this symposium was proposed by Dr D. P. R. Muller, Professor 0. H. Wolff and Professor J . T. Harries

John Harries died shortly after the symposium, on 27th March 1983. His comments in the discussion have been included, edited by David Muller

Editors: Ruth Porter (Organizer) and Julie Whelan

A. T. DIPLOCK (Chairman) Introduction 1

G. W. BURTON, K. H. CHEESEMAN, T. DOBA, K. U. INGOLD and T. F. SLATER Vitamin E as an antioxidant in vitro and in vivo 4 Discussion 14

R. L. WILLSON Free radical protection: why vitamin E, not vitamin C, p-carotene or glutathione? 19 Discussion 37

A. T. DIPLOCK The role of vitamin E in biological membranes 45 Discussion 53

A. T. QUINTANILHA and L. PACKER Vitamin E, physical exercise and tissue oxidative damage 56 Discussion 61

H. J. KAYDEN Tocopherol content of adipose tissue from vitamin E- deficient humans 70 Discussion 85

J. S . NELSON Neuropathological studies of chronic vitamin E deficiency in mammals including humans 92 Discussion 99

V

vi CONTENTS

D. P. R. MULLER, J. K. LLOYD and 0. H. WOLFF Vitamin E and neurological function: abetalipoproteinaemia and other disorders of fat absorption 106 Discussion 117

General discussion Problems of defining vitamin E deficiency 122

J. E. LAFUZE, S . J. WEISMAN, L. M. INGRAHAM, C. J. BUTTERICK, L. A. ALPERT and R. L. BAEHNER The effect of vitamin E on rabbit neutrophil activation 130 Discussion 141

N. N. FINER, K. L. PETERS, R. F. SCHINDLER and G. D. GRANT Vitamin E and retrolental fibroplasia: prevention of serious ocular sequelae 147 Discussion 159

H. M. HIlTNER and F. L. KRETZER Vitamin E and retrolental fibropla- sia: ultrastructural mechanism of clinical efficacy 165 Discussion 182

M. L. CHISWICK, M. JOHNSON, C. WOODHALL, M. GOWLAND, J. DAVIES, N. TONER and D. SIMS Protective effect of vitamin E on intraventricular haemorrhage in the newborn 186 Discussion 197

C. H. McMURRAY, D. A. RICE and S. KENNEDY Experimental models for nutritional myopathy 201 Discussion 218

M. J. JACKSON, D. A. JONES and R. H. T. EDWARDS Vitamin E and skeletal muscle 224 Discussion 234

Final general discussion Circadian rhythms in clinical studies 240 Roles of lipid peroxidation 241 Vitamin E and ageing 246

A. T. DIPLOCK Chairman’s closing remarks 249

Index of contributors 251

Subject index 253

Participants

S. R. AMES 61 Biltmore Drive, Rochester, New York 14617, USA

R. L. BAEHNER Department of Pediatrics, Indiana University School of Medicine, Section of Pediatric Hematology-Oncology , James Whitcomb Riley Hospital for Children P132, 1100 West Michigan Street, Indiana- polis, Indiana 46223, USA

M. L. CHISWICK Neonatal Medical Unit, North Western Regional Peri- natal Centre, St Mary’s Hospital, Whitworth Park, Manchester M13 OJH, UK

J. R. COOKE Laboratory of the Government Chemist, Department of Industry, Cornwall House, Stamford Street, London SE1 9NQ, UK

A. N. DAVISON Department of Neurochemistry, The National Hospital, Queen Square, London WC1 3BG, UK

A. T. DIPLOCK Department of Biochemistry, Guy’s Hospital Medical School, London Bridge, London SE1 9RT, UK

R. H. T. EDWARDS Department of Medicine, University College London, School of Medicine, The Rayne Institute, University Street, London WClE 655, UK

N. N. FINER Neonatal Intensive Care Unit, Royal Alexandra Hospital, 10240 Kingsway, Edmonton, Alberta T5H 3V9, Canada

*J. T. HARRIES Institute of Child Health, 30 Guilford Street, London WClN IEH, UK

G. A. HIGGS Department of Prostaglandin Research, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, UK

*Died 27th March 1983.

vii

viii PARTICIPANTS

H. M. HITTNER Pediatric Ophthalmology Associates, Doctors Center, 7000 Fannin, Suite 2250, Houston, Texas 77030, USA

K. U. INGOLD Division of Chemistry, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada

M. J. JACKSON Department of Medicine, University College London, School of Medicine, The Rayne Institute, University Street, London WClE 655, UK

H. J. KAYDEN Department of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA

J. K. LLOYD Department of Child Health, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK

C. H. McMURRAY Department of Agriculture, Veterinary Research . Laboratories, Stormont, Belfast BT4 3SD, Northern Ireland

D. P. R. Muller Department of Child Health, Institute of Child Health, 30 Guilford Street, London WClN lEH, UK

J. S . NELSON Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, USA

L. PACKER Membrane Bioenergetics Group, Lawrence Berkeley Labora- tory, Department of Physiology-Anatomy , University of California, Ber- keley, California 94720, USA

W. A. PRYOR Departments of Chemistry and Biochemistry, Louisiana State University, Baton Rouge, Louisiana 70803, USA

T. F. SLATER Department of Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, UK

H. WEISER Physiological Laboratory, Central Research Units, F. Hoff- mann-La Roche & Co., CH-4002 Basle, Switzerland

A. WENDEL Department of Biochemistry, Institute of Physiological Chemistry, University of Tubingen, D-74000 Tubingen 1, West Germany

PARTICIPANTS ix

R. L. WILLSON Department of Biochemistry, Brunel University, Ux- bridge, Middlesex UB8 3PH, UK

0. H. WOLFF Institute of Child Health, 30 Guilford Street, London WClN lEH, UK

This Page Intentionally Left Blank

Introduction

A. T. DIPLOCK

Department of Biochemistry, Guy’s Hospital Medical School (University of London), London SEl 9RT, UK

I983 Biology of vitamin E. Pitman Books, London (Ciba Foundation symposium 101) p 1-3

The chief purpose of this symposium is to discuss recent research on the biology, biochemistry and physiology of vitamin E. There may appear to be a dichotomy between those of us concerned with the fundamental biological role of vitamin E and those interested in the clinical applications of this vitamin, and I shall revert to this point in a moment.

Approximately 60 years have elapsed since .Herbert Evans first described vitamin E, and in the intervening time, research on this topic has been marked by tremendous controversy. Two controversies have been upper- most: on the fundamental research side, a controversy over whether the only role of vitamin E is as an antioxidant, or whether it has some other function; and, on the clinical side, whether vitamin E has any therapeutic use in human beings. We have recently seen great progress on both these aspects, and this progress has often depended on advances made in parallel fields. On the question of the basic role of the vitamin, for example, much has been learned from the study of oxygen radicals in biology which has been useful in putting vitamin E into context with other substances that together provide a protec- tive mechanism against oxygen toxicity. On the clinical side, the role of vitamin E has become clearer as more sophisticated techniques, such as high performance liquid chromatography, have become available for measuring the tocopherols, and for elucidating the pathology of conditions in which an inadequacy of tissue vitamin E content is believed to be involved. So, as I indicated, our principal purpose is to come together as basic scientists, and as clinicians concerned with the therapeutic use of vitamin E and, if a gap exists, to try to bridge that gap. We have to be prepared to admit our several limitations, caused by our coming from a range of different disciplines each with its own specialized nomenclature. Basic scientists often feel diffident

2 DIPLOCK

about expressing opinions on topics that seem to be more concerned with clinical aspects and clinicians equally feel diffident about expressing opinions on the basic science. I suggest that we put these hesitations aside and proceed on the basis of accepting our several limitations without fear of criticism and in a spirit of trying to help one another to understand the various problems. If we can do this, we shall make progress.

The symposium begins with aspects of the basic science of vitamin E and will move through the biochemical and physiological to the more clinical aspects. We shall therefore begin with the basic chemistry, and Keith Ingold will discuss his work on the rate constants for reactions between peroxyl radicals and both tocopherols and some model compounds. He will also tell us about interesting new work on the levels of vitamin E found in hepatomas and in normal hepatocytes.

Robin Willson will consider the free radical properties of tocopherol using the fast-reaction pulse radiolysis technique, and will compare vitamin E with other cellular constituents such as ascorbate, p-carotene and glutathione. He will also discuss the mechanism of ascorbate augmentation of the activity of vitamin E as an antioxidant.

In my chapter, I hope to take the discussion forward to the more bioche- mical aspects and to give an overview of how I see the role of vitamin E, and then to concentrate on the function of vitamin E in biological membranes, including our work on model systems, which we believe gives an indication of how vitamin E may fulfil part of its role.

Lester Packer will take as his theme the idea that vitamin E deficiency exacerbates oxidative damage due to endogenous energy metabolism in biological membranes. He will show that vitamin E deficiency produces several biochemical and morphological changes indicative of increased oxida- tive damage in vitamin E deficiency during exercise.

We shall move to the more clinical aspects, with two chapters specifically on abetalipoproteinaemia. Herb Kayden will describe his technique for measuring tocopherol in needle biopsy samples of human adipose tissue, and we shall hear about differences seen in untreated patients with abetalipop- roteinaemia and how the tocopherol levels are affected by massive therapy with vitamin E. David Muller will describe work done here in London on similar patients, emphasizing the neurological abnormalities in these patients. He will bring us to the view that vitamin E has an essential role in normal neurological function. Closely related to this, Jim Nelson will tell us about studies on the neuropathology of chronic vitamin E deficiency, both in animal species such as rats and monkeys, and in children with congenital biliary atresia. He will discuss the possibility of a direct role of vitamin E deficiency in the pathogenesis of the lesions seen.

We shall have two contributions on a different clinical condition, namely

INTRODUCTION 3

retrolental fibroplasia. Neil Finer will describe his work with low birth weight, premature infants, where he finds a significant lowering of the incidence of the cicatricial, or scarring, form of the disease in infants who had received supplementary vitamin E. Helen Hittner will tell us about her work, also on retrolental fibroplasia, particularly her ultrastructural studies of the retinal lesions that develop in this condition, and the effect of early vitamin E supplementation upon them.

Two chapters will consider the role of vitamin E in the prevention of nutritional and other myopathies. Cecil McMurray will discuss his work on young ruminants given vitamin E-deficient and selenium-deficient diets. He has interesting evidence of the formation of lipofuscin-like pigments in heart Purkinje fibres, and he has made considerable strides in characterizing this pigment. Malcolm Jackson will consider the role of vitamin E in protecting against exercise-induced damage in experimental animals, and his work will in many ways link up with that of Lester Packer, described earlier in the symposium.

Finally, we have two chapters about interesting new clinical effects of vitamin E. Malcolm Chiswick has been investigating the incidence of in- traventricular haemorrhage in very premature babies. He has evidence that mortality from intraventricular haemorrhage appears to be less in babies given vitamin E. Robert Baehner will describe his work in vivo on neutrophils and will show that certain chemoattractants cause neutropenia and the seq- uestration of neutrophils, leading to blockage of the lung microvasculature with consequent respiratory distress. He will show evidence for the reversal of these events by vitamin E.

This brief outline of the symposium may give some indication of its breadth, and of the considerable research interest that now exists in vitamin E and its biological role.

Vitamin E as an antioxidant in vitro and in vivo

G. W. BURTON*, K. H. CHEESEMANt, T. DOBA*, K. U. INGOLD* and T. F. SLATERt

'Division of Chemistry, National Research Council of Canada, Ottawa, Ontario, Canada KIA OR6 and f Department of Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, U K

Abstract. Measurements of the absolute rate constants for the reaction with peroxyl radicals of a, /3, y and 8-tocopherol and several model compounds are described. The peroxyl radicals were obtained either by the autoxidation of styrene or by the flash photolysis of di-t-butyl ketone in an oxygen-saturated environment. The kinetic data are discussed in stereoelectronic terms. Vitamin E and total lipid-soluble, chain-breaking antioxidant concentrations in some normal and cancerous tissues have been measured. In human blood plasma and erythrocyte ghost membranes vitamin E is the major, and possibly the only, chain-breaking antioxidant. Lipid extracts of Novikoff ascites hepatoma cells contain considerably more vitamin E relative to lipid than do extracts of normal rat liver. These tumour lipids contain relatively fewer highly unsaturated fatty acids and are present at lower lipidiwet tissue ratios than the normal liver lipids. A number of unresolved problems relating to the action of vitamin E in vivo are discussed.

1983 Biology of vitamin E. Pitman Books, London (Ciba Foundation symposium 101) p 4-18

Autoxidation, which is the more or less spontaneous reaction which many organic compounds undergo with atmospheric oxygen at ambient tempera- tures, is a free radical chain reaction. The rate of an autoxidation can generally be reduced by the addition of certain compounds known as antioxidants. For convenience, antioxidants are divided into two classes. Preventive, or primary, antioxidants reduce the rate of initiation of free radical chains, most commonly by converting the free radical-producing hydroperoxidic products of prior autoxidation, ROOH, to innocuous pro- ducts, such as the corresponding alcohol, i.e.,

+ ROO', RO', HO' Preventive ROOH sh!i;/ Jniti:tk RoH ' antioxidant processes

4

VITAMIN E AS ANTIOXIDANT 5

Chain-breaking, or secondary, antioxidants trap the chain-propagating peroxyl radicals, ROO’, and thereby reduce the length of the autoxidation chains. The majority of chain-breaking antioxidants, AH, are phenols or diarylamines and their mechanism of action can be described by reactions 1 and 2 .

ROO’ + A H --+ ROOH + A’

ROO’ + A’ - ROOA

Each molecule of A H stops two oxidation chains because the resonance- stabilized antioxidant radical, A’, is too unreactive to continue the chain.

Vitamin E is a hydrophobic, peroxyl radical-trapping, chain-breaking antioxidant found in the lipid fraction of living organisms. Its principal (only?) function is to protect the lipid material of an organism from the undesirable effects of uncontrolled, spontaneous autoxidation.

The objective of our studies has been to correlate the biological activities of tocopherols with their detailed individual chemical structures and reactivities. We have investigated the tocopherols and also a large number of model compounds using the techniques of physical organic chemistry. We have also embarked on a programme to study vitamin E in normal and cancerous tissue.

(1)

(2)

Kinetic studies on vitamin E and related compounds

The relative and absolute effectiveness of chain-breaking antioxidants de- pends primarily on their reactivity towards peroxyl radicals; that is, on the rate constant for reaction 1, k l . (Reaction 2 is very rapid and does not affect the kinetics.) The larger the magnitude of k l the better will be the antioxidant activity. Kinetic studies on the inhibition of the autoxidation of styrene by a wide variety of synthetic phenols have shown that kl can be enhanced, relative to kl for phenol itself, by substitution of the aromatic ring by various electron-donating groups (Howard & Ingold 1963). These results suggest that the optimum substitution pattern for a phenolic antioxidant would be one with a methoxy group in the 4-position and methyl groups at the other four positions-that is, 2,3,5,6-tetramethyl-4-methoxyphenol (TMMP), a com- pound that was not, in fact, examined in the original study but one with a basic structure not too dissimilar to that of a-tocopherol (a-T) (see Table 1). On the basis of the methyl substitution pattern of the different tocopherols, the results of Howard & Ingold (1963) further suggest that antioxidant activities should decrease along the series: a-T > P-T 2 7-T > ST.

If vitamin E’s primary role in biological systems is that of a lipid-soluble,

6 BURTON ET AL

TABLE 1 Absolute values of k l for some phenols (in M-I s-l units)

AntioxidanP klb klC

a-Tocopherol

P-Tocopherol

Ho*R

Ho7@R

y-Tocopherol

&Tocopherol

Pentamethylhydroxy- chroman (PMHC)

2.4 x 106

1.7 X lo6

1.6 X lo6

6.5 x 105

2.1 x 106

2.6 X lo6

-

7 . 1 x 105

3.3 x 105

2.1 x 106

2.1 x 105 2.8 x los

Butylated hydroxytoluene (BHT)

1.2 x 104 2.4 x 104

aR = phytyl, C16H33. CBy laser flash photolysis of di-t-butyl ketone at 23°C.

bBy inhibited autoxidation of styrene at 30°C.

chain-breaking antioxidant, then the individual tocopherols would be ex- pected to have large kl values in comparison with those of most synthetic phenols. This we have shown to be the case, using Howard & Ingold's (1963) inhibition of styrene autoxidation method to measure kl (Burton & Ingold

VITAMIN E AS ANTIOXIDANT 7

1981). Some of our results are presented in Table 1. It is clear that some earlier claims that vitamin E is a poor antioxidant in vitro are without foundation. Such claims appear to have been based on qualitative experi- ments in which chain-breaking antioxidant activities could not be properly compared because the rates of chain initiation were not properly controlled. The results were, therefore, overly susceptible to the effects of minor impurities, such as catalysis by trace metals.

It should be noted that the order of antioxidant activities of the tocopherols ( a > p 2 y > 6) is the same as that of their biological activities (Century & Horwitt 1965). Moreover, a-T and the structurally related model compound, pentamethylhydroxychroman (PMHC), are considerably better (100 X ) traps for peroxyl radicals than butylated hydroxytoluene (BHT), the phenolic antioxidant most widely used in commerce.

We have recently developed a completely different technique for measur- ing kl values. The pulse from a nitrogen laser is focused on a sample in the cavity of an electron spin resonance (ESR) spectrometer which contains di-tert-butyl ketone and a phenol in an oxygen-saturated hydrocarbon sol- vent. Peroxyl radicals are formed in an essentially instantaneous process by the reaction sequence

(t-Bu)?C=O 5 t-Bu. + t-BuC*=O 2 t-BuOO. + t-BuC(O)OO.

The decay of these radicals by reaction with the phenol can be monitored by kinetic electron spin resonance (ESR) spectroscopy. It follows first-order kinetics from which the value of kl can be calculated (Table 1). The agreement with the results obtained by the styrene autoxidation method is satisfactory, considering the difference in the nature of the peroxyl radicals and the possible experimental errors in the two techniques.

We have invoked stereoelectronic factors to explain the fact that TMMP has only about 10% of the reactivity of a-T or PMHC (Burton & Ingold 1981). In TMMP the methoxy group is perpendicular to the plane of the aromatic ring, as has been shown by X-ray crystallography. In this position the p-type lone pair on the ethereal oxygen cannot stabilize the corresponding phenoxyl radical. However, in a-T and PMHC the second ring holds the ethereal oxygen in such a position that its p-type lone pair makes an angle of about 74' with the aromatic ring. In this position, the lone pair's orbital can overlap with the orbital containing the unpaired electron and so stabilize the phenoxyl radical. The more stable the phenoxyl radical, the weaker will be the 0-H bond in the phenol, and the weaker this bond, the more readily it will be cleaved by an attacking peroxyl; that is, the more effective it will be as an antioxidant. a-T, PMHC and related compounds are therefore extremely efficient antioxidants.

8 BURTON ET AL

TABLE 2 Relative k l values for some phenols

Antioxidant (k,lkl*T)a (kilk,

1.

2.

3.

4.

H o J b ' L I

0.8

0.6

1.3

0.7

0.8

1.1

aBy inhibited autoxidation of styrene at 30°C. bBy laser flash photolysis of di-t-butyl ketone at 23°C.

Although a-T and PMHC are the most efficient chain-breaking, phenolic antioxidants known, the question remains as to whether or not they really do have the optimum structure for this activity. We have begun a programme to measure kl values for some related compounds (Table 2). The stereoelec- tronic arguments outlined above provide a simple rationale for the observed kinetic behaviour. To be specific, in (1) (Trolbx C) the electron-withdrawing C02H group hinders stabilization of the phenoxyl by the ethereal oxygen's p-type lone pair. In (2) the 3p-type lone pair on sulphur will overlap with the 7~ system of the phenoxyl less well than would a 2p-type lone pair on oxygen. In (3) we believe the peri-interaction between the N-ethyl group and the 8-methyl group twists the saturated ring so that the nitrogen's lone pair is poorly oriented with respect to the preferred position perpendicular to the aromatic ring. For these reasons, (l), (2) and (3) are poorer antioxidants than a-T and PMHC. In contrast, (4) is a slightly better antioxidant because its five-membered ring holds the ethereal oxygen's p-type lone pair closer to the preferred perpendicular position than does the six-membered ring of a-T and PMHC.

VITAMIN E AS ANTIOXIDANT 9

In summary, our kinetic experiments show that the tocopherols are outstanding chain-breaking antioxidants and that the order of their reactivi- ties is the same as that of their biological potencies. Their high reactivity can be explained in stereoelectronic terms. One antioxidant that is slightly more efficient than a-T has been discovered.

Studies on vitamin E and related compounds in biological systems

Vitamin E is an outstandingly effective antioxidant in v i m and there is ample evidence that it protects against lipid peroxidation in vivo (Machlin 1980, Tappel 1980). However, other lipid-soluble materials might also function as chain-breaking antioxidants in living organisms. Their importance, relative to vitamin E, remained a matter for speculation until we developed a procedure for the quantitative measurement of the concentration of all lipid-soluble, chain-breaking antioxidants in biological systems (Burton et a1 1982, 1983a). This procedure is based on the peroxyl radical titration method of Mahoney et a1 (1978). A lipid extract is added to a hydrocarbon (generally styrene in our experiments) which is undergoing a thermally initiated autoxidation at 30 "C under carefully controlled conditions. The chain-breaking antioxidants that were present in the lipid extract inhibit autoxidation for a sharply defined time, known as the induction period. The total molar concentration of all chain-breaking antioxidants can be calculated from the duration of the induction period, there being no requirement for any knowledge of their type or chemical structure. For each lipid extract the concentration of vitamin E was also determined by a quantitative analysis for a, p, y and &tocopherols, using high pressure liquid chromatography (HPLC).

These procedures were applied to human blood plasma and erythrocyte ghost membranes obtained from several donors (Table 3) (Burton et a1 1982,

TABLE 3 Concentration of tocopherols [TI and of total antioxidant [AH] minus tocopherols in human plasma and erythrocyte ghost membranes (concentrations in pmole per litre of plasma or packed red blood cells)

Donor Ghosts

[TI [AHI-LTI [TI IAHl-[Tl

1 46 1 5 1 2 33 0 7 1 3 15 1 I 1 4 30 0 4 0 5 22 3 5 0 6 19 5 6 1 I 28 0 4 0

10 BURTON ET AL

1983a). Vitamin E is certainly the major, and is quite probably the only, chain-breaking antioxidant in human blood. This does not rule out the possibility that compounds functionally, if not necessarily structurally, related to vitamin E may play an important role in other human tissues.

Lipid peroxidation, as measured by the thiobarbituric acid (TBA) test for malonaldehyde, is much reduced in many cancer tissue samples in compari- son with adjacent normal tissue (Ahmed & Slater 1981, Dianzani 1982, McBrien 8c Slater 1982). We have now examined the composition and autoxidation of the lipids extracted from the livers of normal male Wistar rats and from Novikoff ascites hepatoma cells grown for seven days in the peritoneal cavity of such rats. Direct monitoring of oxygen absorption during the thermally initiated autoxidation of the lipid extracts at 30°C in an inert solvent (chlorobenzene) shows that the tumour tissue contains more chain- breaking antioxidant relative to lipid than the normal tissue (see Fig. 1). Furtheimore, the tumour lipids autoxidize more slowly than the normal tissue after the end of the induction period. The lower oxidizability* of the tumour lipids can be attributed to the lower proportion of highly unsaturated fatty acids they contain as compared with the fatty acids found in lipids from normal tissue (Wood 1979, Ahmed & Slater 1981) (see Table 4), since lipid oxidizability is determined almost exclusively by the concentration of polyun- saturated fatty acids. (It should be noted that malonaldehyde, which is the

TABLE 4 Composition of lipid extracts from normal rat liver and Novikoff ascites hepatoma cells (values are averages of duplicate measurements on four individual normal livers and on four samples of Novikol asdtes hepatoma cells: errors correspond to one standard deviation)

Normal liver Novikoff cells

[a-T + yT]/lipid (mM/kg)a [Antioxidant]/lipid (rnM/kg)b Total lipid/wet tissue (weight %) CholesteroMipid (weight %) Fatty acid composition (%)' 16 : 0

18:O 18: 1 18:2 20:4 22 : 6

1.9 t 0.3 2.6 t 0.4 2.5 t 0.3 9.6 t 0.3

18.4 k 0.6 17.7 t 0.9 11.5 k 0.6 19.4 k 1.2 15.5 t 0.7 6.3 t 0.4

5.5 t 0.5 6.9 k 0.8 1.0 t 0.1

18.7 f 1.3 13.8 f 0.5 17.0 f 0.7 20.0 k 0.3 29.5 f 1.0 9.6 k 0.6 1.9 t 0.7

a[a-T]/[yT] = 27.8. bCalculated as a-T. CChain length : number of double bonds.

'This is a kinetic term, defined as being equal to k , / (2kt)~M-k~, where kp and 2k, are the rate constants for the reactions of peroxyl radicals with the organic substrate (ROO' + RH .--, products) and with each other ( 2 R 0 0 ' .--, products). At a controlled rate of chain initiation, oxidizability is proportional to the rate of oxygen absorption after the end of the induction period.

VITAMIN E AS ANTIOXIDANT 11

Time (hours)

FIG. 1. Thermally initiated, direct oxidation of lipids extracted from normal rat livers and from Novikoff ascites hepatoma cells in chlorobenzene (2.0 ml) at 3OOC. Peroxyl radicals, generated at a steady rate of 1.2 x 10-8mol h-’ by the decomposition of 2,2’-azo-bis-(2-propionitrile) (3.63 x mol), attack polyunsaturated fat after stoichiometric consumption of antioxidant. Oxygen uptake is measured with a pressure transducer. Lipid was extracted with chloroform- methanol (2: 1). Upper: normal whole rat liver lipid (6.6mg). Lower: Novikoff hepatoma lipids (4.7 mg) .

oxidation product measured by the TBA test, is formed only from fatty acids having three or more methylene interrupted C==C double bonds.) The major chain-breaking antioxidant which induces the induction periods shown in Fig. 1 is vitamin E (see Table 4).

The data in Table 4 also show two results in agreement with earlier observations: (i) the tumour tissue contains a considerably smaller proportion of lipid than does the normal tissue (Bergelson 1972, Steele & Jenkins 1973); (ii) the tumour tissue lipids contain a higher proportion of cholesterol than the normal tissue (Bergelson 1972, Van Hoeven & Emmelot 1973, Wood 1979).

The decreased total lipid content and fraction of fatty acids that are highly unsaturated, plus the increased concentration of vitamin E and cholesterol (which is an ‘inert’ diluent insofar as the autoxidation of polyunsaturated fatty acids is concerned), all have a similar effect, since they all make cancerous tissue more resistant to autoxidation than normal tissue. It is difficult to avoid the conclusion that such a coordinated change must have a common end: the survival, growth and spread of the tumour at the expense of the host organism (Dianzani 1982, Burton et a1 1983b).

12 BURTONETAL

Some unresolved problems relating to vitamin E in vivo

Despite very extensive studies on vitamin E (Machlin 1980) there are still some problems relating to its chemical structure, physical location, and mode(s) of action.

Three structural problems concern us. First, although a-tocopherol is a superb chain-breaking antioxidant, our model compound studies do suggest that structurally related but slightly more active compounds might be prepared. Will the best of our model compounds, after modification with an added hydrocarbon tail to give them appropriate lipophilicity and membrane- philicity, show improved vitamin E activity? Second, our studies throw no light on the fact that 2R, 4’R,8’R-a-T (natural a-T) is biologically more active than the synthetic form, all-rac-a-T (Century & Horwitt 1965, Ames 1971). It has been reported (Desai 1980) that the natural isomer of a-T is stored and retained in the body better than the unnatural isomers. Is this due to natural a-T having a greater solubility in biological membranes? Third, it has been suggested that vitamin E plays a structural role in biological membranes as a consequence of an attractive hydrophobic interaction between the methyl groups in its phytyl tail and ‘pockets’ provided by the double bonds of arachidonic acid (Diplock & Lucy 1973). The low ratio of vitamin E to arachidonic acid (1 : 500 for erythrocyte membranes) was recognized as a strong argument against such a structural role. A later suggestion (Maggio et a1 1977) that the interaction between the vitamin and the membrane occurred on a dynamic basis between one molecule of vitamin E and a number of polyunsaturated molecules seems unlikely to be important at the ratios of vitamin E to arachidonic acid found in biological membranes. As an alternative, could the incorporation into vitamin E of a phytyl chain with its methyl branches simply be a consequence of the biosynthetic preference of plants for molecular architectures based on isoprene units? Also, could the phytyl tail confer a greater solubility in the more fluid, as opposed to the more rigid, regions of the biomembrane (cf. Maggio et a1 1977), in comparison to a straight-chain, saturated hydrocarbon tail,? (Autoxidation in an uninhibited system would occur more readily in the fluid regions than in the semicrystal- line regions.)

The time-averaged location of vitamin E within a biological membrane is still unknown. There is evidence that vitamin E preferentially orients its long axis parallel to the hydrocarbon chains in a phospholipid bilayer, but the average depth of the head group and the ease with which the molecule can invert (i.e., change from a ‘head-up’ to a ‘head-down’ position) have not been determined. Another problem relating to the physical location of vitamin E in an organism has to do with the quite different vitamin E/lipid ratios found in different tissues-for example, plasma lipids as compared to erythrocyte

VITAMIN E AS ANTIOXIDANT 13

membrane lipids, and normal liver cell lipids compared to Novikoff hepatoma lipids. Could the wide variation in the symptoms of vitamin E deficiency between species be a consequence of small changes in its solubility in the various membranes of different animals? Also, the potential role of other lipid-soluble, chain-breaking antioxidants in ‘vitamin E deficiency’ diseases should not be overlooked.

A number of in vitro experiments have shown that the tocopheroxyl radical formed from vitamin E by removal of the phenolic hydrogen can be reduced back to the starting tocopherol by vitamin C and some other reagents (Packer et a1 1979, Niki et a1 1982). There is a need to check whether this vitamin Chitamin E interaction mode also occurs in vivo, when the tocopheroxyl radical is buried within a biological membrane and the ascorbic acid is in the surrounding aqueous phase. If vitamin C does exert such a direct repairing effect on oxidized vitamin E in vivo it would imply that a greater number of free radical chains are initiated in the lipids of the human body than would be indicated by our daily requirement for vitamin E, namely about 10 mg/day, which is nominally capable of trapping about 5 X 10-5 mole of radical per day. Finally, the role played by vitamin E in reducing the ease of autoxidation of certain tumour lipids deserves detailed investigation.

Acknowledgements

This work was supported by a grant from the National Foundation for Cancer Research. We gratefully acknowledge outstanding technical assistance by L. Hughes, D. A. Lindsay, M. Slaby and A. Webb. This paper is publication NRCC no. 22482.

REFERENCES

Ahmed SM, Slater TF 1981 Lipid peroxidation in microsomal fractions obtained from some rat and mouse tumours. In: Slater TF, Garner A (eds) Recent advances in lipid peroxidation and tissue injury. Brunel Printing Services, London, p 177-194

Ames SR 1971 Isomers of alpha-tocopheryl acetate and their biological activity. Lipids 6:281-290 Bergelson LD 1972 Tumor lipids. Prog Chem Fats Other Lipids. 13:l-59 Burton GW, Ingold KU 1981 Autoxidation of biological molecules. 1. The antioxidant activity of

vitamin E and related chain-breaking phenolic antioxidants in vitro. J Am Chem SOC

Burton GW, Joyce A, Ingold KU 1982 First proof that vitamin E is major lipid-soluble, chain-breaking antioxidant in human blood plasma. Lancet 2:327

Burton GW, Joyce A, Ingold KU 1983a Is vitamin E the only lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membranes? Arch Biochem Biophys

103:6472-6477

221 :281-290

14 DISCUSSION

Burton GW, Cheeseman KH, Ingold KU, Slater TF 1983b Lipid antioxidants and products of lipid peroxidation as potential tumour protective agents. Biochem SOC Trans 11:261-262

Century B, Honvitt MK 1965 Biological availability of various forms of vitamin E with respect to different indices of deficiency. Proc Fed Am SOC Exp Biol 24:906-911

Desai ID 1980 Assay methods. In: Machlin LJ (ed) Vitamin E: a comprehensive treatise. Marcel Dekker, New York, p 67-98

Dianzani MU 1982 Biochemical effects of saturated and unsaturated aldehydes. In: McBrien DCH, Slater "F(eds) Free radicals, lipid peroxidation and cancer. Academic Press, London,

Diplock AT, Lucy JA 1973 The biochemical modes of action of vitamin E and selenium: a

Howard JA, Ingold KU 1963 The inhibited autoxidation of styrene. Part 111. The relative

Machlin LJ (ed) 1980 Vitamin E: a comprehensive treatise. Marcel Dekker, New York Maggio B, Diplock AT, Lucy JA 1977 Interactions of tocopherols and ubiquinones with

monolayers of phospholipids. Biochem J 161:lll-121 Mahoney LR, Korcek S , Hoffman S, Willermet PA 1978 Determination of the antioxidant

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McBrien DCH, Slater TF (eds) 1982 Free radicals, lipid peroxidation and cancer. Academic Press, London

Niki E, Tsuchiya J, Tanimura R, Kamiya Y 1982 Regeneration of vitamin E from a-chromanoxyl radical by glutathione and vitamin C. Chem Lett, p 789-792

Packer JE, Slater TF, Willson RL 1979 Direct observation of a free radical interaction between vitamin E and vitamin C. Nature (Lond) 278:737-738

Steele W, Jenkin HM 1973 Lipids and lipid metabolism of Novikoff rat hepatorna cells. In: Wood R (ed) Tumor lipids: biochemistry and metabolism. Am Oil Chem SOC Press, Champaign, Illinois, p 215-224

Tappel AL 1980 Measurement of and protection from in vivo lipid peroxidation. In: Pryor WA (ed) Free radicals in biology. Academic Press, New York, vol 4:l-47

Van Hoeven RP, Emmelot P 1973 Plasma membrane lipids of normal and neoplastic tissues. In: Wood R (ed) Tumor lipids: biochemistry and metabolism. Am Oil Chern SOC Press, Champaign, Illinois, p 126-138

Wood R 1979 Distribution of dietary geometrical and positional isomers in brain, heart, kidney, liver, lung, muscle, spleen, adipose and hepatoma. In: Emken EA, Dutton HJ (eds) Geometrical and positional fatty acid isomers. Am Oil Chem SOC Press, Champaign, Illinois,

p 129-158

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DISCUSSION

Diplock: Were the rate constant studies done with all-racemic tocopherols? Ingold: Yes, because we were looking at reactions that could not be

affected by the presence or absence of chirality in the antioxidant molecule. Packer: In those kinetic studies, butylated hydroxytoluene (BHT) was 240

times less effective as an antioxidant than a-tocopherol. That is surely not as great a difference as you would expect in biological systems?

VITAMIN E AS ANTIOXIDANT 15

Ingold: I agree that there might be an even bigger difference in biological systems, especially since BHT has little or no vitamin E activity. I was specifically talking here about how rapidly BHT trapped peroxyl radicals, relative to a-tocopherol. The same reactivity ratio will presumably apply in vivo, where the vitamin E activity of BHT would be much less, because it will probably partition less favourably than a-tocopherol into the sites where it is needed in the cell.

Wendel: The second-order rate constants for the Cu/Zn-containing super- oxide dismutases are about 2 x 108 M-1 s-l, and the rate constant for the glutathione peroxidase reaction is about 1 X 108 y-1 s-1. These rates are several orders of magnitude faster than your value for a-tocopherol. What do these different kinetic parameters mean for the biological functions of these various molecules, in terms of affecting the initiation phases of radical chain reactions?

Ingold: It means that the molecules you mention are extremely effective at doing what they are supposed to do, since the maximum rate constant for a bimolecular reaction is around lo9, Such high rate constants tell us that biology has gone as far as it can; the enzyme molecule is reacting at essentially every encounter with the appropriate molecular species, As to why vitamin E is slow by comparison with these enzymes, it might be possible to design a molecule much more reactive than vitamin E, but such a molecule almost certainly either would react directly with oxygen, to generate free radicals, or would itself be a free radical. Thus it would act as an initiator of peroxidation, rather than as an antioxidant.

Pryor: When asking questions about the effectiveness of an inhibitor such as vitamin E, one must ask: what reactions can the radicals in the system undergo, other than reacting with the inhibitor? The molecule that is inhibit- ing a given reaction has to be effective enough to block the competing reactions; it doesn’t need to be any better than that. The two milieus discus- sed above are very different. The superoxide anion is a very reactive species and superoxide dismutase rapidly scavenges it in water, a medium in which vitamin E is not soluble. However, the peroxyl radicals in the lipid bilayer have a very different choice: they can abstract an allylic hydrogen atom’or add to a double bond of a polyunsaturated fatty acid, or they can react with the antioxidant, vitamin E. In the natural bilayer system, thefusteststep is the one that we wish to occur, namely the reaction of the peroxyl radical with the antioxidant. So tocopherol is good enough as it is! There is no reason for Nature to attempt to make it better.

Dr Ingold, in the experiments in which you added biological samples to a test solution and measured oxygen consumption (Table 3), why did you choose styrene as your substrate? If you use a different substrate, would it make any difference?

16 DISCUSSION

Zngold: We have used styrene, tetralin, cyclohexene and other hydrocar- bons. We see no difference in the results. Styrene is the molecule of choice for this study because it is one of the most reactive of organic molecules towards the peroxyl radical. It also can be easily obtained in high purity in large amounts, in contrast to, say, linoleic acid.

Pryor: Have you tested linoleic acid, or any other polyunsaturated fatty acid?

Zngold: No; however, I see no reason why linoleic acid should give different results in this experiment.

Pryor: Nor do I, but we have thought about this problem, and we decided that if Nature uses the polyunsaturated fatty acids, perhaps we should as well! It would be comforting to obtain the same series of antioxidant reactivities using a biological substrate-for example, a polyunsaturated fatty acid.

Zngold: The minor disadvantage of almost all the non-biological substrates except styrene, which would be a major problem with the biological systems, is that hydroperoxides are products of autoxidation and they will be sensitive to traces of iron, which are difficult to avoid in biological systems.

Pryor: There is one problem with styrene, however. Instead of forming a hydroperoxide, styrene forms a styrene-peroxide polymer with oxygen. This is an unusual kind of reaction. So it is very different from what occurs in the biological system.

Zngold: However, in the peroxyl radical titration for total antioxidant one is looking at the competition between a peroxyl radical reacting with the antiox- idant and a peroxyl radical reacting with an organic substrate.

Pryor: Nevertheless, the two kinds of substrate, styrene and fatty acid, should be compared in the same study.

Edwards: Vitamin E is generally said to be oriented in the lipid bilayer of biological membranes with its more hydrophilic end in the polar region, adjacent to the aqueous surrounding medium. But, if it is less hydrophilic than has been assumed, could the vitamin E molecule be acting as a dowel, sunk deeper among the lipid molecules of the membrane and thereby pre- venting the separation of the two layers? Perhaps freeze-fracture studies or membrane dissolution studies could indicate whether, in vitamin E-deficient animals, there is a greater tendency for the membrane bilayers to separate. Is there a difference in the freeze-fracture pattern in rancid tissues and non- rancid tissues? If there were a structural difference, we could establish whether the failure of antioxidant protection in the deficiency is primarily due to the punching of holes right through the membrane, attacking both parts of the bilayer, or whether the dissolution of membrane integrity is due to separation of the two leaflets of the lipid bilayer.

Ingold: All we know about the location of the vitamin E molecule is that it orients itself with its long axis parallel to the long axes of the phospholipid