Section of Medicine, Experimental Medicine & Therapeutics with Section of Pathology
89
Degkwitz E & Staudinger H (1974) In Vitamin C. Ed. G G Birch & K Parker. Applied Science Publishers, London; p 161 Edgar J A (1974) Nature, London 248, 136 Fielding A M & Hughes R E (1975) Experientia 31, 1394 Ginter E (1974) In Vitamin C. Eds. G G Birch & K Parker. Applied Science Publishers, London; p 179 (1975) The Role of Vitamin C in Cholesterol Catabolism and Atherogenesis. Veda, Vydaatelstvo Slovenskej Academie Vied, Bratislava, Czechoslovakia Grimble R F & Hughes R E (1967) Experientia 23, 362 Hodges R E, Hood J, Canham J E, ;auberlich H E & Baker E M (1971) American Journal of Clinical Nutrition 24, 432 Hughes R E (1964) Nature, London 203, 1068 (1973) In Molecular Structure and Function of Food Carbohydrate. Eds. G G Birch & L F Green. Applied Science Publishers, London; p 108 (1974) In Vitamin C. Eds. G G Birch & K Parker. Applied Science Publishers, London; p 68 (1976) Journal ofHuman Nutrition 30, 315 (1977) Food Chemistry (in press) Kefalides N A (1973) International Review of Connective Tissue Research 6, 63 Lewin S (1974) In Vitamin C. Eds. G G Birch & K Parker. Applied Science Publishers, London; p 221 Murray D & Hughes R E (1976) Proceedings of the Nutrition Society, in press Pauling L (1970) Vitamin C and the Common Cold. Freeman, San Francisco (1974) Proceedings of the National Academy of Sciences of the USA 71, 4442 Reid K B M (1974) Biochemical Journal 141, 189 Rhead W J & Schrauzer G N (1971) Nutrition Reviews 29, 262 Schlegel J V, Pipkin G E, Nishimura R & Schultz G N (1970) Journal of Urology 103, 155 Stick H F, Karim J, Moropatnick J, & Lo L (1976) Nature, London 260, 722 Stone I (1972) The Healing Factor. New York Tappel A, Fletcher B & Deamer D (1973) Journal ofGerontology 28, 415
1960). However, two important pieces of research stimulated the search for more biologically active metabolites of vitamin D. These were the observation of a time lag between the administration of vitamin D and the resulting physiological response (Carlsson 1952) and, later, that actinomycin D (an antibiotic which, when added to bacterial cultures, inhibits DNA-directed RNA synthesis) inhibited the action of vitamin D (Zull, Czarnowska-Misztal & DeLuca 1965). The discovery of metabolites was initially hampered by lack of availability ofnuclear magnetic resonance measurements and mass spectrometry (Napoli 1975). However, these chemical problems were overcome and in the 1960s DeLuca's group demonstrated the existence of biologically active metabolites of vitamin D3 (Lund & DeLuca 1966). It was established that vitamins D3 is metabolized to 25 hydroxy vitamin D3 (25 OH D3) in the liver (Blunt et al. 1968, Bhattacharyya & DeLuca 1973), and in the kidney 25 OH D3 is further hydroxylated to 1,25 dihydroxy vitamin D3 (1,25 diOH D3) (Fraser & Kodicek 1970) or 24,25 dihydroxy D3 (24,25 diOH D3) (Ghazarian & DeLuca 1974). As a result of intense chemical and biochemical research a plethora of vitamin D metabolites has been isolated, and many synthetic analogues have been made. Chemically the vitamin D3 (cholecalciferol) molecule has the potential for hydroxylation at further positions, so giving opportunities for discovering other biologically active metabolites. Biological studies have shown that an elegant vitamin D metabolic pathway now exists (see Fig 1), so that skin and dietary sources of vitamin D
Dr Angela Fairney (Department of Chemical Pathology, St Mary's Hospital Medical School, London W2 1PG)
7- Dehydro- ultraviolet radiation
DIET
cholesterol
25 OH D3
Current Status of Vitamin D Metabolism In the past decade major advances have been made in our understanding of the biochemistry of vitamin D. The information gained has greatly enhanced our understanding of many diseases associated with abnormalities of calcium homeostasis.
Biochemistry Biologically active forms of vitamin D were thought for many years to be obtained by endogenous skin synthesis and from the diet. The main vitamin D-rich foods were eggs, fish, margarine and cod-liver oil (McCance & Widdowson
1_4
VITAMIN D V
24,25 diOH D3
1,25 diOH D3
1,24,25 triOH D3
bone
I ntestine
Fig I Vitamin D metabolism
90
Proc. roy. Soc. Med. Volume 70 February 1977
are converted to biologically active metabolites in the kidney. In addition, trihydroxy metabolites are known to exist, the exact biological purpose of which is unknown in man. The speed at which novel hydroxylated relatives ofvitamin D appear is very perplexing. However, hypothetical biochemical explanations for many clinical calcium problems now exist. There are two aspects of vitamin D metabolism that deserve further mention. First, the control of the hydroxylation steps in the liver and kidney had been hotly debated. Many factors have been shown to regulate the renal tubular production of 1,25 diOH D3, e.g. calcium, phosphate parathyroid hormone, calcitonin and 1,25 diOH D3 itself. Hypocalccmia increases the formation of 1,25 diOH D3 whereas at normal levels and in hypercalcaemia the formation of 1,25 diOH D3 is decreased, so that 24,25 diOH D3 becomes the major metabolite of vitamin D formed (Napoli 1975). Second, the structural conformation of the metabolites of vitamin D in vivo has recently been shown to have biological significance (Norman et al. 1975). It is emphasized that in solution there is a rapid equilibrium between the two A ring chair conformations of the molecule, and in addition the plane of the hydroxyl group is relevant to the biological structure of the molecule.
Clinical Aspects The clinical application of the current state of vitamin D metabolism may be considered from three points of view. These are measurement of the vitamin and its metabolites, study of the etiology of calcium disorders, and therapy. Measurement of vitamin D and its metabolites: Attention has been concentrated on the hepatic metabolites of vitamin D (these being the major circulating forms of the vitamin) and subsequently the development of assays for 1,25 diOH D3. Measurement of 25 OH D3 is comparatively easy using competitive protein binding techniques (Haddad & Chyu 1971). These assays, in which different sources of binding protein may be used, e.g. rachitic rat kidney, rachitic rat serum or normal rat kidney preparations, all have limitations of specificity. In general these assays cannot differentiate between 25 hydroxy vitamin D2 and 25 hydroxy vitamin D3 (Bowling & Fairney, personal communication), and care is needed in the interpretation of the results due to seasonal variation in random values of 25 OH D3 (McLaughlin et al. 1974). A clinically suitable assay for 1,25 diOH D3 has yet to be documented. The present known assay (Brumbaugh et al. 1974) has produced useful research information but it requires a large volume of plasma and is very laborious,
rendering it unsuitable for routine use by the clinical biochemist. In general the assayist still has four big problems, i.e. lack of specificity, inadequate supplies of metabolites to enable novel assay methods to be investigated, lack of labelled metabolites with high specific activity and the tedium of present methods. Etiology of calcium disorders: The etiology of many conditions may now be considered as a defect at different stages in the vitamin D pathway. There may be a lack of available vitamin D, as in Asian immigrants and in malabsorption; a defect in hepatic hydroxylation, as in chronic biliary cirrhosis and anticonvulsant osteomalacia; and a defect in renal hydroxylation such as that proposed to account for the bone disease of chronic renal failure. Although this approach seems to clarify the etiology of many disorders, the results of treatment in small groups of patients, together with assay results to date, suggest that in some conditions the defect may be at more than one site in the pathway. Therapy: Sadly, the therapeutic use of vitamin D metabolites has been greatly hampered by inadequate supplies of the synthetic compounds. Low levels of 1,25 diOH D3 have been confirmed in renal failure (Haussler 1975), and in a small number of patients therapeutic 1,25 diOH D3 has been shown to improve the bone disease (Brickman et al. 1972). To circumvent the lack of available 1,25 diOH D3 lac hydroxy vitamin D3 has been used therapeutically in the hope that the patient's own liver will provide the hydroxylation in the 25 position. This compound is more easily synthesized than 1,25 diOH D3 but has not quite realized its original potential. Summary In summary one can record that the biochemist has discovered a beautiful metabolic pathway for vitamin D which is probably as yet incomplete. Subsequently the medicinal chemist has provided numerous synthetic analogues. However, delays in the diagnosis and treatment of vitamin D disorders, due to lack of specific assays and inadequate supplies of therapeutic metabolites, still unfortunately exist. It is hoped that these problems will be rectified as soon as more plentiful supplies of the appropriate metabolites are available. REFERENCES Bhattacharyya M H & DeLuca H F (1973) Journal of Biological Chemistry 248, 2969 Blunt J W, DeLuca H F & Schnoes H K (1968) Biochemistry 7, 3317 Brickman A S, Coburn J W & Norman A W (1972) New England Journal of Medicine 287, 891 Brumbaugh P F, Haussler D H, Bursac K M & Haussler M R (1974) Biochemistry 13, 4091
Section of Medicine, Experimental Medicine & Therapeutics with Section of Pathology Carlsaon A (1952) Acta physlologica scandinavica 26, 212 Fraser D R & Kodlcek E (1970) Nature, London 228, 764 Ghazaribn J G & DeLuca H F (1974) Archives of Biochemistry and Biophysics 160, 63 Haddad J G & Chyu K J (1971) Journal of Clinical Endocrinology and Metabolism 33, 992 Haumler M R (1975) Vitamin D and Problems Related to Uramic Bone Disease. De Gruyter, Berlin; p 25 Lund J & DeLuca H F (1966) Journal of Lipid Research 7. 739 McCance R A & Widdowson E M (1960) Composition of Foods. HMSO, London McLaughlin M, Fairney A, Lester E, Raggatt P R Brown D J & WIlls M R (1974) Lancet i, 536 Napoli J L (1975) Annual Reports in Medical Chemistry 10, 298 Norman A W, Okamura W H & Wing R M (1975) In: Calcium Regulating Hormones. Ed R V Talmage. Excerpta Medica, Amsterdam; p 362 Zull J E, Czarnowska-Misztal E & DeLuca H F (1965) Science 149, 182
Dr T L Dormandy (Department of Chemical Pathology, Whittington Hospital (Archway Wing), London N19)
Vitamin E and Antoxidant Activity Autoxidation and Antioxidants Within the last thirty years vitamin E has been hailed as an elixir of youth and dismissed as a tonic for tired sheep. Between these extremes almost every shade of opinion has been expressed. One reason for such disagreement is the ignorance which still surrounds biological autoxidation and the mechanisms which protect against it. Autoxidation (or peroxidation, as it is increasingly called) means the nonenzymic interaction between unsaturated compounds, mainly polyunsaturated lipids and molecular oxygen. Orthodox biochemistry is so firmly rooted in enzymology that, until comparatively recently, such nonenzymic processes were tacitly but resolutely ignored. Yet polyunsaturated lipids are essential constituents of all cells; and, in the presence of oxygen and ubiquitous trace catalysts, they are intrinsically autoxidizable. Their actual autoxidation depends on the random generation of highly reactive free radicals derived from the reduction of the oxygen molecule, 02, Conditioned as we are to the continuous and predictable working of enzymic systems, the randomness of this event (comparable in some respects to the randomness of genetic mutations) remains an intellectual barrier as well as a technical difficulty. In the darkness and the cold of a refrigerator a lump of butter will remain 'fresh' for a long time; but even there, sooner or later, it will develop signs of rancidity. Rancidity means
91
autoxidation, not so much the primary formation of lipid peroxides but their secondary breakdown into many, still imperfectly characterized, fragmentation products. In contrast to the generation of the initial free radicals this fragmentation is highly exergonic, virtually spontaneous and irreversible. In a biological system it is also wasteful and destructive, since there is no known mechanism whereby the energy set free can be used for biological work. If biological - i.e. enzymic oxidation sustains living cells like a finely tuned internal combustion engine, autoxidation is like dropping a lighted match into the petrol tank. In contrast to the lump of butter in the refrigerator, we spend a lifetime exposed to light and other forms of high-energy irradiation, incubating ourselves at 37°C and more or less in perpetual, if not always productive, motion. Since we do not on any massive scale go rancid until a few hours after death we must assume that we are actively preserved, that every cell in our bodies is shielded by an extremely efficient antioxidant mechanism or several antioxidant mechanisms (Barber & Bernheim 1967, Dormandy 1969). It is in this context that the role and importance of vitamin E must be assessed. Vitamin E in Vitro and in Animals There is no doubt whatever that when vitamin E is added to highly autoxidizable mixtures in vitro whether the mixture is a simple lipid emulsion or homogenized tissue - it behaves as an efficient antioxidant or preservative. In this respect it is not, of course, unique among biological compounds: under varying conditions many phenolic substances, trace elements and enzymes act as autoxidation inhibitors. Nor are the tocopherols as powerful as some of the synthetic preservatives widely used in the chemical and food industry (Witting 1975). But they are nontoxic and readily available. The question is: are they essential? If there can be no doubt about the antioxidant potency of the tocopherols in vitro, it is only marginally less certain that vitamin E deficiency is the specific cause of well defined syndromes in animals. Between species the clinical manifestations differ, ranging from sterility in sheep and rats to encephalomalacias in chicks, muscular dystrophies in cows and demyelination in horses (Wolbach & Bessey 1942, Pappenheimer 1943, Olcott & Mattill 1941). Strong experimental evidence suggests, moreover, that in all these syndromes the vitamin deficiency is associated with an increased susceptibility to autoxidation of the animal as a whole and in particular of the tissue or organ clinically affected. In short, the clinical manifestations and the biochemical abnormality are almost certainly causally linked. Even in animals, however, vitamin E is not the only anti-
89
Degkwitz E & Staudinger H (1974) In Vitamin C. Ed. G G Birch & K Parker. Applied Science Publishers, London; p 161 Edgar J A (1974) Nature, London 248, 136 Fielding A M & Hughes R E (1975) Experientia 31, 1394 Ginter E (1974) In Vitamin C. Eds. G G Birch & K Parker. Applied Science Publishers, London; p 179 (1975) The Role of Vitamin C in Cholesterol Catabolism and Atherogenesis. Veda, Vydaatelstvo Slovenskej Academie Vied, Bratislava, Czechoslovakia Grimble R F & Hughes R E (1967) Experientia 23, 362 Hodges R E, Hood J, Canham J E, ;auberlich H E & Baker E M (1971) American Journal of Clinical Nutrition 24, 432 Hughes R E (1964) Nature, London 203, 1068 (1973) In Molecular Structure and Function of Food Carbohydrate. Eds. G G Birch & L F Green. Applied Science Publishers, London; p 108 (1974) In Vitamin C. Eds. G G Birch & K Parker. Applied Science Publishers, London; p 68 (1976) Journal ofHuman Nutrition 30, 315 (1977) Food Chemistry (in press) Kefalides N A (1973) International Review of Connective Tissue Research 6, 63 Lewin S (1974) In Vitamin C. Eds. G G Birch & K Parker. Applied Science Publishers, London; p 221 Murray D & Hughes R E (1976) Proceedings of the Nutrition Society, in press Pauling L (1970) Vitamin C and the Common Cold. Freeman, San Francisco (1974) Proceedings of the National Academy of Sciences of the USA 71, 4442 Reid K B M (1974) Biochemical Journal 141, 189 Rhead W J & Schrauzer G N (1971) Nutrition Reviews 29, 262 Schlegel J V, Pipkin G E, Nishimura R & Schultz G N (1970) Journal of Urology 103, 155 Stick H F, Karim J, Moropatnick J, & Lo L (1976) Nature, London 260, 722 Stone I (1972) The Healing Factor. New York Tappel A, Fletcher B & Deamer D (1973) Journal ofGerontology 28, 415
1960). However, two important pieces of research stimulated the search for more biologically active metabolites of vitamin D. These were the observation of a time lag between the administration of vitamin D and the resulting physiological response (Carlsson 1952) and, later, that actinomycin D (an antibiotic which, when added to bacterial cultures, inhibits DNA-directed RNA synthesis) inhibited the action of vitamin D (Zull, Czarnowska-Misztal & DeLuca 1965). The discovery of metabolites was initially hampered by lack of availability ofnuclear magnetic resonance measurements and mass spectrometry (Napoli 1975). However, these chemical problems were overcome and in the 1960s DeLuca's group demonstrated the existence of biologically active metabolites of vitamin D3 (Lund & DeLuca 1966). It was established that vitamins D3 is metabolized to 25 hydroxy vitamin D3 (25 OH D3) in the liver (Blunt et al. 1968, Bhattacharyya & DeLuca 1973), and in the kidney 25 OH D3 is further hydroxylated to 1,25 dihydroxy vitamin D3 (1,25 diOH D3) (Fraser & Kodicek 1970) or 24,25 dihydroxy D3 (24,25 diOH D3) (Ghazarian & DeLuca 1974). As a result of intense chemical and biochemical research a plethora of vitamin D metabolites has been isolated, and many synthetic analogues have been made. Chemically the vitamin D3 (cholecalciferol) molecule has the potential for hydroxylation at further positions, so giving opportunities for discovering other biologically active metabolites. Biological studies have shown that an elegant vitamin D metabolic pathway now exists (see Fig 1), so that skin and dietary sources of vitamin D
Dr Angela Fairney (Department of Chemical Pathology, St Mary's Hospital Medical School, London W2 1PG)
7- Dehydro- ultraviolet radiation
DIET
cholesterol
25 OH D3
Current Status of Vitamin D Metabolism In the past decade major advances have been made in our understanding of the biochemistry of vitamin D. The information gained has greatly enhanced our understanding of many diseases associated with abnormalities of calcium homeostasis.
Biochemistry Biologically active forms of vitamin D were thought for many years to be obtained by endogenous skin synthesis and from the diet. The main vitamin D-rich foods were eggs, fish, margarine and cod-liver oil (McCance & Widdowson
1_4
VITAMIN D V
24,25 diOH D3
1,25 diOH D3
1,24,25 triOH D3
bone
I ntestine
Fig I Vitamin D metabolism
90
Proc. roy. Soc. Med. Volume 70 February 1977
are converted to biologically active metabolites in the kidney. In addition, trihydroxy metabolites are known to exist, the exact biological purpose of which is unknown in man. The speed at which novel hydroxylated relatives ofvitamin D appear is very perplexing. However, hypothetical biochemical explanations for many clinical calcium problems now exist. There are two aspects of vitamin D metabolism that deserve further mention. First, the control of the hydroxylation steps in the liver and kidney had been hotly debated. Many factors have been shown to regulate the renal tubular production of 1,25 diOH D3, e.g. calcium, phosphate parathyroid hormone, calcitonin and 1,25 diOH D3 itself. Hypocalccmia increases the formation of 1,25 diOH D3 whereas at normal levels and in hypercalcaemia the formation of 1,25 diOH D3 is decreased, so that 24,25 diOH D3 becomes the major metabolite of vitamin D formed (Napoli 1975). Second, the structural conformation of the metabolites of vitamin D in vivo has recently been shown to have biological significance (Norman et al. 1975). It is emphasized that in solution there is a rapid equilibrium between the two A ring chair conformations of the molecule, and in addition the plane of the hydroxyl group is relevant to the biological structure of the molecule.
Clinical Aspects The clinical application of the current state of vitamin D metabolism may be considered from three points of view. These are measurement of the vitamin and its metabolites, study of the etiology of calcium disorders, and therapy. Measurement of vitamin D and its metabolites: Attention has been concentrated on the hepatic metabolites of vitamin D (these being the major circulating forms of the vitamin) and subsequently the development of assays for 1,25 diOH D3. Measurement of 25 OH D3 is comparatively easy using competitive protein binding techniques (Haddad & Chyu 1971). These assays, in which different sources of binding protein may be used, e.g. rachitic rat kidney, rachitic rat serum or normal rat kidney preparations, all have limitations of specificity. In general these assays cannot differentiate between 25 hydroxy vitamin D2 and 25 hydroxy vitamin D3 (Bowling & Fairney, personal communication), and care is needed in the interpretation of the results due to seasonal variation in random values of 25 OH D3 (McLaughlin et al. 1974). A clinically suitable assay for 1,25 diOH D3 has yet to be documented. The present known assay (Brumbaugh et al. 1974) has produced useful research information but it requires a large volume of plasma and is very laborious,
rendering it unsuitable for routine use by the clinical biochemist. In general the assayist still has four big problems, i.e. lack of specificity, inadequate supplies of metabolites to enable novel assay methods to be investigated, lack of labelled metabolites with high specific activity and the tedium of present methods. Etiology of calcium disorders: The etiology of many conditions may now be considered as a defect at different stages in the vitamin D pathway. There may be a lack of available vitamin D, as in Asian immigrants and in malabsorption; a defect in hepatic hydroxylation, as in chronic biliary cirrhosis and anticonvulsant osteomalacia; and a defect in renal hydroxylation such as that proposed to account for the bone disease of chronic renal failure. Although this approach seems to clarify the etiology of many disorders, the results of treatment in small groups of patients, together with assay results to date, suggest that in some conditions the defect may be at more than one site in the pathway. Therapy: Sadly, the therapeutic use of vitamin D metabolites has been greatly hampered by inadequate supplies of the synthetic compounds. Low levels of 1,25 diOH D3 have been confirmed in renal failure (Haussler 1975), and in a small number of patients therapeutic 1,25 diOH D3 has been shown to improve the bone disease (Brickman et al. 1972). To circumvent the lack of available 1,25 diOH D3 lac hydroxy vitamin D3 has been used therapeutically in the hope that the patient's own liver will provide the hydroxylation in the 25 position. This compound is more easily synthesized than 1,25 diOH D3 but has not quite realized its original potential. Summary In summary one can record that the biochemist has discovered a beautiful metabolic pathway for vitamin D which is probably as yet incomplete. Subsequently the medicinal chemist has provided numerous synthetic analogues. However, delays in the diagnosis and treatment of vitamin D disorders, due to lack of specific assays and inadequate supplies of therapeutic metabolites, still unfortunately exist. It is hoped that these problems will be rectified as soon as more plentiful supplies of the appropriate metabolites are available. REFERENCES Bhattacharyya M H & DeLuca H F (1973) Journal of Biological Chemistry 248, 2969 Blunt J W, DeLuca H F & Schnoes H K (1968) Biochemistry 7, 3317 Brickman A S, Coburn J W & Norman A W (1972) New England Journal of Medicine 287, 891 Brumbaugh P F, Haussler D H, Bursac K M & Haussler M R (1974) Biochemistry 13, 4091
Section of Medicine, Experimental Medicine & Therapeutics with Section of Pathology Carlsaon A (1952) Acta physlologica scandinavica 26, 212 Fraser D R & Kodlcek E (1970) Nature, London 228, 764 Ghazaribn J G & DeLuca H F (1974) Archives of Biochemistry and Biophysics 160, 63 Haddad J G & Chyu K J (1971) Journal of Clinical Endocrinology and Metabolism 33, 992 Haumler M R (1975) Vitamin D and Problems Related to Uramic Bone Disease. De Gruyter, Berlin; p 25 Lund J & DeLuca H F (1966) Journal of Lipid Research 7. 739 McCance R A & Widdowson E M (1960) Composition of Foods. HMSO, London McLaughlin M, Fairney A, Lester E, Raggatt P R Brown D J & WIlls M R (1974) Lancet i, 536 Napoli J L (1975) Annual Reports in Medical Chemistry 10, 298 Norman A W, Okamura W H & Wing R M (1975) In: Calcium Regulating Hormones. Ed R V Talmage. Excerpta Medica, Amsterdam; p 362 Zull J E, Czarnowska-Misztal E & DeLuca H F (1965) Science 149, 182
Dr T L Dormandy (Department of Chemical Pathology, Whittington Hospital (Archway Wing), London N19)
Vitamin E and Antoxidant Activity Autoxidation and Antioxidants Within the last thirty years vitamin E has been hailed as an elixir of youth and dismissed as a tonic for tired sheep. Between these extremes almost every shade of opinion has been expressed. One reason for such disagreement is the ignorance which still surrounds biological autoxidation and the mechanisms which protect against it. Autoxidation (or peroxidation, as it is increasingly called) means the nonenzymic interaction between unsaturated compounds, mainly polyunsaturated lipids and molecular oxygen. Orthodox biochemistry is so firmly rooted in enzymology that, until comparatively recently, such nonenzymic processes were tacitly but resolutely ignored. Yet polyunsaturated lipids are essential constituents of all cells; and, in the presence of oxygen and ubiquitous trace catalysts, they are intrinsically autoxidizable. Their actual autoxidation depends on the random generation of highly reactive free radicals derived from the reduction of the oxygen molecule, 02, Conditioned as we are to the continuous and predictable working of enzymic systems, the randomness of this event (comparable in some respects to the randomness of genetic mutations) remains an intellectual barrier as well as a technical difficulty. In the darkness and the cold of a refrigerator a lump of butter will remain 'fresh' for a long time; but even there, sooner or later, it will develop signs of rancidity. Rancidity means
91
autoxidation, not so much the primary formation of lipid peroxides but their secondary breakdown into many, still imperfectly characterized, fragmentation products. In contrast to the generation of the initial free radicals this fragmentation is highly exergonic, virtually spontaneous and irreversible. In a biological system it is also wasteful and destructive, since there is no known mechanism whereby the energy set free can be used for biological work. If biological - i.e. enzymic oxidation sustains living cells like a finely tuned internal combustion engine, autoxidation is like dropping a lighted match into the petrol tank. In contrast to the lump of butter in the refrigerator, we spend a lifetime exposed to light and other forms of high-energy irradiation, incubating ourselves at 37°C and more or less in perpetual, if not always productive, motion. Since we do not on any massive scale go rancid until a few hours after death we must assume that we are actively preserved, that every cell in our bodies is shielded by an extremely efficient antioxidant mechanism or several antioxidant mechanisms (Barber & Bernheim 1967, Dormandy 1969). It is in this context that the role and importance of vitamin E must be assessed. Vitamin E in Vitro and in Animals There is no doubt whatever that when vitamin E is added to highly autoxidizable mixtures in vitro whether the mixture is a simple lipid emulsion or homogenized tissue - it behaves as an efficient antioxidant or preservative. In this respect it is not, of course, unique among biological compounds: under varying conditions many phenolic substances, trace elements and enzymes act as autoxidation inhibitors. Nor are the tocopherols as powerful as some of the synthetic preservatives widely used in the chemical and food industry (Witting 1975). But they are nontoxic and readily available. The question is: are they essential? If there can be no doubt about the antioxidant potency of the tocopherols in vitro, it is only marginally less certain that vitamin E deficiency is the specific cause of well defined syndromes in animals. Between species the clinical manifestations differ, ranging from sterility in sheep and rats to encephalomalacias in chicks, muscular dystrophies in cows and demyelination in horses (Wolbach & Bessey 1942, Pappenheimer 1943, Olcott & Mattill 1941). Strong experimental evidence suggests, moreover, that in all these syndromes the vitamin deficiency is associated with an increased susceptibility to autoxidation of the animal as a whole and in particular of the tissue or organ clinically affected. In short, the clinical manifestations and the biochemical abnormality are almost certainly causally linked. Even in animals, however, vitamin E is not the only anti-
