Biochemical effects of human injury

TIBS - April 1983

142 the title. For example, the oli- (oligomycin-insensitive) and oxi- (cytochromes oxidase) mutants in Saccharomyces have been mapped in mitochondrial DNA and the loci more recently sequenced2'L Thus the genes in mitochondrial DNA have been related to the proteins of certain subunits. The mere existence of their genes in mitochondrial DNA indicates that these proteins are important for the enzyme, and the inactivation of the enzyme by mutation nails down their role as subunits. Similar studies on the nuclear genes will no doubt show which of the cytoplasmic proteins are true subunits. With bacterial ATP synthase, an enzyme very similar to the mitochondrial one, genetic manipulation to over-produce the complex and the complete nucleotide sequence of the unc-operon (see Ref. 27) show clearly that there are eight different subunits in this enzyme. Five of these arrange the F1-ATPase as described above; the rest, a, b and c, assemble the F0 in a stoichiometry 1:2:10 (Ref. 27). Thus the E. coli ATP synthase is composed of a total of 22 polypeptides. It is intriguing how well the architecture of the ATP synthase is preserved in evolution. The corresponding enzymes from bacteria, chloroplasts and mitochondria have very much the same design. Comparison of the bacterial and mitochondrial enzymes would suggest that cytochrome oxidase has much more drastically changed its structure during evolution. A characteristic feature of respiratory complexes is their asymmetry, both struc-

tural and functional, with respect to the membrane sides. This makes them dissimilar to the soluble multi-enzyme structures or, for example, to viruses. In these there is usually a principle of symmetry that makes the structure, however complex it may be, comprehensible. The subunits in the complexes must, of course, be assembled into specific, closed three-dimensional structures. How does this happen? How do the twenty or so NADH dehydrogenase subunits find each other in a co-ordinated way to make up the enzyme? It seems likely that we shall soon understand how the organelle proteins are imported into a mitochondrion. The next stage of questioning will be even harder.

Acknowledgements I am grateful to Drs J. E. Walker and M. Wikstr6m for comments on the manuscript and to the Academy of Finland for general support.

References 1 Hatefi, Y. (1976) in Enzymes o f Biological Membranes (Martonosi, A., ed.), pp. 3-41, John Wiley, New York 2 Heron, C., Smith. S. and Ragan, C. I. (1979) Biochem J. 181,435--443 3 Marres, C. A. M. and Slater, E. C. (1977) Biochim. Biophys. Acta 462,531-548 4 Weiss, H. and Kolb, M. J. (1979) Eur. J. Biochem. 99, 139-149 5 Wikstr6m, M., Krab, K. and Saraste, M. (1981) Cytochrome Oxidase - A Synthesis, Academic Press, London 6 Ludwig, B., Prochaska, L. and Capaldi, R. A. (1980) Biochemistry 19, 1516-1523.

Biochemical effects of human injury Roger Smith and Dermot H. Williamson Accidental injury in man produces widespread biochemical changes. Recent work pin-points the importance o f alterations in protein synthesis in the regulation o f muscle mass. Severe accidental injury is a leading cause of death in young people in the Western world. Its immediate effects, such as fracture and blood loss, are obvious and mechanical; why, therefore, should injury be of interest to biochemists? First, because it leads to protein loss and muscle wasting which hinder recovery and survival; second, because the proper treatRoger Smith is at the Nuffield Department o f Orthopaedic Surgery, Nuffield Orthopaedic (.'entre, Oxford OX3 7LD, U.K. and Dermot H. Williamson is at the Metabolic Research Laboratory, Nuffield Department o f Clinical Medicine, Radcliffe Infirmary, Oxford OX2 6HE, U.K.

ment of trauma requires knowledge of its biochemical effects; and third, because accidental injury provides an opportunity to study the results of severe stress in man. When human beings are injured they must rapidly alter their metabolism to cope with impending immobility and starvation. Adequate and appropriate fuel has to be provided for the tissues whilst protein loss is minimized. These related and often conflicting requirements are controlled by the integrated action of hormones and by the effects of one metabolite upon another. This account will concentrate on the causes of post-traumatic protein loss after

, 1983.ElsevierSciencePublishersB k . Amsterdanl XXXX XXXX/~3/$Ult)tt

7 Schatz, G. (1979)FEBS Lett. 103,203--211 8 Poyton, R. O. and Schatz, G. (1975)J. Biol. Chem. 250, 752-761 9 Rubin, M. S. and Tzagoloff, A. (1973) J. Biol. Chem. 248, 4269-4274 10 Tanford, C. and Reynolds, J. (1976) Biochim. Biophys. Acta 457, 133-170 11 Merle, P., Jarausch, J., Trapp, M., Scherka, R. and Kadenbach, B. (1981) Bioehim. Biophys. Acta 669,222-230 12 Poyton, R. O. and Schatz, G. (1975)J. Biol. Chem. 250, 762-766 13 Kadenbach, B. and Merle, P. (1981)FEBS Lett. 135, 1-11 14 Saraste, M., Penttil~i,T. and Wikstr6m, M. (1981) Eur. J. Biochem. 115,261-268 15 Foster, D. L. and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015 16 Downer, N. W., Robinson, N C. and Capaldi, R. A. (1976) Biochemistry 15, 2930-2936 17 Merle, P. and Kadenbach, B. (1980) Eur. J. Biochem. 105,499-507 18 Kadenbach, B., Hartmann, R., Glanville, R. and Buse, G. (1982) FEBS Lett. 138, 236-238 19 Ludwig, B. (1980)Biochim. Biophys. Acta 594, 177-189 20 Winter, D. B., Bruyninckx, W. J., Foulke, F. G., Grinich, N. P. and Mason, H S. (1980)J. Biol. Chem. 255, 11408-11414 21 Deatherage, J., Henderson, R. and Capaldi, R. A. (1982)J. Mol. Biol. 158, 487-500 22 Fuller, S. D., Capaldi, R. A. and Henderson, R. (1982) Biochemistry 21, 2525-2529 23 Frey, T. G., Chan, S. H. P. and Schatz, G. (1978) J. Biol. Chem. 253, 4389--4395 24 Capaldi, R. A., Darley-Usmar. V., Fuller, S. and Millett, F. (1982)FEBS Lett. 138, 1-7 25 Casey, R. P., Thelen, M. and Azzi, A. (1980) J. Biol. Chem. 255, 3994-4(]00 26 Tzagoloff, A. (1982) Mitochondria, Plenum Press, New York 27 Walker, J. E., Saraste, M. and Gay, N. J. (1983) Bioehim. Biophys. Acta (in press)

accidental injury (uncomplicated by bums or sepsis) and the factors which appear to influence it. It is necessary to consider briefly the known biochemical effects of trauma and to appreciate some important points about injury research. To elucidate the effects of human injury we need to amplify the information gained from animals by direct studies on man. Such investigations are often done in the midst of life-saving activities, and the results cannot have the clarity expected of elective experiments. Further, trauma is often associated with partial starvation, loss of mobility, and sepsis, so that the biochemical effects cannot be attributed to injury alone. Within such limitations recent investigations have yielded information of considerable interest to the biochemist and to the practising physician.

The main biochemical effects of injury After injury there is a short phase (24 h) when body temperature and energy production fall and endogenous metabolic fuels, particularly glucose and fatty acids, are

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mobilized. During this phase, necrobiosis and death may occur; in survivors this is followed by a period when metabolic processes accelerate, but protein loss increases. These catabolic processes are eventually replaced by anabolism and recovery over a period of days, weeks or even monthsL The biochemical changes associated with injury clearly differ with time, and should be interpreted accordingly. The overall biochemical effects of injury are best understood from work on human starvation 2 where the liver can be regarded as a central transformer (Fig. 1) between the supply and utilization of fuel by the tissues, the direction and flux of which is mediated by the endocrine system. In short-term starvation the liver converts amino acids (predominantly alanine) 3 released from muscle protein to provide glucose for the nervous system, particularly the brain; and also converts fatty acids derived from adipose tissue stores to ketone bodies. The use of ketone bodies by the brain as an alternative to glucose may be important after injury (see Fig. l). The effects of injury resemble and include those of short-term starvation but they differ in the magnitude of the hormonal changes. Injury increases the plasma concentrations of cortisol, glucagon, catecholamines and other stress hormones while that of insulin is inappropriately low for the prevailing glucose concentration. This tips the balance strongly in favour of catabolism and promotes gluconeogenesis, lipolysis and proteolysis, leading to muscle wasting and the utilization of fat. Protein loss after injury The loss of nitrogen is normally greatest between the second and fourth days after injury and is proportional to its severity. It is also greater in previously well-nourished young subjects than in the poorly nourished elderly, and is prolonged and exaggerated by sepsis and burns. For many years after the pioneering observations of Cuthbertson4, it was assumed that the protein was lost from skeletal muscle, and that this loss was due to an increase in rate of catabolism (proteolysis). Whilst the first assumption seems to be true (in man at least), the second is probably not. Since nitrogen excretion represents the balance between the rates of protein synthesis from amino acids and the rate of catabolism (Fig. 2) there are several diifferent situations in which net loss of protein could occur (Fig. 3). A number of studies on the effects of feeding, fasting, exercise" and immobilization7, have all shown that the main determinant of muscle mass and protein turnover is the rate of protein synthesis,

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(a) proteolysis, (b) gluconeogenesis, (c) lipolysis and (d) ketogenesis. The brain (and nervous tissues) utilize ketone bodies after injury and starvation (interrupted arrow).

which appears to be far more labile and sensitive to change than proteolysis. It should come as no surprise that after elective surgery such as skin grafting 8, abdominal operations 9 or total hip replacement l°, synthesis is reduced whereas catabolism remains unchanged (Fig. 3), but to attribute this to the direct effect of the operation is probably incorrect. Certainly after abdominal operation, food intake is drasticaUy reduced, and after any major opera-

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tion there is loss of mobility, both of which decrease the rate of protein synthesis. After total hip replacement in man the reported changes in synthesis and catabolism depend on changes in nutrition, and few observations have been made in the injured human where the pre-traumatic intake has been completely maintained. In rats it has been directly shown" that after femoral fracture, protein synthesis is increased in all tissues (excluding lung)

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144 provided that adequate nutrition continues, whereas post-operative feeding with 5% dextrose (as often occurs in man) reduces the fractional synthetic rate of all tissues. In rats, the fractional rate of protein synthesis in the gastrocnemius muscle is reduced by a 7-day period of hind limb immobilization, and when mobility is resumed returns to control values within 6 h (Ref. 7). Four days after immobilization ceased, synthesis rates doubled and were further increased by exercise. These findings emphasize that protein loss after immobility is attributable to a reduction in protein synthesis, and suggest that the mechanism controlling protein synthesis in skeletal muscles rapidly responds to major changes in their contractile activity. Equally important is the severity of injury and its effects on protein synthesis and catabolism. Clague TM has analysed the existing data on protein turnover after injury. Where the nutritional intake remains the same, both protein synthesis and catabolism increase along with trauma severity, but catabolism increases more than synthesis (Fig. 3d). In the more usual postoperative state - where nutrition is poor synthesis is below normal; but with an increase in the severity of injury, breakdown and synthesis increase disproportionately (Fig. 3c). Finally if an increased intake is given post-operatively, synthesis rate increases towards the elevated catabolic rate (not shown).

3-Methylhistidine The major source of protein loss after injury appears to be voluntary muscle. Clinically this is suggested by the degree of muscle wasting; and biochemically by the increased excretion of creatine and intracellular ions such as zinc and magnesium. However, the fractional contribution of muscle protein to total protein loss after injury in man is not known, and studies on animals, where there is initially a considerable loss of liver protein, may not be applicable to man. Until recently no attempt had been made to measure muscle synthesis directly in man, but it is possible to estimate the catabolic rate of myofibrillar protein from the excretion of 3- methylhistidine (3-MeH) (Fig. 2) 13. This amino acid comes from the post-translational methylation of peptidebound histidine in actin and myosin; since 3-MeH cannot be reutilized in protein synthesis and is excreted unchanged in the urine without further metabolism, its excretion rate indicates the rate of myofibrillar protein breakdown, provided that the diet is meat-free ~4. When expressed in relation to creatinine, itself proportional to muscle mass, 3-MeH excretion can be regarded as

11BS - A p r i l 1 9 8 3

quently excreted more 3-MeH and nitrogen in the urine than the hyperketonaemic subjects. There was no clear difference between individuals who reacted in these two different ways, although it later appeared that the normoketonaemic patients were (c) (d) often the most severely injured, and study of a larger miscellaneous group of patients tended to confu-m these findings 17. In normal subjects, starvation leads to hyperketonaemia, and this response appears to be suppressed in some injured patients TM. If insulin concentrations were higher after more severe trauma this would tend to decrease lipolysis, circulating fatty acid concentrations and subsequent S C S C S C S C ketonaemia. However, the relationship (cI) (b) (c) (d) between fatty acid and glycerol concentraFig. 3. Proposed and described variations in the tions (as an index of lipolysis) and ketonerates of protein synthesis (S) and catabolism ((9 body concentrations is not close, and the after surgical operation or accidental injury. Before degree of ketonaemia is probably more operation, (a) S and (" are equal. After operation, net protein loss could occur with (b) an increase in determined by hepatic events. Research on C only, (c) a decrease in S on(v, or (d) a disproporanimals suggests that the conversion of tionate increase in C over S. These alternatives are fatty acids to ketone bodies in the liver is discussed in the text. The upper diagram is modified suppressed by at least two of the stress horfrom M. B. Clague (Ref. 12). mones - adrenaline and vasopressin t8'2°, a measure of the fractional myofibrillar which increase after injury. This is in addicatabolic rate. The interpretation of 3-MeH tion to the control of ketogenesis by excretion data is not without difficulties, changes in the glucagon/insulin ratio. The especially in animals where non-muscle relationship between normoketonaemia sources appear to be important, but it is and increased nitrogen loss may thus be interesting to find that after an elective spurious, both resulting from the severity of orthopaedic procedure such as a total hip the injury. Alternatively it may be an inbuilt replacement, 3-MeH excretion hardly individual characteristic. There are several ways in which hyperalters, which is compatible with the unaltered post-operative breakdown rate of total ketonaemia might reduce protein loss. One body protein, as determined isotopically TM. way is that ketone bodies might be used as a In contrast, where accidental injury is respiratory fuel and thus minimize the need severe, or where tourniquets have been to catabolize protein~L Alternatively, used (as in knee replacement) ''~, the ketone bodies may have a direct effect on increased excretion of 3-MeH may be proteolysis, or on the catabolism of amino acids (e.g. those with branched chains), derived from irreversibly damaged muscle. thus preventing depletion of the intracellular pool. The role of ketone bodies By necessity, pre- and post-injury studies on protein turnover can only be Branched-chain amino acids One biochemical change after severe done under the controlled conditions of elective operations. However, the most accidental injury is a considerable increase severe protein loss follows accidental injury in the circulating concentrations of the and the results of biochemical measure- branched-chain amino acids, leucine, ments on such patients, mainly victims of isoleucine and valinez2. In contrast to other road-traffic accidents, have been of particu- amino acids, the branched-chain amino lar interest; inter alia they have suggested acids are initially metabolized in muscle, that the degree of protein breakdown may and their circulating concentration may be related to the initial changes in ketone- increase when there is net muscle breakdown or when their uptake into muscle is body metabolism. An early study ~'~showed that within 24 h blocked. Thus the concentrations of of accidental injury the circulating concen- branched-chain amino acids in the blood tration of ketone bodies (acetoacetate and reflect events in muscle and they are 3-hydroxybutyrate) were either increased increased, for example, in normo(hyperketonaemia) or normal (nor- •ketonaemic injury. Work which has received considerable moketonaemia). The normoketonaemic subjects had higher initial blood concentra- attention suggested that, in vitro, leucine tions of glucose, alanine and branched- itself was capable of increasing proteinchain amino acids (see below), and subse- synthesis rateZa: this has not been confirmed

145

T I B S - A p r i l 1 983 in vivo 24. Whether or not synthesis can be

in Duchenne muscular dystrophy3°. Whereas the authors had no reason to doubt the reduced rate of muscle-protein synthesis found by direct measurement, they found it difficult to explain the increase in 3-MeH in this disorder and considered that it might come from sources other than skeletal muscle. However, the predominantly muscular origin of 3-MeH in man is supported by observations that 3-MeH excretion was only 25 % of normal in a severely wasted Clinical considerations patient with no detectable muscle sl. In theory, protein loss could be preIf, as seems likely, protein balance is vented, either by increasing the rate of determined largely by changes in protein synthesis or by reducing the rate of break- synthesis, and if the major protein involved down or both. Clinically, this is most often is that of muscle, the key question is: what attempted by providing adequate nutrition controls muscle-protein synthesis (both and by maintaining mobility. Where myofibrillar and sarcoplasmic)? The rapid neither of these can be achieved and where muscle wasting caused by immobilization nitrogen loss is severe, the biochemical emphasizes one important factor. We need studies just described suggest two further to know how mechanical forces stimulate possibilities, namely, the infusion of ketone muscle synthesis, and how the use of musbodies to decrease protein catabolism or of cles increase their mass. Is it entirely a probleucine to increase protein synthesis. In lem of synthesis; or does exercise also practice, these approaches have not proved reduce muscle breakdown (and 3-MeH clinically useful. However, in long-term excretion) as the results of Rennie et al.'; starvation the administration of 3-hy- imply? An important contribution may be droxybutyrate reduces nitrogen loss 2'~ that of blood flow, particularly in the two and the excretion of 3-methylhistidine27; extremes of immobilization and exercise. and the same may be true after injury. It has The way in which mechanical stress influalso been suggested that the infusion of ences the activity of the muscle-forming amino acid spares protein more effectively cells is as obscure as its effect on the cells of than the infusion of glucose because another mechanically important tissue, ketonaemia is not abolished and insulin sec- bone. There are several useful analogies retion is not stimulated. Recently, the use of between these tissues which have both a solution enriched with branched-chain mechanical and biochemical functions. amino acids appeared to decrease post- First, their mass is increased by use; operative nitrogen loss 28. second, synthesis and breakdown (or, for bone, formation and resorption) are closely Problems of protein synthesis coupled; and third, the nature of the link In man, the overall rate of protein syn- between cellular activity and mechanical thesis may be estimated using a variety of stress is unknown. labelled amino acids which give similar Such a link between the use of muscle results2L Where it is possible to examine a and its rate of protein synthesis could be variety of tissues, as in animals, the indi- biochemical. Thus it has been suggested vidual synthetic rates of the proteins they that branched-chain amino acids produced contain can also be measured. In man, this during protein breakdown can stimulate the can only be done on biopsy material, and formation of muscle protein; or that exerpreliminary results using 13C-labelled cising or stimulated muscle produces leucine 5 show that the marked increase in somatomedin-like growth factors. Cerwhole body synthesis on feeding largely tainly it appears that insulin can promote reflects a doubling of protein synthesis in protein synthesis, and here the wellmuscle. According to these results muscle established resistance to insulin after injury in fed man contributes to more than half of and the changes in hormone delivery the whole body protein synthesis. In a associated with alterations in blood flow detailed study of the effect of exercise on may play a role in the net loss of muscle protein turnover'; it was found that total pro- protein. tein synthesis decreases and total protein breakdown increases; however, measure- Future ments of 3-MeH in muscle, plasma and Not all severe injury and its complicaurine suggested that the fractional rate of tions can be prevented, If we accept that myofibrillar breakdown was decreased. excessive post-traumatic loss of protein preSome of the difficulties in interpreting data judices survival, we can try to suppress the relating to muscle-protein synthesis and metabolic response to injury or to selecbreakdown are illustrated by a similar study tively reduce protein utilization. The increased appears to have little relevance after injury since both the extracellular and the apparent intracellular concentration of the branched-chain amino acids are already high 2s. Further, the increase in the intracellular concentration is proportionately greater than that in the plasma, so that the gradient between the inside and outside of the cell is increased.

former is proportional to the severity of injury and the hormonal response which it evokes, and there is some evidence that the response may be suppressed by the use of synthetic opiates or by extensive epidural anaesthesia; in this respect we have much to learn about the role of endorphins and enkephalins in injury. The ways of manipulating the biochemical response to injury remain largely empirical, but recent research has emphasized the central importance of protein synthesis in determining protein mass. We have indicated some of the ways in which such synthesis may be regulated. We now need to establish how protein synthesis and proteolysis are controlled in the hope that this will lead to more effective methods of maintaining the protein mass after trauma.

References 1 Kinney, J. M. (1977) in Nutritional Aspects of Care in the Critically 111 (Richards, J. R. and Kinney, J. M.. eds), pp. 9.5--133, Churchill Livingstone,Edinburgh 2 Cahill, G. F. (1970) New Engl. J..~4ed. 282, 668-675 3 Snell, K. (1980)Biochem. Soc. Trans. 8, 205-213 4 Cuthbertson,D. P. (1976) in Metabolism and the Response to Injury (Wilkinson,A. W. and Cuthbertson, D., eds), pp. 1-34, Pitman Medical, London 5 Rennie, M. J., Edwards, R. H. T., Halliday, D., Matthews, D. E., Wolman, S. L. and Millward, D. J. (1982)Clin. Sci. 63,519-523 6 Rennie, M. J., Edwards, R. H. T., Krywawych. S., Davies, C. T. M., Halliday, D., Waterlow, J. C. and Millward, D. J. (1981) Clin. S¢i. 61, 627-639 7 Tucker, K. R., Seider, M. J. and Booth, F. W. (1981)J. Appl. Physiol. Respir. Environ. Exercise Physiol. 51, 73-77 8 Kien,C. L., Young, V. R., Rohrbaugh,D. K. and Burke, J. F. (1978)Metabolism 27, 27-34 9 O'Keefe, S. J. D., Sender, P. M. and James, W. P. T. (1974) Lancet ii, 1035-1037 10 Crane, C. W., Picou, D., Smith, R. and Waterlow, J. C. (1977)Brit. J. Surg. 64, 129--133 11 Stein, T. P., Leskiw, M. J., Wallace, H. W. and Oram-Smith,J. C. (1977)Amer. J. Physiol. 233, E348-E355 12 Clague, M. B. (1981) in Nitrogen Metabolism in Man (Waterlow, J. C. and Stephen, J. M. L., eds), pp. 525-539, AppliedSciencePublications, Londonand New Jersey 13 Young, V. R., Haverberg, L. N., Bilmazes, C. and Munro, H N. (1973) Metabolism 22, 1429-1436 14 Elia, M., Carter, A., Bacon, S., Winearls, C. G. and Smith, R. (I 981 ) Brit. Med. J. 282, 351-354 15 Threlfall, C. J., Stoner, H. B. and Galasko, C. S. B. (1981)J. Trauma. 21,140-147 16 Smith, R., Fuller, D. J., Wedge, J. H., Williamson, D. H. and Alberti,K. G. M. M. (1975)Lancet ii, 1-3 17 Stoner, H. B., Frayn, K. N., Barton,R. N., Threlfall, C. J. and Litlle, R. A. (1979) Clin. Sei. 56, 563-573 18 Birldmhn,R. H., Long, C. L., Fitkin, D. L., Busnardo, A. C., Geiger,J. W. and Blakemore,W. S.

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(1981)J. Trauma 21,513-519 19 Sugden, M. C.. Watts, D. 1. and Marshall, C. E. (1982) Biochem. J. 204, 749-756 20 Williamson,D. H., Ilic, V., Tordoff,A. F. C. and Ellington, E. V. (1980) Biochem. J. 186, 621~fi24 21 Robinson. A. M. and Williamson, D. H. (1980) Physiol. Rev. 60, 143-187 22 Wedge, J. H.. De Campos, R., Kerr, A., Smith, R., Farrell, C. R., llic, V. and Williamson, D. H. (1976) ('/in. Sci. Mol. Med. 50, 393-399

23 Buse, M. G. and Reid, S. S. (1975)J. Clin. Invest. 56, 1250-1261 24 McNurlan, M. A., Fern, E. B. and Garlick, P. J. (1982 ) Biochem. J. 204,831-838 25 Askanazi, J., Elwyn, D. H., Kinney, J. M., Gump, F. E., Michelsen,C. B. and Stinchfield, F. E. (1978)Ann. Surg. 188. 797-803 26 Sherwin R. S., Hendler, R. G. and Felig, P. (1975)J. Clin. Invest. 55, 1382-1390 27 Pawan,G. L. S. and Semple,S. J. G. (1983)Lancet i, 15-17

28 Cerra. F. B., Upson, D., Angelico,R., Wiles, C.. Lyons, J., Faulkenbach, L. and Paysinger. J. (1982)Surgery 92, 193-199 29 Halliday, D. and Rennie, M. J. (1982)(7in Sci. 63,485--496 30 Rennie, M. J., Edwards, R. H. T., Millward. D. J., Wolman,S. L., Halliday, D. and Matthews, D. E. (1982)Nature (London) 296, 165-167 31 Ailing, E. G., Bernhardt, W., Janzen, R. W. C. and Rothig. H. J. (1981) Biochem. J. 2(XL 449~-52

50 Years Ago From ignose to hexuronic acid to vitamin C R. ElwynHughes In 1840 George Budd, a much neglected prepared several grams of crude calcium figure in the history of nutritional thought, ascorbate during the nineteen twenties, but wrote that scurvy resulted from a'lack of an can-ied his work no further because of lack essential element which it is hardly too san- of financial support s . guine to state will be discovered by organic The purest of these early 'concentrates' chemistry or the experiments of physiol- still contained much impurity 4, and it was ogists in a not too distant future '1. not until 1932 that Waugh and King pubBut little happened to fulfil Budd's lished their paper 'Isolation and identificaprophecy until the beginning of the twen- tion of vitamin C' which included a tieth century, when the work of Lunin and photograph of vitamin C crystals 9. (The Gowland Hopkins led to a more general 'antiscorbutic factor' had, by now, on acceptance of the concept of accessory food Drummond's suggestion, became known as factors. In 1912 Funk introduced his 'vit- 'vitamin C'. ) amine hypothesis' in which he attributed These early attempts to isolate and scurvy to the absence of an 'anti-scurvy vit- characterize vitamin C were paralleled by amine '~. By the 1920s the 'anti-scurvy vit- two separate, but nevertheless highly relevamine' was known as 'C Factor' or 'the ant, developments in other areas. Tillmans antiscorbutic substance ':~, and the period and Hirsch, German government chemists, 1920-1930 witnessed a series of attempts to extensively studied the capacity of lemon juice preparations to reduce the redox dye isolate the pure principle 4. Foremost in these early studies was S. S. 2,6-dichlorophenolindophenol and they Zilva at the Lister Institute, London. The claimed that the reducing power of their essential feature of Zilva's procedure was preparations was always in proportion to precipitation of the antiscorbutic factor with their antiscorbutic potency. Zilva disagreed basic lead acetate after removal of the bulk with this and claimed that he had frequently of the organic acids with calcium carbon- obtained fractions of high reducing power ate. Zilva applied his technique to a vari- which were devoid of antiscorbutic potency ety of sources such as lemon juice and and he explained the German results by swede tissue and succeeded in concentra- suggesting that reducing substances ting the antiscorbutic factor some 200-300 'protected the antiscorbutic principle' but times:"". Other workers were similarly were not necessarily identical with it. Zilva occupied, notably Bezssonoff in France appears to have been temporarily diverted and King in the USA 7-8. King has stated that from his main endeavour at this point by a during this period 'many investigators had somewhat unnecessary attempt to disprove abandoned or failed to publish their work the German claims 1°. The other significant development was for various reasons' and he referred specifically to Karl Link of Wisconsin who had Szent-Gyorgyi's isolation of hexuronic acid. Albert Szent-Gyorgyi, a Hungarian R. E. Hughes is at the Department o f Applied Biol- biochemist working on plant respiration ogy, University o f Wales Institute o f Seience and systems at Groningen, Holland, became interested in a reducing compound present 7~'chnology, ('ardiff ('Fl 3NU, U.K. ,

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in his preparations. Gowland Hopkins invited him to Cambridge to extend his studies. Whilst there in 1927, SzentGyorgyi isolated the 'Groningen reducing agent' in a crystalline form from oranges, lemons, cabbages and adrenal glands H. He proposed to name his crystalline sample 'ignose' - thus indicating its apparent relationship to sugars whilst at the same time underlining his ignorance of its true nature. But Harden, the editor of the B i o c h e m i c a l J o u r n a l at the time, according to SzentGyorgyi 'did not like jokes and reprimanded me'. A second suggestion 'godnose' was judged to be equally unacceptable. Szent-Gyorgyi finally agreed to accept Harden's suggestion 'hexuronic acid' - ' s i n c e it had 6 Cs and was acidic '82. Hexuronic acid was a strongly reducing compound. So too, according to Tillmans and Hirsch, was the antiscorbutic substance (vitamin C). The suggestion that hexuronic acid and vitamin C were in reality the sarae substance appeared in print in 1932, in papers by both Tillmans and Hirsch 83 and by Waugh and KingL There is little doubt, however, that the idea had been first mooted some years previously. Who exactly first made the suggestion is somewhat u n c l e a r - e v e n the main participants in the drama were apparently later uncertain and confused. According to King it was E. C. Kendall in 19298; according to Gowland Hopkins (reported by King) it was L. J. Harris in 19288; Harris himself in 1953 pleaded forgetfulness 84 - and he had, in any case, already attributed the idea to Tillmans and Hirsch2; and Hirst (a member of the team involved in chemical studies on the structure of vitamin C) named Waugh and King ~5. Gowland-Hopkins had already, in 1928, sent a sample of Szent-Gyorgyi's hexuronic acid to Zilva for comments on its vitamin C potency. According to King, Gowland Hopkins was disturbed because Zilva had never reported the evidence of his tests but had only told them (i.e. the Cambridge biochemists) that the product was not vitamin C 8. By 1932 however, the evidence in