vitamin c its chemistry

7/22/2019 Vitamin C Its Chemistry

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VITAMIN CI ts Chemistry and Biochemistry


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Royal Society of Chemistry Paperbacks

VITAMIN CIts Chemistry and Biochemistry

MICHAEL B. DAVIESJOHN AUSTINDAVID A. PARTRIDGEDepartment o f Applied ScienceAnglia Polytechnic, Cambridge


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ISBN 0-85186-333-7A catalogue record for this book is available from the British Library

he R oyal Society of Chemistry 1991All Rights ReservedNo part of this book may be reproduced o r transmitted in any f o r mor by any means - graphic, electronic, including photocopying,recording, taping or information storage and retrieval systems-without written permission f r o m Th e Royal Society o ChemistryPublished by The Royal Society of Chemistry,Thomas Graham House, Science Park, CambridgeCB4 4WFTypeset by Servis Filmsetting Limited, Manchesterand printed by Th e Bath Press, Lower Bristol Ro ad, Bath


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ForewordbyProfessor M. Stacey CBE, FRS, DSc., C.Chem., FRSCThe steady output of publications on the chemistry, biochemistry, andmedical applications ofthe still mysterious vitamin C has kept alive mymemory ofthose exciting days of60 years ago. I t was the time ofchangefrom the test-tube chemistry of organic chemical structure sobrilliantly practised by Sir Robert Robinson and his pupils to theapplication of physical techniques such as ultraviolet spectroscopy,optical rotatory dispersion and X-ray crystallography. Biochemistrywas just beginning to emerge as a separate science due to men likeProfessors Gowland Hopkins, A . Harden, and Harold Raistrick.

It is not always appreciated that Professor Haworth owed much ofhis success in the carbohydrate field to the development ofmicroanaly-sis by Pregl. In 1925 H. D. K . Drew was sent from Birmingham toPrague to learn the techniques and to bring back the famousKuhlmann balance and other apparatus. 5 milligrams was all that wasnow needed for the analysis of a methylated sugar and this was ofenormous help with the meagre supplies of vitamin C. Anotherinvention of Haworth was the formation of a team, which he called asyndicate whereby a crash programme could be carried out. Eachresearcher had to drop his own research topic and concentrate on aparticular stage in the synthesis or degradation The tension in thelaboratory was due to the fact that the Professors knew that their rivalsin Europe, notably Professors Karrer, Reichstein, and Micheel, hadmaterial and were coming very close to the correct structure. Haworthanticipated the potential value of X-ray crystallography and in 1928had recruited C. G. Cox (now Sir Gordon) to work on carbohydratestructures. With vitamin C, Cox quickly showed that the carbon andoxygen atoms lay in the same plane, confirming that it was, indeed,from his earlier studies, a carbohydrate.

In the summer of 1933I spent six weeks in the laboratories ofBDH inGraham Street, London, exploring with their chemists the possibility

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vi Forewordof an industrial-scale process for the vitamin and indeed the halfki logram we made is s ti ll in ou r museum How ever , a long cam e theremarkable five-stage synthesis of Reichstein a nd Grussner , whe re thekey s tage i s the b io log ica l ox ida t ion of~sorb i to lo L-sorbose. O n emust pay t r ibute to the br i l l iant chemists a n d chem ical engineers of th eRoche Co m pan y who put this process o n to the enormous industr ialscale an d w here the process still holds its own . A few years ag o I h a d t heg re at thrill of seeing a silo holding 30 tons of glucose a nd mo untains of2 cwt polythene bags of beautifully crystall ine vitamin C awai t ingexport . Product ion continues to grow.

For th is book the authors have ranged far an d wide to br ing u p toda te a ll recent advances concern ing th e v itamin . T h e chap ter on thehistory of scurvy is fascinat ing. O n e wonders why the conquest of th isdisease took so long when the answ er was star ing the doctors in th eface, for so little of the vita m in is needed to prev ent t he disease. All th echa pters a re well researched a n d struc tures set forth in a c lea r mann er .O f grea t im por tance an d in terest is the s tudy o f t h e medica l aspects.

If al l the claims for the biological functions an d cu rative prope rties ofthis simple yet mysterious molecule are true, then surely we havediscovered something ne ar t o the Elixir of Life so long sought by theancient a lchemists How ever , the authors gave us a warn ing tha t insome cases the vi tamin ca n be harmful , especially in megadoses. T h ebiological systems of humans do show wide var iat ions and what issauce for the goose is not alway s sauce for the g and er

I t has been a pleasure to read the manuscr ip t and I warmlycom m end it , especially to stude nts seeking inspiration.M aur ice S taceyBirmingham 1991


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Acknowledgements

N o book is ever wri t ten without the combined help of a large num be r ofpeople. W e would like to tha nk o u r wives and families for puttin g u pwi th us dur ing the wr i t ing and Margare t Haw, who typed themanuscript, for her patience in deciphering our almost i l legiblescr ibbles and for not complaining as we continued to al ter themanuscr ipt even at the very last moment. b 'e would like toacknowledge the help of those who gave their t ime to read themanuscr ipt and provide very valuable comments: Dr Roger Mor-t imer , D r I an Fiddes and Paul Lowing; and the he lp of ou r colleaguesat work, pa rticularly Joh n H ud son for valua ble discussions on thehistorical aspects. We are also grateful to Dr Edwin Constable forvaluable advice on the pre parat io n of diagrams.Special mention must be given to Professor hla uric e Stacey, CB E,FRS, whose first-hand know ledge of the events surro un din g aspects ofvi tamin C in the 1930s provided us with a fascinating insight intodevelopments and personalit ies at that t ime.I n ad ditio n, the advice and assistance of th e following ind ivid ual s isgratefully appreciated: P eter Carter (Scher ing L t d ) ) Clive Co ndu itan d D avid Godfrey (Ro che Products Lt d) , Pau l Skel ton (Univers i tyChemical Laborator ies, Cambridge) and Bruce Robertson andCarol ine Robinson (Media Product ion, Anglia Polytechnic) .

Finally, we must acknowledge the countless \vorkers in vitamin Cchemistry a nd biochemistry over the past century o r so and the manyautho rs of the hundreds o fpa pe rs a n d books which we have consultedin ou r work on vitamin C chemistry and in the prep arat ion o f this book.If we have forgot ten to mention anyone who has helped weapologise, b u t are , nevertheless, still gra tefu l. : h y mistakes in this bookare ofcourse ent irely du e to the authors, though we have str iven to keepthem to a n abso lu te min imum


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ContentsForeword by Professor M. Stacey CBE, FRSAcknowledgementsChapter 1IntroductionChapter 2History of Vitamin C and I t s Role in the Preventionand Cure of ScurvySuggested Causes of ScurvyJames LindSir G ilbert BlaneScurvy on L andExplorat ion of the Arctic an d A ntarct icScurvy in C hi ldrenT h e Even ts Lead ing u p to the Discovery of Vitamin CChapter 3Discovery and Structure of Vitamin CIsolation

Stru cture E lucidat ionStru ctura l Detai l by In strum ental M ethods

Chapter 4Synthesis, Manufacture, and Further Chemistry ofVitamin CSynthet ic M ethods for L-Ascorbic Acid

Further Chemis try of L-Ascorbic A cidDehy droascorbic Acid an d i ts Derivatives

Chapter 5Biochemistry of Vitamin C

Biosyn thesis

V

vii1

71 1141617202122

26263036

48485766

7475

ix


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ContentsXVitamin C in FoodOxidation and HydroxylatiollReducing PropertiesElectron TransportTissue LocationIntake, Excretion, and Catabolism

Chapter 6Medical Aspects of Vitamin CDeficiency

Maintenance o f HealthTherapeutic Use'Toxic Eff'ec sVeterinary Use

Chapter 7Inorganic and Analytical Aspects of Vitamin CChemistryAnaly tical Chemistry

Inorganic ChemistryRedox Reactions of Vitamin CAscorbate Oxidase

BibliographySubject Index

808290919494

9798

100103111113

115115123128145147149


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Chapter 1Introduction

Everyone has heard of vi tamin C. T he re can be few s imple organicmolecules which have excited such universal interest. At least p ar t ofthe reaso n for this has been the g ene ral intere st in t he beneficial effectsof all vi tamins and o the r t race substances on hu m an hea lth w hich hasdeveloped in recent years along with concern on the effects of othersubstances, particularly additives, on those who consume foodcontaining them. We know that v i tamins are essent ial to our well-being and because of this they ha ve excited an interest an d curiositywhich has resulted in many of them being attr ibuted with disease-healing and health-giving properties which they could not possiblyhave , Vi tamin C has itselfbeen said to have almost magical propertiesby som e writers an d it is useful to get a picture of the chemistry a n dbiochemistry of th is en igmat ic com pou nd.

V i t a m i n C is different. I t is different from the o ther v itamins a n d weshall see in th e course of this book th at i ts chemistry a n d biochem istrysingle i t out am ongst molecules in m an y im po rta nt ways, Vitam in C isubiqui tous. I t is found throughout the plant and animal kingdoms,wh ere i ts roles ar e often not known o r are poorly understood. T h esyn thetic vitam in is very w idely used as a food add itive a n d thereforeh as a n E num ber (E3 00) . However , un l ike m any o ther addi t ives , fewpeople would object to its presence in foods. T h er e is no d o u bt th at i tsanti-o xida nt p roperties confer stabili ty o n foods to which it has beena d d e d .

V i t a m i n C has been the subject offreq uen t controversy, even beforei ts natu re h ad been establ ished. I ts role (as a constituent of f rui ts an dvegetables) in th e cure an d p revention ofscurvy was widely de ba ted forhund reds of years. I ts very existence was dou bte d by m an y even asrecent ly as the the beginning of the twentieth century. There werequa rrels over w ho was t he first to discover i t . Even today there is muchcontroversy abo ut the exac t ro le of the v i tamin in h um an hea l th a ndthere i s no t even agreement over the amount of the v i tamin which

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2 Chapter 1needs to be consumed for optimum well being, with various authoritiesrecommending amounts varying from 30 mg to 10 g per day. The rolein the relief of cold symptoms, in the improvement of quality of life forcancer patients and in other medical areas are all topics for intensediscussion. The biochemistry of L-ascorbic acid in mammals is verypoorly understood, so that it is not even clear what the biochemical roleof the vitamin is in such systems. Although the chemical structureof L -ascorbic acid has been unequivocally established by single crystal X-ray diffraction, the structure of its very important two-electronoxidation product, dehydroascorbic acid, has not been finally estab-lished, since it has not yet proved possible to isolate crystals, or indeedthe pure compound, as a solid.

Vitamin C is chemically the simplest of the vitamins and for thisreason was among the first to be isolated, characterised, and purifiedand to have its structure determined. *More vitamin C is producedindustrially than any other vitamin, or indeed all the other vitaminsput together. It is one of the few pure chemical compounds which istaken routinely by human beings in gram quantities (a possiblechallenger is sugar). It appears to have no harmful effects even in theselarge amounts and it is a medicine which it is a pleasure to take,especially in the form of fruit or vegetables.

It may be thought that the chemistry of th is simple molecule wouldno longer hold any surprises after the vast amount of research that hasbeen carried out over the years. However, conferences on aspects ofvitamin C chemistry still attract large numbers of workers in the fieldand new aspects ofthe chemistry are always being revealed. The reasonfor the continued interest in the chemistry of L-ascorbic acid lies in thefact that despite it being such a simple molecule, its ene-diol structureprovides it with a highly complex chemistry. Thus it has a verycomplicated redox chemistry involving comparatively stable radicalintermediates which is heavily modified by the acidic properties of themolecule. It has been known for many years that L-ascorbic acid iseasily oxidised by dioxygen. Although the first product ofthis process isdehydroascorbic acid, which still has antiscorbutic properties, thefurther oxidation by oxygen produces compounds which are notreadily converted back to L-ascorbic acid, and the vitamin is effectivelydestroyed. The mechanisms of the reactions involved are still largelyunknown, although they have been widely studied. There has beenmuch recent work on the interactions of vitamin C with metal ions,particularly transition metal ions. This has unearthed a rich vein ofchemistry involving L-ascorbic acid as both a redox companion and as


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Introduction 3a complexing agent; indeed the reaction of L-ascorbic acid with oxygenand other oxidising agents is catalysed by transition metal ions,especially copper(II), so that sometimes solutions are stabilised by theaddition of EDTA, which complexes the metal ions and arrests thecatalysis. It appears that vitamin C may not always act alone in itsbiochemical processes, but may act synergistically with other sub-stances, of which vitamin E may be a typical example.

Research into vitamin C chemistry appears to have reached a kindofsteady state. In the years 1969, 1979, and 1989, there were about thesame number ofpapers published each year on aspects ofthe chemistryof L-ascorbic acid. Thus the extent of the work on this compound hasbeen remarkably constant over the past twenty years and there is nosign of a diminution of interest yet.

The development of analytical techniques to detect and determinevitamin C has been crucial in the understanding of the presence andstability of the compounds in nature. At first, biological techniqueswere used and these gradually became replaced by chemical methodswhich were more sensitive, more selective, and easier to carry out.Today the analysis is largely centred around the use of high-performance liquid chromatography and there are many successfulmethods available. However, the technique is still limited by the factthat detection of dehydroascorbic acid in the presence of L-ascorbicacid still has a comparatively low sensitivity by virtually all detectiontechniques. This makes the determination of amounts of dehydro-ascorbic acid in plants and animals much more difficult than for L-ascorbic acid. The same applies to further oxidation products andthere is a need for further work in the development of detectionmethods for these compounds and to investigate the extent and kineticsof oxidation of L-ascorbic acid in fruits and vegetables by determiningthe amounts of all oxidation products and the oxidation pathways bywhich these products are formed.

We are familiar with the use of ascorbic acid in pharmaceuticalpreparations. Although it is often found as the pure compound, in mostcases it is present with a wide variety of other substances, which areoften present simply to make the vitamin more palatable. However,much ascorbic acid is used for less well-known purposes. Muchresearch has been carried out on the effects of ascorbic acid on variousaspects of plant growth. It has been found to have effects upongermination and root growth. Spraying with ascorbic acid has beenfound to be effective in the protection ofplants against the worst effectsof ozone in the atmosphere produced by photolytic action on polluted


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4 C'hupter Iair, particularly in big cities. *Most domestic and farm animals are ableto synthesise their own vitamin C, but even so this is sometimesreinforced by additional ascorbic acid. Fish are unable to synthesiseascorbic acid and the results of' a vitamin C deficiency in fish arecollectively known as 'broken back syndrome'. Thus, fish with a lowdietary intake of vitamin C commonly suffer from distortions of thevertebral column, impaired collagen production, poor growth, andother symptoms. The widespread increase in aquaculture has meantthat large amounts of ascorbic acid are used in the breeding andrearing of fish.When L-ascorbic acid is used in the food industry, we can broadlydivide the applications into two categories.( 1 ) It is frequently used as an additive to foods where i t enhances thenutritious qualities. I t may be added to restore loss ofvitamin due

to the food processing or to increase the natural amount of thevitamin present. In either case the term nutrification has beenused to describe i t s addition. Thus, L-ascorbic acid is added tofruit juice to fortify that which is naturally present or it may beadded to artificial fruit drinks to improve taste and thenutritiousness ofthe drink.

(2) L-Ascorbic acid may also be used as a food additive incircumstances where i t is not expected to provide any increase inthe nutritious nature ofthe food, but where i t is present to preventoxidation, as a preservative, to increase acidity, as a stabiliser, oras a flour improver. It is very often used as an additive for all thesepurposes.Red meat has its characteristic colour because of the myoglobin and

similar iron complexes which are present. This red colour is enhancedby the addition ofnitrite as part of the curing process. This is due to thereaction of nitrogen monoxide with the myoglobin. The addition ofascorbic acid to meat also improves colour, flavour and odour, as wellas lowering the amount of nitrite which has to be used in curing.Almost as a side effect i t has been found that ascorbic acid, alone or inco-operation with tocopherol when used in meat curing, inhibits theformation of nitroso compounds, which are believed to be carcino-genic, while not interfering significantly with the inhibition by nitriteof the very dangerous C'lostridium botulinuin micro-organism.

L-Ascorbic acid is very widely used in bread baking, where it ispresent as a 'flour improver'. I n practice, this means that the additionof L-ascorbic acid improves the bread texture and the size of theresulting loaf, the dough has greater elasticity, increased gas retention,


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Introduction 5and improved water absorption. Furthermore the addition means thatstorage time can be saved, since flour to which I,-ascorbic acid has beenadded behaves rather like flour which has been matured over time. I t iscomforting to know that the products of the decomposition of I,-ascorbic acid in bread making are carbon dioxide, L-threonic acid, and2,3-diketogulonic acid and N O T oxalic acid In many countries, i t isthe only flour improver which is allowed. il'here others are used, i t isinteresting that they are all oxidising agents such as potassiumhromate. L-Ascorbic acid is the only reducing agent used for suchpurposes. The exact mode of'action of'the vitamin C is still a mystery. I tis clear that during the dough mixing operation all the L-ascorbic acidis converted into dehydroascorbic acid and this remains stable in themixture. Perhaps the best-known process using L-ascorbic acid as anadditive in bread making is the so-called Chorleywood process.

Some countries ha\re a legal maximum for the amount ofL-ascorbicacid used as a flour improver. These may be as high as 200 mg kg- ' inCanada, or as low as 20 mg kg-I in Uruguay. hlany other countries,however, such as the United Kingdom leave the amount added toGood Manu fac u r ng Prac ic e.

Other areas where 1,-ascorbic acid has fbund uses in industrialprocesses are, for example, in polymerisation reactions, in photo-graphic developing and printing, in metal technology, and even inintravaginal contracepti iw. Most of these applications involve the useof the reducing properties of' L-ascorbic acid i n some way.

L-Ascorbic acid is found all over the plant \$.orld,often in quite largequantities and distributed throughout the plant. The biochemistry ofvitamin C in plants is \very poorly understood. ;I view which seems tobe accepted generally is that in some way 1.-ascorbic acid is merely asecondary product of plant metaholism. It seems curious that such aubiquitous compound in plants should be there almost incidentally asa by-product of other processes, though i t is fortunate for thosecreatures tha t have lost their ability to synthesise the vitamin that i t isso

There is evidence that tartaric acid in grapes has L-ascorbic acid as amajor precursor. Thus, immature grapes fed ivith L-ascorbic acidlabelled with l 4 C : at C 1 were found to ha\.e 72 ,, of the 14C in thecarboxyl carbon of' artaric acid. Th e indications are that in L-ascorbicacid C- 1 to C-4 end up as tartaric acid; indeed, if C-6 is labelled withI4Cnone ofit appears in the tartaric acid. Hoi\.ever, the process in vinesis by no means simple and depends on a number offactors , includingthe extent of development of the plant, and is different fbr different


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6 Chapter 1parts ofthe plant. In a similar way, oxalate has been found as a possibleproduct of biosynthetic routes in some plants such as the geranium.Many plants are capable of accumulating comparatively largeamounts of oxalic acid, sometimes even as crystals of calcium oxalate.Perhaps the best known oxalate accumulator is rhubarb. It seemslikely that in these plants as well, the oxalic acid produced has L-ascorbic acid as a precursor. The biosynthesis of L-ascorbic acid itselffrom D-glucose in plants occurs by a process quite different from theroute believed to take place in those animals which are able tosynthesise it. In plants, the biosynthetic route is thought to involve theoxidation of C-1 of glucose, epimerisation of C-5, and retention of theC-6 hydroxymethyl group.The process of photosynthesis, which is so vitally important to theexistence of virtually all life of Earth, is now known to be anextraordinarily complex process which is still only poorly understood.It is known, however, that although dioxygen is essential to thedevelopment of all life, nevertheless high concentrations have anadverse effect on a number of important biochemical processesincluding photosynthesis. In this case, high concentrations ofdioxygeninhibit the development of chloroplasts. In the process of illuminationof chloroplasts, damaging oxygen-containing species such as hydrogenperoxide and singlet oxygen may be formed. It is suggested that thedamaging effects of these species is limited by the presence of L-ascorbicacid in plants, which acts to suppress these highly oxidising molecules.

The arguments will continue over the biochemical role of L-ascorbicacid in animals, over the reasons for its large concentrations in plants,and over who was the first to discover and isolate it. Interest in itschemistry will continue and there is certainly much to discover.However, for most people, its role in medicine will remain the mostinteresting and controversial area. There is much conflicting evidenceconcerning the amount we should be taking. Does it help to relieve thesymptoms of the common cold? Does it have a role in cancer therapy?Perhaps the best position to take up is to stay with what is known forcertain. Vitamin C is clearly vital to the production of collagen and isimportant in wound healing. For this reason alone it is probably wise toensure that we receive amounts rather higher than those recom-mended for the suppression of scurvy. Although there is not muchevidence for serious effects on taking very large amounts, perhaps it isunwise to take tens of grams at a time of any compound. We are oftenasked how much should be taken daily. The above is the nearest wewould give to an answer


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Chapter 2History of Vitamin C and ItsR ole in the Prevention and Cureof Scurvy

Nothing emphasises the importance of vitamin C to human beingsmore than the effect ofbeing without i t for a relatively short time. Just afew months deprivation produces the particularly unpleasant andultimately fatal disease, scurvy. I t is hard for u s today to appreciate thefear with which this mysterious disease was regarded, particularly byseafarers in the middle ages. Although i t was only one disease amongmany which afflicted sailors, i t seemed to flare up for no apparentreason, particularly on long sea voyages, which were becoming moreand more common from the fourteenth century. It was not uncommonto lose more than half the crew on such a journey. Vasco da Gama losthalf his complement of men when he first rounded the Cape between1497 and 1499 and scurvy continued to take i t s toll ofsea travellers forfour hundred years after this time.

What is this disease? A textbook definition would be somethinglike: A disease which Produces haemorrhaging into tissues, bleeding gums, looseteeth, anaemia andgeneral weakness. However, contemporary descriptionsofindividual cases bring home to us the unpleasantness ofscurvy. ThusThomas Stevens wrote from a ship travelling from Lisbon to Goa in1579:

. . . heir gums wax gre at , an d swell , . . . heir legs swell, an d all the b odybecometh sore, and so benum bed , t ha t t hey can no t s t i r hand norfoot, and they die for weakness, or fall into f luxes an d agues, a n d d iethereby. . .Father Antonia de la Ascension in 1602 wrote in his diary while on. . . T h e fi rs t symptom they not ice is a pain in the whole body whichmak es it sensitive to touch . . . all the bo dy, especially from th e waistdown becomes covered with purple spots . . . T h e sensitiveness of t he

expedition along the coast of California:

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8 Chapter 2bodies of these sick pcoplc is so grcat that . . . t h c best aid which can bcrendered them is not even to touch thc bcdc-lothcs . . . the upper andlower gums ofthe m o u t h in the inside ofth c m o u t h and outside the teeth,become swollen to such a size that ne i t he r tccth nor t hc molars can bebrought together. lhc teeth become so loose and without support thatthey move while moving the head . . . hcir natural vigour fails them andthey die all of a sudden, while talking.W e now know th at scurvy is a deficiency disease an d it must ha ve

existed th rou gho ut all history. H owe ver, i t was really the very lo ng seajourn eys of the early ,Middle Ages which d rew atten tion to i t, thoug han y disasters which affected th e food supp ly, such as sieges, would alsoresul t in a n upsurge of scurvy. I t would ap pe ar , however , th at we ca nidentify at least three ma jor factors which con tribu ted to th e failure torecognise the c ure for scurvy.( I ) Th e Lack of Communication in Ancient 11edicine

I t seems likely that the fact t ha t co nsum ption of citrus fruits couldcu re scurvy was known or suggested very ear ly on. W inslow an dDuran-Reynals have quoted a th ir teenth century Spanish medicalt ract recomm ending o ran ge an d lemon juice as being beneficial toscurvy sufferers. Sim ilar state m ents ar e to be found ove r the next fourcentur ies, but w ere not widely available , nor does there ap pe ar to hav ebeen a custom ofw idely read ing the works of medical experts elsewherein the world , even when they were avai lable . I t took a n incredibly longtime for something which seems obvious toda y to become accepted bythe m edical establ ishment .(2) T h e Instability of Vitamin cW e will examine the chemistry of t h e oxidat ion ofvi tam in C in laterchapters. I t has been known for a very long time t hat t he antiscorbuticeffects of fresh fruits an d vegetables diminish after the t ime th at theyhave been harvested. This is because of loss of vitamin C d u e t o avariety of oxidative processes involving oxygen from the air . Thiscon tribute d to the confusion over th e efficacy of fruit , vegetables a n dfrui t ju ices in the prevention an d c ur e ofsc ur vy , even as la te as thebeginning of the twentieth century.(3) Distribution of Scurvy Amongsi the Population

Sc urvy was perceived to be a disease of a relatively na rrow section ofthe population and was thought not to afflict the rich and famous.However , we shal l see la ter that there is evidence of its a p p e a r a n c edur ing the win ter months am on g the ar is tocracy an d indeed i t mayeven have affl ic ted m embers of th e the R oyal Household, tho ugh i t wasnot recognised at the t ime. I t tended not to be a disease of cities. Th is


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History of Vitamin c and ih H o l e in he Yrecentioii and Ciue ojSr ury 9and a lack of understanding ofthe pathology meant that this was not afashionable disease that captured the interest and the imagination ofthe medical establishment.

One of the earliest accounts of cures of scurvy is to be found inHakluyts Principal Navigations which was published in 1600. Thiswas referring to events which took place on Jacques Cartiersexpedition to Newfoundland in 1535.

Some did lose all their strength . . .others also had all their skins spottedwith spots ofblood o fa purple colour: then did i t ascend to their ankles,thighs, shoulders, arms and necks. Thei r mouths became stinking, theirgums so rotten that all the flesh did rot off, even to the roots of the teeth,which almost all fall out. O ur Captain, considering our estate and howsickness was increased and hot amongst us, one day \vent forth from thefort, and walking upon the ice saw a troop of those countrymen comingfrom Stadacona. amongst which was Domagaia. who not ten or twelvedays before, had been very sick with that disease, and had his kneesswollen as big as a child of two years old, all his sinews shrunk together,his teeth spoiled, his gums rotten and stinking. Our Captain, seeing himwhole and sound, was marvellous glad, hoping to understand how hehad healed himself, to the end he might ease and help his own men. Assoon as they came near he asked Domagaia how he had healed himselche answered that he had taken the .juice and the sap of the leaves of acertain trcc, and had with these hcalcd himself. Ihcn our Captain askedhim if any were to had thereahout . . . Domagaia straight sent twowomen to fetch some ofit, who brought ten or twelve branches ofi t, andthen showed how to use i t and that is, to take the bark and leaves andboil them together, then to drink the said decoction every other day . . .The tree in their language is called Ammeda or Hannedcw, this thoughtto be the Sassafras tree. Our Captain presently caused some ofthat drinkto be made for his men, but there were none durst taste of i t , except oneor two, who ventured drinking of it: others seeing did the same, andpresently recovered their health and were delivered of their sickness, andwith this drink were clean healed. After this medicine was found andproved to be true, there was much strife about i t . who should be the firstto take it, that they were ready to kill one another. A tree as big as anyoak in France was spoiled and lopped bare, and occupied all for 5 or 6days and it wrought so well, that if all the physicians in Montpelier orLovain had been there with all the drugs of Alexandria, they would notalone so used in one year, as that tree did in six days, for i t did so prevail,that as many as used of it, by the Grace ofGod recovered their health.

The cases which have been quoted and described here are clear-cut.The major problem for any ship-board physician was that the sailorswere subject to a large number of different diseases arising from avariety of causes. These were usually poorly described and therefore itwas often difficult or impossible to give a definite diagnosis or to


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10 Chapter 2distinguish between different diseases. The result was that many casesof scurvy went undiagnosed thus complicating the treatment.

One of the first references in English to the disease of scurvy was inHakluyts Principal1 Navigations, which was first published in 1589,where he records that in a 1582 expedition returning from the Straits ofMagellan two men died of skurvie when nearly home.

Most of the early long-distance exploration by sea was carried out bythe Spanish and Portuguese. However, British exploration began inearnest in the sixteenth century, epitomised by Drakes circumnaviga-tion of the world in 1577-1579. Like the Portuguese and Spanishsailors, the British were plagued by scurvy. Again there are passingreferences to the effectiveness of various fruits and herbs in thetreatment of the disease. Thus the journals of the voyage ofcircumnavigation undertaken by Thomas Cavendish in 1586 referfrequently to the importance offresh fruit and there were relatively fewcases ofscurvy on this voyage. We can get an idea ofthe length of time ittook to encounter scurvy on a sea voyage from the account of hisvoyage in 1593, provided by Sir Richard Hawkins. They began thevoyage in April and by the time they reached the Equator in August,some of the crew had gone down with scurvy. By October, there wereonly four healthy individuals amongst the crew. The situation was thensaved in Brazil, a Portuguese colony at that time, where oranges andlemons were purchased. Although Hawkins recommended orangesand lemons as a cure for the disease, he nevertheless considered thatdrinking dilute (very) sulphuric acid was helpful, but most of all air ofthe land, considering that the sea was the natural place for fishes andland the natural place for man

Although by no means generally accepted in Britain, it is clear thatthe notion that fruit juices helped in the treatment of scurvy wasgradually becoming better known. The journal of Sir James Lancas-ters voyage to Sumatra in 1601 is quite unequivocal about the value oforanges and lemons:

. . . the reason why the generals men stood b etter in he alth was this; hebro ugh t to sea with him bottles of the juice of lemons, which h e gave toeach on e, as long as it would last, three spoonfuls every m or n in g. . .Bythis means the general cured many of his men. . .

Later in the journey he repeated the process by calling at the Bay ofAntongile to refresh our men with oranges and lemons, to clearourselves of the diseases.

Among the victuals recommended to be carried on expeditions


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History of Vitamin C nd its Role in the Preuention and Cure of Scurvy 1 1funded by the East India Company, lemon water was included. Evenin 1626 some argued that tamarind was of more value than other freshfruit in the treatment of scurvy, though fruit juice continued to besupplied to ships of the East India Company.

SUGGESTED CAUSES OF SCURVYI t is worth pausing at this point in the consideration ofthedevelopmentof a cure for scurvy to consider what people believed to be the cause ofthe disease. This is not intended to be an exhaustive treatment of thistopic, but to highlight some of the early theories.

One of the earliest suggestions was that scurvy was a disease of thespleen. Even in the late ,Middle Ages the ideas about the operation ofthe body and the causes of it going wrong were very different fromthose which we hold today, although as today they were believed to beabsolutely correct, little having changed from the time of the AncientGreeks, whose approach to medicine was held in the greatest respect.Hippocrates of Cos lived from 460 to 377 BC and was a contemporaryof Socrates. The teachings of the Hippocratic School of Medicine werebrought together as a collection of sixty texts which are known as theHippocratic Corpus. Although much of the contents can be attributedto Hippocrates, there are nevertheless many contributions from otherscholars including Hippocrates son-in-law, Polybios. A characteristicofGreek medicine was that they only had a very hazy knowledge ofthestructure and functions of the various parts of the human body. Theywere particularly unclear about the nature and functions of the vitalorgans and, ofcourse, knew nothing about the circulation of the blood.They were familiar with the fact that the human body secreted fluids.It was obvious to them that these had something to do with disease;excessive bleeding caused illness and ultimately death, a running noseis a feature of the common cold and other illnesses, people often vomitwhen they are ill, and so on. They therefore formulated a theoryincorporating these fluids or humours. The body was thought tocontain and be influenced by four humours:

bloodblack bileyellow bilephlegmThe balance between these humours governed the health of the

individual. Each humour had associated with it a property:


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12Blood - sanguine or hopefulBlack bile - melancholy or gloomyYellow bile - choleric or irasciblePhlegm - phlegmatic or even temperedThese humours were considered to originate and be replenished byvarious organs: blood by the heart, black bile by the spleen, yellow bile

by the liver, and phlegm by the brain. Lastly they had associated withthem a physical property: blood - hot and wet, black bile - cold anddry, yellow bile- hot and dry, phlegm- hot and wet, as represented inFigure 2.1 These in their turn were considered to be related to the fourelements, earth, air, water, and fire.

SANGUINEICHOLERICBLOOD

PHLEGMIMELANCHOLIC PHLEGMATIC

Figure 2.1 T h e theocy oJthef0ur humoursTerms relating to the idea of these humours are of course still used

today and we talk of people being melancholic),phlegmatic, bilious,sanguine, while someone who is ill tempered is called liverish. It wasbelieved that someone suffering from a disease such as scurvy had aswollen and hard spleen, so that its function was impeded. This meantthat the black bile could not be purified in the recycling process andthis would result in the symptoms ofscurvy. Although we recognisetoday that this theory has no validity, i t also suffers from thedisadvantage that it does not suggest a cure, nor does it provide a


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HiJtory o j ' Vilurnin C' mid it., Role in he Prevention cind (. lire o j Sccurzy 13process leading to a cure. However, since black bile was thought to beassociated with melancholy, presumably by improving the dispositionof the sufferer this would go some way towards curing the disease, andthe provision ofgood clean air and pleasant surroundings, for exampleremoving the unpleasantness of being crowded with other people on asmall ship, would help to relieve the symptoms.

We take for granted today the value of chemistry in both thetreatment of disease and the understanding ofthe causes of ill health.In the sixteenth and seventeenth centuries, chemistry as we know i ttoday scarcely existed but one or two ideas about the interactionsbetween substances which we would find acceptable today weregradually emerging from the darkness of alchemy. On e of these wasthat some substances could be classified into acids and alkalies. Therewas no definition such as we have today, but i t was known that therewere acidic and alkaline substances which had corrosive effects uponthe human body. It was assumed that the process of digestion, forexample, involved an acid-alkali reaction. Lt'hen the digestive processwas not working properly, it would introduce, say, acid into the bodyand produce certain symptoms associated with an excess of acid. Suchdiseases could then be treated using alkaline preparations. Similarly,other types of digestive imbalance might result in a n alkalinecondition, when administration of acid would be prescribed. Scurvywas regarded as a mixture of these conditions, since the ulcers whichwere characteristic ofthe disease were thought to be an att ribute of anacid condition, while the evil-smelling breath was a feature of analkaline condition. Thus, both acid and alkaline medicines had to beused together with bleeding, which was generally regarded as beingbeneficial in a wide variety of ailments and as extensively used overseveral centuries, often with great enthusiasm Indeed the use ofbleeding in the treatment ofdisease continued right into the middle ofthe nineteenth century.

In many ways, scurvy was a disease of Empire. Those countries withterritorial ambitions, such as Spain, Portugal, and Britain, had todevelop powerful navies for exploration, to extend their empires and todefend their far-flung colonies. The British Navy began operating inthis role at the end of the seventeenth century. From this time, scurvybegan to be a major problem for the Navy. The war with Spain, whichbegan in 1740, required the British Navy to extend itself over greatdistances in capturing Spanish ships bringing treasure from SouthAmerica and her colonies elsewhere. The experiences were similar tothose of Portuguese and Spanish sailors. After a few months at sea, menstarted to suffer the symptoms of scurvy. There are many references in


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14 Chapter 2this context to the efficacy offresh fruit and vegetables in the treatmentof the disease, but there was also much confusion and a widespreadassumption that good things such as fresh air, fresh meat, temperateweather were effective in its treatment. The standard medicine whichwas frequently prescribed for treatment was the so-called elixir ofvitriol which was essentially dilute sulphuric acid containing ethanoland sugar, made palatable by the addition of flavouring. I t is easy forus to see today, with the benefit of hindsight, that against thisbackground of confusion within which there was enough knowledge toprovide a cure for scurvy, there was a need for a clear-cut scientificdemonstration of the effectiveness offresh fruit and vegetables. That is,what was needed was what we would call today a clinical trial.Something very close to this was to be provided by James Lind.

JAMES LINDIn the eighteenth century the normal way to become a surgeon was tobe apprenticed. James Lind was born in 17 16, in Edinburgh. However,his ancestors came from Dalry which was in Ayrshire. It is aremarkable coincidence that today Roche Products have a factory atDalry manufacturing many thousands of tonnes of L-ascorbic acidevery year. At the age of fifteen Lind became apprenticed to a localsurgeon. He learned his trade in the same way as an apprentice to say acarpenter would become skilled, by watching, listening, and graduallybeing allowed to do more and more of the practical surgery. When hewas 23 in 1739, he joined the Royal Navy and served as a surgeonsmate. He became a full surgeon at the age of 30 on H M S Salisbury andvery soon he was faced with cases ofscurvy with outbreaks occurring in1746 and 1747. It was during the second outbreak that he set up whatcan only be described as the much-needed clinical trial to test theeffectiveness of the various treatments of scurvy in use at that time.

He chose twelve men who had contracted scurvy and fed each thesame diet throughout the day. He then applied six different treatmentsto the men, so that groups of two would each have identical treatment(Table 2.1). The result was that, although there were only enoughoranges and lemons to carry out the treatment for six days, thosetreated in this way quickly recovered, to such an extent that they wereable to resume normal duties. For the men provided with cider, at theend of two weeks the putrefaction of their gums, but especially theirlassitude and weakness somewhat abated. The other treatments had,as we now with the gift of hindsight would expect, little or no effect


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History of Vitamin C nd its Role in the Prevention and Cure aJScurvy 15Table 2.1 A summary of James Linds rial of cariousPossible cures o r scurvyGroup Treatment1 One quart of cider23456

25 drops of elixir of vitriol three times a dayTwo spoonsful of vinegar before mealsHalf a pint of sea waterTwo oranges and one lemonA medicinal paste containing a variety of substances such asgarlic, mustard seed

upon the condition of the men or the course of the disease. James Lindconcluded from this experiment that oranges and lemons were themost effectual remedies for this distemper at sea. In 1748 Lind left theNavy and went back to Edinburgh, where in 1750 he was elected aFellow of Edinburghs Royal College of Physicians. Then, in 1758 hebecame physician to the new naval hospital at Haslar in Gosport, nearPortsmouth, which is still a working naval hospital today. Thebuilding of these naval hospitals was itself a response to the everincreasing problem of sickness in the Royal Navy at that time.

In 1753 Lind published his famous book entitled:A Treatise on the Scurvy in three parts containing An Enquiry into theNature, Causes and Cure, of that Disease together with A Critical andChronological View of what has been published on the Subject.A second edition was published in 1757.The book gives an excellent account of the previous knowledge of

scurvy, but his theories of the cause of the disease are difficult tointerpret. Carpenter has pointed out that even in the 1953 symposiumcelebrating the two hundredth anniversary of the Treatise, no attemptwas made to summarise the theory, which is based on ideas concernedwith clogging of the pores of the skin, which were themselvesconsidered the major means of getting rid of unwanted humours andvapours.

Lind made recommendations concerning what antiscorbutic vic-tuals should be carried on ships, but even he remained unconvincedthat green vegetables and fruit could actually prevent scurvy. Hecertainly recommended that ships should carry an extensive store ofgreen vegetables as well as oranges and lemons.

Nobody in authority listened to Lind, however. He did not seem tohave influence in the right places and there was a considerable delaybefore Gilbert Blane, a man who did have the right influence, camealong.


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16SIR GILBERT BLANE

Chapkr 2

The influence of the British Navy in the development of the means oftreatment and prevention of scurvy was further enhanced by theappointment in 1781 of Gilbert Blane as Physician to the British Fleetby Admiral Rodney. Blane was born in 1749 and was a graduate ofEdinburgh and Glasgow Medical Schools. After obtaining his M. D . ,he practised as a physician and gained a considerable reputation. Thisresulted in his friendship with Admiral Rodney. He spent ten monthswith the fleet before preparing and sending a memorandum to theAdmiralty concerning the casualty rate in the fleet. In the year before,1600 men died, but only 60 of these died as a result of enemy action.Indeed there were only 12000 men in the fleet altogether While therewere many fatal diseases, there is no doubt that many of those deathscould be directly attributed to scurvy. Blane stated at the time:

.. Scurvy, one ofthe principal diseases with which seamen are afflicted,may be infallibly prevented, or cured, by vegetables and fruit,particularly oranges, lemons or limes

The memorandum recommended that arrangements be made tocarry fresh fruit . . . every fifty oranges may be regarded as a hand tothe fleet.

Having returned to London with Admiral Rodney in 1782, Blanewent back to the West Indies for a further two years and after that didnot return to sea again.

But still the confusion continued. Despite Blanes unequivocalstatement concerning the effectiveness of fresh fruit and despite thegreat weight of practical evidence in its favour, the Board of Sick andHurt Seamen rejected this and other suggestions by physicians that theprovision of fresh fruit would solve the scurvy problem. Othernutritional solutions continued to be championed. Beer, molasses, freshbread, all found their proponents. In the nature of medical trials andparticularly uncontrolled trials, these and other remedies sometimesproduced apparent cures. Even Gilbert Blane on his return to civilianlife, writing in Observations on the Diseases of Seamen, wasconvinced of the antiscorbutic activity of other foods, including,particularly, molasses.

It is fortunate for the history of the cure of scurvy that Gilbert Blanebecame physician to the Household of the Prince of Wales. This gavehim a social status and friendships which allowed him to have hisstrong views about the effectiveness of fruit in the cure of scurvylistened to in the right places. He was now able, through his friendship


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James Lind ( 1716-1 794)(Wellcome Institute Library, London) Sir GilbertBlane F(Royal College o


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L.L. MOSSSatercolour of the daib dislribulion o lime ju ice during lhe 187Scott Polar Institute, Cambridge)


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History o f Vitamin c crticl i1.r Kolu iti /lie Yrrrwilioti ( i t i d (.i/tu c /.Smrry 17with Adm iral Gar de ne r , to get the Sick ar id H u rt Board to approve theidea of carry ing lemon juice o n a ship ofthc fleet ( i l A 4 SS u f d k ) , whichwas sent to the East Indies. N o men died of the scurvy on tha t voyage.T h e very few w ho cont rac ted i t were cured quickly by encouragingthem to dr ink orange juice. W hen, in 1795, Blane became acomm issioner to the Board, i t authorised tha t lemon juice be a regularissue to the British Nav y. T h e chosen allowan ce was three-quarters ofa n ounce per da y , an d f rom 1796 the inc idence of scurvy drop ped offdram atical ly . Scurvy was el iminated f rom the navy as a major problemfrom then on . This was two years a f te r the dea th ofJ am es Lind an d jus tab o u t fifty years after his famous clinical tr ial which dem ons trated th eeffectiveness of oran ges an d lemo ns in curin g scurvy Before his d e a thin 1834,Gilber t Blane had been m ade a Fellow o f t h e Royal Socie tya n d received a knighthood in 1812.

T h e scientif ic relationship between Lind an d Blane is a n interest ingone. Lind died in 1795 with l it tle or no recognition. H e ha d ret iredfrom H asla r eleven years before. H e ha d n o influence on th e victualingprocedu res of the Royal Na vy. B lane, how ever, was able to recognisethe most important aspects of Linds and other peoples work.Fu rtherm ore, because of his social stand ing a n d his social contacts, h ewas able to br ing Linds ideas to the r ight people an d the a pp ro pri atecommittees, so th at they could be applied to the nutri t ion policies o ft h eRoyal Navy. Both must be regarded highly in the history of t h eprevention and cure of scurvy. Many people l ived who otherwisewou ld h ave died because of the l ives of Lind an d Blane.Largely because of Lind an d Blane scur\- j. had been conq uered atsea. This should be the end o f t h e s to ry , bu t in the n ine teen th cen turyevents were happe ning on shore which w ould b r ing the curse ofscurvyinland.

SCURVY ON LANDI n Nor thern Europe , dur ing the win ter m onths , frui t and vegetab leshav e always been in short supply. I t is l ikely t hat throu gho ut history,th e peoples of the British Isles must hav e bee n on the edge ofsufferingfrom scurvy or have suffered from w ha t is now known as pre-clinicalscurvy during the winter months. This s i tuat ion may have beenexacerbated in Christian countries by the very str ict adherence toLe nte n fastin g in th e six weeks before E aste r. Lthen Ea ster w as early,this would hav e occurred just as fresh vegetables were beco mingavai lable af ter the winter . Fur thermore the ar is tocrat ic people


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18 C'huper 2regard ed vegetables as food which was f i t only for the lower classes,because they grew in the 'dirty' soil. In some ways, therefore, thearistocracy were more prone to the possible ravages ofscurvy as thewinter wore on. In other words, scurvy on land was very much aseasonal disease, appearing in winter and beginning to disappear inspring. These Facts and others have been pointed out by SusanMaclean Kybett in reasoning that the mystery il lness suffered byHe nry V I I I in the last ten years ofhis life was probably scurvy, brou ghto n by a large intake offbod w hich did not contain an y vi tamins dur in gthe winter m onths and very str ic t Lenten fasting in the Spring. K ybetthas pointed out tha t the English h ad a diet which relied heavily on themeat of birds and animals and, indeed, were afraid of eat ing frui t ,which m an y believed caused skin eruptio ns a n d fevers. D urin g th e lastten years of his life, Henry became extremely fat, but suffered badlyfrom leg ulcers, fevers, spots on his body, swings of mood, and othersym ptoms which are st rongly reminiscent of those produce d by scurvy.It is on e of the curious features of'scurvy th at the pa tien t m ay be well-fed in terms ofqu an ti ty of food a nd hence be over-weight and yet bedesp erately ill from the disease.

Hen ry d ied on the 28 th Janu ary 1547; the time of th e year may besignif icant . I t may he that scurvy made a major contr ibut ion to hisde ath . H enr y would not h ave been alone in suffering from the diseaseand there are records of other ar is tocrat ic people also having thesymptoms, bo th in H enry 's t ime an d la te r . Th us , it seems v ery likelytha t James I a nd his wife both suffered f rom scurvy an d no d ou bt i trem aine d u ndiagn osed am on g the countless illnesses suffered bypeople in the Northern Hem isphere throu gho ut the ages. I t is not unt i lthe nineteenth century, however , tha t we begin to get a clear pictureab ou t the occurrence ofscurvy on land as the focus moved aw ay fromthe incidence of scurxy at sea.

Although historically potatoes were introduced into the WesternWorld comparat ively recent ly , the nutr i t ional importance of thepotato has frequently been underestimated. ,Many of us think ofpotatoes as the food which has to be c ut dow n on in a diet to lose weightbecause of their h igh carbohydrate content . Carpenter and otherautho rs have pointed ou t the im portan ce of this humb le vegetable inthe prevention an d cure of sc ur vy . Prisons in the nineteenth centurywere, next to hospitals, places to keep away from if you wished toremain heal thy. At that t ime i t was part of the philosophy of thetreatm ent of cr iminal behaviour (a n d perhaps still is) that the wrong-do er should suffer, not just by loss oflib erty , but by th e depriv ation of


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History (f itamin c and ils liola in lhe Yreuenlion and C u r e oJSctirL:p 19com fort an d good food. As a dire ct result of this th ere were spasm odicoutbreaks of scurvy in all British prisons at this time. Any prisongove rnor in the early ninetee nth centu ry found himself in th e difficultdi lemma of t rying to provide a diet which would he sufficient toprevent disease, but which would not be regarded by the generalpublic as being too luxurious Go vernm ent cuts are not just aphenom enon of the la te twentieth century an d per iodically governorswere ordered to cut back even on the meagre rations which theysupplied to those in their care. When this happened, one of the firstthings to be curtailed w as potatoes. I t becam e clear that this policy wasactually allowing the development of scurvy in British prisons.Analysis of th e food provision in prisons at th at t ime, using the reportsm ad e by the Inspectors ofPrisons, show ed that, in gaols where potatoeswere not a pa rt of the diet, scurvy was to be foun d. As a result of thispotatoes were reco m m ende d as pa rt of every prisoner's diet . This wasacceptable because the potato was a very cost-effective crop andch ea pe r th an green vegetables. It was also for this reason t ha t thepota to had become a vital part of the diet of the poorer people ofI reland a t this time.T h e Sum m er of 1845 was par t icular ly cold a nd wet even for theBritish Isles. Indeed the whole of Northern Europe was d a m p th atyea r. Dur ing tha t Sum mer a disease began to attack potatoes whichturn ed the leaves black a n d pro duce d tubers kvhich were deformed a nddiscoloured. W hen they were stored, they rapidly rot ted. Encou ragedby the da m p wea ther , the disease spread throughout the whole ofNo rthern Europe. In 1845, vir tual ly half the pota to crop for that ye arwas destroyed. Wh at m ade things even worse was tha t the cro p failedagain in 1846. This resulted in a terrible famine, which hit Irelandpart icular ly badly. Potatoes were a staple food, so th at th e first effect ofthe famine was widespread starv ation. Th is weakened people, leavingthem vulnerable to var ious diseases such as typhoid, and manyconsequently died. Thing s were also very ba d in mainland Bri ta in , bu tnot as terr ible as in I reland, where the populat ion was much moredepe nden t upon the potato . Soon scurvy began to app ear . Despi te theevidence available abou t the use of potatoes in prisons, the app ear an ceof scurvy cam e as a surprise to th e m edical establishment.

O n e of the problems with theories is th at even wh en they ar e wrong,bu t ar e accepted, they guide an d sometimes govern pract ice. O n etheory pu t forward at the t ime of the p otato fam ine was tha t scurvy wascaused by a lack of protein. T his arose from a n errone ous guess abo utwhat common feature was possessed b y the various foods known, or


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20 Chapterbelieved, to have antiscorbutic properties. The result, when the theorywas acted upon, was that the wrong foods were recommended in someprisons. The consequence of this was, of course, scurvy. Despite yetanother wrong interpretation of the cause ofscurvy, it at least now wasgradually becoming accepted that there was some substance in thefood which, if it were not present, would produce the disease. Th at is,in modern terminology, scurvy was a deficiency disease. A theory alsoprevalent at this time that scurvy was caused by the absence ofpotassium in some foods arose from similar thinking.

Meanwhile, a normal potato harvest was produced in 1848. Thatyear, scurvy virtually disappeared. The famine had had the mostappalling social consequences, particularly in Ireland, which are stillfelt today after nearly 150 years.

The supply of foods is always a major problem during wars and eachof the skirmishes which spasmodically broke out during the nineteenthcentury had its crop of deaths from scurvy. In the American civil war,25 percent of the captured soldiers who died in Andersonville prisondied of scurvy. Scurvy killed many in the siege of Paris in 1870 andthere was a very large outbreak of the disease during the Crimean war(1854-1856). There were the usual episodes of failure to deliver theright food to the right place during that war, as in all wars. Among thenumerous diseases suffered by the soldiers looked after by FlorenceNightingale, scurvy accounted for many deaths while the food supplysituation was difficult.

Scurvy remained a problem even into the twentieth century andwould continue to be so until the antiscorbutic essence in foodstuffs wasrevealed and isolated.

EXPLORATION OF TH E ARCTIC AND ANTARCTICThe last great areas of exploration a t the end of the nineteenth centuryand the beginning of this century were the Arctic and Antarctic. Therewas much activity in the exploration of the Arctic throughout thenineteenth century. Typical of such expeditions was to journey aroundCape Horn, up the American Coast and along the Northern coast ofAlaska. Such a journey was undertaken by the ship Investigator, whoseexperiences have been described in detail by Carpenter. This and otherships became stuck in ice, often for several years. I n many such cases,scurvy often struck, sometimes even when they had what was regardedas an antiscorbutic diet. Indeed, by the time of Scotts first expeditionto the Antarctic, the idea that scurvy was caused by the primary


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History of Vitamin C and its Hole in the Preuention and Cure of S c u r y 21decomposition products of meat, so-called ptomaines, had becomewidely accepted. Indeed the provisions which were carried on thisexpedition were carefully chosen to make sure that the levels ofptomaines remained at a minimum. This expedition set off in 1901, atthe beginning ofthe twentieth century By September 1902, there werethe first signs of scurvy. At this stage, fruit juice and vegetables wereprovided. The men recovered. During an attempt to get as far south aspossible that same year, Scott and Shackleton began to show thesymptoms ofscurvy from which they recovered when they returned. Atthe time, their recovery was attributed to the consumption offresh sealmeat.

In 1910, Scott set off on his famous and ill-fated expedition to theSouth Pole. Fruit juice was carried, but the men were not ordered toconsume it. Fresh meat was carried in abundance at the beginning.There was no doubt that during the expedition, Lieutenant EdwardEvans (later Admiral Lord Evans), contracted scurvy, from which helater recovered.There appears to be no evidence that scurvy contri-buted to the deaths of Scott, Evans, Oates, and Wilson, though it hasbeen suggested that the circumstances of Evanss death were such thatscurvy could have been a contributory factor. Such a suggestion at thattime would have been regarded as scandalous.

SCURVY IN CHILDRENIn the second half of the nineteenth century, there was an increasingtendency to wean children away from mothers milk early. The milkwas replaced by rusks and condensed milk. Many such childrenbelonged to wealthy families who would be shocked to hear that theiroffspring were prone to scurvy as a result of a poor diet. Children withscurvy suffered great pain and couldnt bare to have anyone touchtheir limbs. A little girl ofjust under two years old under the care of DrThomas Barlow, who was registrar at the Sick Childrens Hospital,died in March 1874. A post rnortern examination indicated that theentire periosteum of the femur was separated from the bone and thespace between was filled with a blood clot. Similar effects wereobserved with other bones. This was diagnosed as being due to scurvyand the disease of infantile scurvy became frequently known asBarlows Disease. Many cases were found in Britain, America, andGermany, frequently superimposed on rickets and sometimes mistakenfor congenital syphilis. It is interesting that one of the most obviousfeatures of scurvy, bleeding gums, was not present in many cases of


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22 Chapter 2infantile scurvy because of the absence of teeth. Barlow treatedchildren with scux-vy with raw minced beef, fresh cows milk, andorange juice and an improvement was usually observed in a very shorttime.Much of the failure to conquer scurvy completely, even at thebeginning of this century, was due to the continuing reluctance, despitethe evidence, to recognise it as a deficiency disease. Until the commonfactor which the various antiscorbutic foods possessed could beidentified, isolated, and shown to be effective in the cure of scurvy, thearguments about the causes of the disease, and which foods were activeagainst it, would remain. More than a quarter of the twentieth centurywas to pass before this was to occur. However, during these twenty-fiveyears, scientists were gradually unravelling the mystery of the elusiveantiscorbutic factor.

TH E EVENTS LEADING UP TO TH E DISCOVERY OFVITAMIN CIt was well into the first decade of the twentieth century before it wasfinally recognised that there was a substance or substances in fruit andvegetables which was essential to the well-being ofhumans. One majorproblem in the investigation of scurvy was that it is not possible toinduce the disease in many laboratory animals such as rats. We nowknow that the reason for this is that these animals are very proficient inthe biosynthesis oftheir own vitamin C. It was in 1907 that Axel Holst(1860-1931) and Theodor Frolich published a very famous paperconcerning the use of guinea-pigs in the investigation of the causes ofscurvy. Holst was professor of Hygiene and Bacteriology at theUniversity of Christiana in Oslo. The choice of guinea-pigs for thisstudy may look surprising today, when the ubiquitous rat is to be foundin many biological laboratories. However at the end of the nineteenthcentury the rat was regarded as an unpleasant, dirty animal, whereasthe guinea-pig was highly regarded as a childrens pet and was cheapto buy and keep. The latter requirement was particularly important inthe days before the existence of large research budgets which areavailable (at least in theory) today. The choice was ofcourse extremelyfortunate, since the guinea-pig is one of the few species of mammalwhich have lost, or never possessed, the ability to biosynthesise vitaminC. Holst and Frohlich fed their guinea-pigs with the same diet whichthey had previously found had produced polyneuritis in pigeons. Thesymptoms of the disease induced in the guinea-pigs were very different


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Axel Holst (1860-1931)(Dr. R . Forbes, University of Illinois) Harriette Chick (187Dr. N . Todhuntcr,


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Rollling limeju ice in Victorian limes(Cambridge University Library)


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from polyneuritis and included muscle haernorrhaging, brittle bones,and bleeding from the gums. Friihlich had preL-iously worked withchildren suffering from infantile scurvy and recognised the similarity inthe pathological changes exhibited by guinea-pigs to those he had seenin children. This gave him and his co-workers the opportunity to testvarious diets to ascertain the extent of antiscorbutic activity. Thus theywere able to demonstrate the effectiveness of fresh fruit and vegetablesagainst scurvy conclusively. They showed that while fresh potatoesgave protection against scurvy, dried potatoes did not and that, ingeneral, heating fresh fruit and vegetables effectively destroyed theirantiscorbutic activity. The only conclusion which could be drawn fromthis work was that scurvy was produced by a defective diet.Fredrick Gowland Hopkins was the first professor ofbiochemistry a tthe University of Cambridge. He studied carefully the influence ofdiets which are apparently well-balanced, containing purified protein,fat, carbohydrate, arid minerals on the grmvth of ra ts . As we wouldnow expect, the effect was catastrophic. HoweLw, the addition ofonlysmall amounts ofcows milk to the diet resulted in a complete recovery.It was becoming clear that there were a variety of substances whichmust be present in a healthy diet. It remained for Casimir Funk oftheLister Institute in London t o propose in 1912 that diseases such as beri-beri, polyneuritis in birds, dropsy, scurvy, and pellagra are alldeficiency diseases, which are preLrented b the presence of smallamounts of substances containing nitrogen in the diet. He proposedthat all these compounds were, in fkct, amines and that they be referredto as vital amines) abbreviated to vitamines.Like many names whichhave stuck (dehydroascorbic acid is another,, this turned out to be amisnomer and of course the e was dropped and they are now calledvitamins.

Even at this late stage the list of mistaken causes ofscurvy was beingadded to. McCollum, in 1917 proposed that scurvy was not adeficiency disease at all, but was due to constipation, which resulted inbacterial infection through the walls of the caecum, produced by thenature of the diet.

The First World FVar contributed to an acceleration in theunderstanding of the causes ofscurvy in at least two ways. The first wasthat many researchers were to have first-hand experienceof the havocwrought by disease in many campaigns during the war. T he secondwas that as men were recruited for the war effort abroad, women tookon many of the tasks at home previously carried out by men. As a resultofthis a team ofwomen remained at the Lister Institute in London, led


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24 Chapter 2by Harriette Chick, who developed an extensive research programmeto investigate the dietary factors which were required to prevent thedevelopment of beri-beri and scurvy. Perhaps the most importantfeature ofthe experiments carried out a t the Lister Institute a t this timewas that the workers took the greatest care to ensure that a verycarefully balanced diet, containing no antiscorbutic foods, was fed tothe guinea-pigs. This ensured that in all ways except contractingscurvy, the animals remained healthy. This allowed them to estimatethe antiscorbutic efectzveness of foods and thus they were able todemonstrate that lemon juice was powerfully antiscorbutic, freshpotatoes moderately so, and fresh milk only slightly. They were able tosettle once-and-fbr-all the question of the effectiveness of lime juice asan antiscorbutic. It turned out that \..Vest Indian sour limes werecomparatively low in antiscorbutic power, and when the juice waspreserved even this was destroyed. Hence, the problems experienced inmany expeditions that took place at the end of the nineteenth andbeginning of the twentieth centuries were due to the use of preservedWest Indian lime juice. There was thus now a reliable biological assayfor the antiscorbutic Factor. It is no exaggeration to say that the carefulapplication of this method of analysis provided information whichalmost immediately, when applied to food supplies to troops, savedmany lives in some First Plorld War campaigns.The availability of a reliable biological method of assaying theantiscorbutic factor, although slow, was crucial in the eventualisolation of the vitamin. By 1919, .McCollum had named two factorsrequired in their diet for the survival ofrats , Znd B. Drummond, inthat year suggested that the antiscorbutic factor be called watersoluble C. However, the term vitamin for such essential Factors wasbecoming widely adopted and the antiscorbutic factor became knownas vitamin C.

The decade between 1920 and 1930 saw much activity in theinvestigation ofvitamin C. Many attempts were made to isolate thevitamin. The Lister Institute was again at the forefront of the researchand Zilva and his co-workers were able to explore aspects of thechemistry of the vitamin, without actually isolating it. They were ableto show that i t was a reducing agent and used this property, whereappropriate, in analysis instead of the very time-consuming bioassay.

The isolation of the vitamin proved to be quite difficult and therewere a number of near misses among the attempts to prepare vitaminC . Perhaps the saddest of these was Link in the University ofWisconsin, who made crude calcium ascorbate, but was unable to


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H i s t ov o f Vitamin c and its R o l e in h e Preuenlion and Cure o Scurvy 25show its antiscorbu tic prop erties because th e university refused him agrant for the bioassay of the subs tance . Another was when Vedderm a d e a cru de sample of v i tamin C in 1927, but s ince he worked in t heoffice of th e Surgeon General o f th e Uni ted S ta tes Arm y, he was movedto a no th er posi t ion before he was ab le to carry ou t the vital bioassays.

W e come to the en d of the decad e tanta l is ingly c lose to the isolat ionofvi tamin C. T h e next few years were to be filled with the dr am a of th eisola t ion, s t ructure de term inat ion, an d synthesis of vi tamin C, withcontroversy over who ha d been f irst to achieve the isola t ion.


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Chapter 3Discovery and Structure ofVitamin C

ISOLATIONThe years immediately after the First World War saw research into theisolation of the elusive vitamin gaining momentum and, in both theUSA and Europe, the race was really on to reap the tremendousscientific and financial rewards that would surely follow. We have seenthat at the Lister Institute Solomon Zilva and his team were workingfeverishly to extract and purify the vitamin from concentrated citrusjuices but, although they obtained material which was powerfullyantiscorbutic, it was not a pure crystalline substance. Similarfrustrations were being suffered by the leading American group,headed by Charles King at the University of Pittsburgh. One of themajor problems was that, being a sugar-like substance, it wasextraordinarily difficult to separate the vitamin from the many otherdifferent sugars present in concentrated fruit juices. Ironically, despitethe dedicated and painstaking work of these groups, fate was settingthe stage for an unknown Hungarian scientist, Albert Szent-Gyorgyi.

Born in 1893, into an aristocratic family of modest wealth, AlbertImre Szent-Gyorgyi von Nagyrapolt was a charismatic man who, aftera distinctly unpromising school career in his native Budapest, hadqualified as a doctor, his training having been rudely interrupted bysome fairly hair-raising experiences as a soldier in the defeated Austro-Hungarian army during the First World War. Szent-Gyorgyi had thatrare but essential gift of any great researcher, the ability, as he later putit, to see what everyone else has seen but think what no-one else hasthought.

After the war, he travelled across Europe with his wife and youngdaughter, working at various laboratories with some well-knownscientists, and it was during this period that he developed a life-long

26


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Discovery and Structure of Vitamin C 27interest in the embryonic field of biochemistry and, in particular, thesubject of intracellular respiration and the associated oxidation-reduction reactions occurring a t the molecular level in living systems.Frequently short of money, but gaining valuable experience, hearrived in the Dutch University town of Groningen in 1922 where heacquired a post as assistant in physiology to ProfessorH.J. Hamberger.With his eye for detail, he perceived a possible, if unlikely, connectionbetween the bronzing of patients suffering from Addisons disease(caused by defective adrenal gland function) and that of freshly cutpotatoes, apples, and pears. It was known that this latter colorationarose from a disturbed redox process and he subsequently discovered,in the juices of lemons and oranges (which did not turn brown whendamaged) and also in cabbage juice, a strong reducing agent. He thenisolated the juices from the adrenal glands of cows and again found areducing agent. He determined to isolate what he thought might be anew adrenal hormone.

After a briefand unsuccessful attempt to isolate his compound a t SirHenry Dales London laboratories early in 1925, he returned toGroningen and, finding himself out of favour with the new professor,sent his family home to Budapest, resigned his position and wentthrough a period of deep depression. However, a chance meeting at aconference in Stockholm with the now world-famous biochemistProfessor Sir Frederick Gowland Hopkins (who had been impressed byone of his papers) resulted in an opportunity to work in Cambridge,whereupon Szent-Gyorgyi sent for his family and embarked on thework that would make his name.

After much frustration, Szent-Gyorgyi managed to accumulate lessthan a gram of an off-white crystalline substance from the adrenalcortex of cattle (in which it was present in only tiny amounts - about300 mg per kilogram of starting material) and, later from orange juiceand cabbage water. He described the process of extracting hisreducing factor as follows:1. I t was extracted from chilled and minced adrenal cortex by

shaking with methyl alcohol, and bubbling through with carbondioxide to prevent its having contact with oxygen.The filtered extract was then mixed with a solution oflead acetate,which precipitated the reducing factor.The precipitate, separated by further filtration, was suspended inwater, and sulphuric acid was added. The reducing factordissolved, and lead sulphate was precipitated.The filtrate was evaporated to dryness in a vacuum.

2.3.

4.


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28 Chapter 35. The solids were re-extracted with methanol, and steps 2, 3, and 4

were repeated.6. The solids were dissolved in acetone, and when an excess of light

petroleum was added, crystals of the reducing factor graduallyprecipitated.Like a typical reducing agent, the substance decolorised iodine and,

from the combining masses of this reaction, he concluded that itsrelative molecular mass was 88.2 or a multiple thereof. The lowering ofthe vapour pressure of water by the crystals suggested a relativemolecular mass of about 180, pointing to 176.4 as the correct value.Finally, combustion analysis, giving carbon, 4.7 hydrogen,and 54.6% oxygen, enabled the molecular formula C6HBO6 to bededuced.

Hopkins encouraged his protigi to publish this work describingwhat was then believed to be a new hormone with the nature ofa sugaracid. Szent-Gyorgyi, however, incurred some difficulty getting hispaper accepted because, in his first draft, he mischievously called hisnew compound ignose (suggesting a sugar-like substance for which hedidnt know the structure) and, when this was rejected, he resubmit-ted, naming the substance Godnose When the irritated editor finallythreatened not to publish unless an appropriate name were chosen,Szent-Gyorgyi relented, accepting the editors suggestion of hexuro-nic acid and so, in the Biochemical Journal of 1928, this milestone of apaper appeared. N o doubt this amusing anecdote featured promi-nently in many of Szent-Gyorgyis future after-dinner speeches. It isinteresting to note that, in this paper, Szent-Gydrgyi suggested that thereducing properties of fruit juices, of interest to students of vitamin C,could well be due to hexuronic acid. Two and two had not yet been puttogether but the idea that they were one and the same was surelygerminating.

The following year, Szent-Gyorgyi visited the USA and spent sometime at the Mayo Clinic in Rochester, Minnesota where, fortunately,the enormous nearby slaughterhouses furnished a plentiful supply offresh adrenal glands. Szent-Gyorgyi was able to isolate almost 25 g ofhis hexuronic acid, untold riches, and he promptly sent about halfbackto England, to Professor Norman Haworth in Birmingham, forstructure determination. Sadly, this quantity proved insufficient forHaworths group to elucidate the structure, which remained amystery.

Throughout his life, Szent-Gyorgyi seemed to have the good fortune


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30 Chapter 3STRUCTURE ELUCIDATION

The Birmingham group this time had in their possession an amplesupply of Szent-Gyijrgyi's off-white crystalline plates, a compound ofknown molecular formula (C,H,O,), melting point (191 "C), andspecific rotation ( +23" in water). Nevertheless, a fascinating piece ofdetective work, typical ofthe heroic age oforganicchemistry, would beneeded to unravel the structure. Edmund Hirst, who had workedunder Haworth at Durham and subsequently rejoined him inBirmingham, was placed in charge of the work.

When boiled with hydrochloric acid, the cr)rstals gave a quantitativeyield of furfural showing that at least five of the six carbon atomsformed an unbranched chain. Further tests showed ascorbic acid to bea weak, monobasic acid and a strong reducing agent. The first stage ofoxidation (which was easily reversible) could be brought about by, forexample, aqueous iodine, acidified quinone, and molecular oxygen inthe presence of copper salts at pH 5. The product of such oxidations,involving the loss of two hydrogens, was named dehydroascorbic acid(C,H,O,). This reversible oxidation by iodine was analogous to aknown reaction of 2,3-dihydroxymaleic acid (equation 1 ),

HOOC C ( 0 Hj= C(OH1COOH + I,+HOOCCO-COCOOH + 2HI 1 )

suggesting the possible presence of an ene-diol grouping, C ( 0 H ) -=C ( OH ) , and the similar absorption spectra of ascorbic anddihydroxymaleic acids, with a single, strong band at about 245 nm,reinforced this idea. Further supportive evidence for this groupingcame from the rapid reactions with diazomethane, yielding dimethyl-ascorbic acid, and with phenylhydrazine which, after first oxidising theascorbic acid, reacted to give an osazone. These reactions aresummarised in Figure 3.1 and expanded upon in Chapter 4.

The acidity of ascorbic acid initially suggested the presence of acarboxylic acid group but dehydroascorbic acid was shown to be aneutral lactone which actually hydrolysed slowly to a carboxylic acid.The easy, smooth interconversion of ascorbic and dehydroascorbicacids strongly pointed to the former also being a lactone, a viewsupported by the fact that dimethylascorbic acid was a neutralcompound which reacted with sodium hydroxide to give a sodium saltwithout loss of a methyl group, indicating lactone ring-opening(Figure 3.2).


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Sir Norman Haworth (18831950)(Wellcome Inst i tute Library, Lon don) A lb erl Szen1-Cy@yi(Wellcome Inst i tu


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Sir Edmund Hint (1898-1975)(Maurice Stacey, personal collcction I

Nobel Prizewinners 19.37: seated, f r o m l e f l are Roger .liartin du Gard (Lilerature) ,Albert Szent-C+ri;rgyi (Phy~io lo~gynd .liedicine) , Paul harrer and .2nrmanHaworlh ( Ch e m i s t y) , and C'linlon J . Ihr i\.coii (P/fr.\ic..i(National Library of Mcdicinc)


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D i sc o v e ry a n d S t ru c t u re OJ Vitamin C' 31

-c-c-II IIIIC NNHPhC NNHPhI

-c=c-OH OHI I

Figure 3.1 R e a c t i o n s of t h e e n e -d i o l group

It was also known that there were two more alcoholic OH groupspresent, these reacting with acetone (propanone) to give an isopropyli-dene derivative which still contained the two enolic hydroxyls.

Further oxidation of dehydroascorbic acid, using sodium hypoioditein alkaline solution, yielded oxalic acid and L-threonic acid, the latterbeing identified by its conversion into the known substances L-dimethoxysuccinamide and tri-O-methyl-L-threonamide. This estab-lished the stereochemical relationship of natural ascorbic acid to the L-series of sugars and also confirmed that the lactone carbonyl wasdirectly adjacent to the ene-diol grouping (Figure 3 . 3 ) .Evidence was now needed for the size of the lactone ring and thisemerged as a result of another elegant piece of organic chemistry. Itwas known that diazomethane converted ascorbic acid into a di-0-methyl derivative and further methylation with iodomethane andsilver oxide produced a tetra-0-methylated compound which, on


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I

0\C-0: I

0\C-0: II

I - I - II -CH Hxidation HO-C-=c hydrolysis 0-cO-C HI=c rcdudion-c I I

dehydmasmrbic acid(neutral)

ascorbic acid(acidic)

d

Figure 3.2 Lactone ring-opening of ascorbic acid and dehydroascorbic acid


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Discovery and Structure of Vitamin C 33OHc=oI

o d i c acid- 106 hydrolysisof Na*OI.dcbydroy2orbic laclonc -+cid HO CHZOH2,3,4-tri -0-methyl-L-thrconamidc

Figure 3.3 Cleavage of dehydroascorbic acid and establishment of ascorbic acidconjguration

ozonolysis, gave a single, neutral ester. Degradation of this productwith methanolic ammonia yielded oxamide and 3,4-di-O-methyl-~-threonamide, the latter being identified by the fact that it gave theWeerman reaction, characteristic of 2-hydroxyamides. This con-firmed the point ofattachment of the lactone ring as being at C-2 of thethreonamide, equivalent to C-4 of the tetra-0-methylascorbic acid(Figure 3.4).

Ascorbic acid was, therefore, shown to be a y-lactone, convenientlyrepresented as in Figure 3.5 although other tautomeric forms may existin small quantities. The configuration at C-5 is L - (or S-, using theCahn-Ingold-Prelog system). The acidic nature in aqueous solutionderives from the ionisation of the enolic OH on C-3 (pK, 4.25), the


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34 Chapter 3

CONH,CONH2oxamidc

3 , 4 - d i - O - m c l h y l - L - t i &+ Na+NCO'

Figure 3.4 Establishment of lactone ring size of ascorbic acid

0

t.2-HO-Y -lC HO OH6 Ho2O CHZOH345Figure 3.5 Slructure of L-ascorbic acid


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Discovery and Structure of Vitamin CTable 3.1 Physical data f o r L-ascorbic acid

35

Nomenclature

m.pt. ("C)rel.mo1. masspKlSpecific rotationSolubility(g per 100 cm3 at 20C)Density

Hexuronic acid, cevitamic acid, redoxon;L-ascorbic acid, L-xyfo-ascorbic acid,~-threo-2,3,4,5,6-pentahydroxyhex-2-enoiccid-4-lac toneL-threo-hexono-1,4-lactono-Z-ene190-192 (with decomposition)176.142nd (C-2-OH) 11.79[ a ] l s o= +23" in water; [a]1, 8= +49" in methanol33 (water), 3 (ethanol), 1 (glycerol)insoluble in chloroform, benzene, ether, petroleumether, fats and oils1.65 g cm-3

1st (C-3-OH) 4.25

resulting ascorbate anion being delocalised (Figure 3.6).A summary ofthe physical properties of L-ascorbic acid is given in Table 3.1.

The Birmingham group quickly confirmed this structure bydeveloping a synthetic route to L-ascorbic acid. This work, publishedin 1933, was the end-product of much painstaking and laborioussynthetic chemistry and it was therefore singularly appropriate thatNorman Haworth should find himself sharing the same stage as Szent-Gyorgyi in Stockholm in 1937, receiving the Nobel Prize for Chemistryfor his work on 'the structure of carbohydrates and vitamin C'. AlbertS

vitamin c its chemistry

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