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[Bale, Sons & Danielsson, Ltd.] In addition to the publications contained in the list given above, numerous memoirs upon work aided by the Medical Research Council have appeared in Scientific Journals. NATIONAL HEALTH INSURANCE '. MEDICAL RESEARCH COMMITTEE Report on the present state of Knowledge concerning Accessory Food Factors (Vitamines) Compiled by a Committee appointed jointly by the Lister Institute and the Medical Research Committee Approved for Publication by the Governing Body of the Lister Institute and the Medical Research Committee. July 1919 Medical Research Committee. (National Health Insurance.) G The Hon. WALDORF ASTOR, M.P. (Chairman). The Eight Hon. CHRISTOPHER ADDISON, M.D., M.P. The VISCOUNT GOSCHEN, C.B.E. (Treasurer). J. C. BOND, C.M.G., F.E.C.S. (Hon. Colonel). Professor WILLIAM BULLOCH, M.D., F.E.S. HENRY HEAD, M.D., F.E.C.P., F.E.S. Professor F. G. HOPKINS, D.Sc., F.K.S. Major-General SIR WILLIAM LEISHMAN, K.C.M.G., C.B., F.E.S. Professor NOEL PATON, M.D., F.E.S. SIR WALTER M. FLETCHER, K.B.E., M.D., F.E.S. (Secretary). 15, BUCKINGHAM STREET, STRAND, W.C. 2. Lister Institute: Governing Body. Major-General SIR DAVID BRUCE, K.C.B., D.Sc., LL.D., F.E.S. (Chairman). Lieut.-Colonel G. W. ADDISON, E.E. (Hon. Treasurer). Professor F. W. ANDREWES, M.D., F.E.S. Professor W. BULLOCH, M.D., F.E.S. Sir JAMES KINGSTON FOWLER, K.C.V.O., M.D. Lieut.-Colonel the Hon. W. E. GUINNESS, M.P. Professor ERNEST H. STARLING, C.M.G., M.D., F.E.S. Professor C. J. MARTIN, C.M.G., M.B., D.Sc., F.E.S. (Director). CHELSEA, S.W. 1. Committee upon Accessory Pood Factors (Vitamines) appointed by the Medical Eeseafch Committee jointly with the Lister Institute of Preventive Medicine to consider and advise upon the best means for advancing and, co-ordinating the various lines of inquiry into the modes of action of the factors in metabolism which are independent of the provision of energy. Professor F. GOWLAND HOPKINS, D.Sc., F.E.C.P., F.E.S. (Chairman). Miss HARRIETTS CHICK, D.Sc. (Secretary). J. C. DRUMMOND, D.Sc. Professor ARTHUR HARDEN, D.Sc., F.E.S E. MELLANBY, M.D. TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION .... ^,_ ... 1 II. ACCESSORY FACTORS AND GROWTH . . . . 5 Eeferences to Chapter II . , ' . . . 24 III. BERI-BERI AND SCURVY . . . ." . . 25 Beri-beri . . . . . 25 Scurvy v . . . . . . , .38 Eeferences to Chapter III . ,. "V . 48 IV. APPLICATION OF EXPERIMENTAL WORK TO THE PRAC- TICAL PROBLEMS OF HUMAN DIETS v . . 49 Diet of Adults . . . V . . 51 Keferences to Chapter IV . . . . . 67 V. APPLICATION OF EXPERIMENTAL WORK TO THE PRAC- TICAL PROBLEMS OF HUMAN DIETS (continued) . 69 Accessory Factors in the Nutrition of Infants . 69 Keferences to Chapter V ... . . . 80 VI. EICKETS AS A DEFICIENCY DISEASE .... 82 Eeferences to Chapter VI . . . . . 91 VII. PELLAGRA . . / . . . . . 92 Eeferences to Chapter VII . ... . 96 APPENDIX . . . . . , . , 97 INDEX 105 (454) Wt. 4295. 231. 1500. 9/19. O.U.P. (5508) Wt. 36019. 206. 1000. 2/20. O.U.P. (1596) Wt. 8101. 819. 1000. 6/20. O.U.P. 437128 ^MLJFl ACCESSORY FOOD FACTORS CHAPTER I INTRODUCTION. THAT substances playing a prominent part in nutrition could have eluded discovery until the arrival of the present decade seemed a short time ago to be quite unlikely : sufficiently unlikely at any rate to induce scepticism towards the subject of this monograph. Nevertheless, while any recognition of the existence and functions of such accessory food constituents as those now termed vitamines is new to the science of nutrition, the facts already available seem to compel belief in their reality and importance. True, there is at present no knowledge concerning their actual chemical nature, but the study of their functions is progressing on real and objective lines, and has become in certain cases even quantitative. As food constituents the vitamines are characterized by a dis- proportion between the apparent importance of their functions and the amounts in which they are normally consumed. They are present in quantities far too small to constitute any appreciable contribution to the energy supply of the body. Whether they ultimately prove to be structural components of living tissues of which a supply is essential even though quantitatively unimportant, or whether (as is equally possible) they are found to act rather as catalysts in certain normal processes of metabolism, it seems certain, because they function in such small amounts, that they must be put in a nutritive category different from any which would comprise the better-known foodstuffs such as proteins, fats, and carbohydrates. As already admitted their actual nature is unknown, though much information has been acquired as to their distribution and relations as well as clear evidence that at least three members of the class exist, each with its special function in nutrition. What can be said precisely at present is that if minute amounts of certain constituents are removed from natural foods, such foods wholly fail to support nutrition, and grave symptoms of actual disease may supervene. The failure and the symptoms may be prevented or the animal restored to health by the replacement of what was removed from the food, or by adding an equally small supply from other sources. The constituents that can be so removed are (or rather, contain) vitamines. All the nutriticmal phenomena observed in connexion with these substances are due to the influence of amounts of actual material so small as to suggest that their functions are of a nature quite distinct from those of proteins or of foods which supply energy. It is not surprising that common experience should have failed to take note of dietetic factors such as these. The vitamines are always present in natural foodstuffs as instinctively consumed by men and animals. There is evidence 1596 B to suggest that they are formed only in the tissues of plants, whence they pass into the tissues of herbivorous animals and thus become available for carnivora. Their distribution in the tissues of either plants or animals may be partial and irregular, but broadly speaking it is safe to say that the individual always finds a sufficient supply of vitamines in his food so long as that food is reasonably varied and has received no artificial or accidental separation into parts, and so long as no destructive influence has been applied to it. In the observation of phenomena the importance of contributory factors is often overlooked until the effects of their removal come to light. In the case of vitamines and their functions, demonstration, so far as it depended upon observation, first came when commercial adventure or other human enterprise had led to the preparation and consumption of foods in which the natural materials had been fractionated for the sake of taste, appearance, or convenience, or treated (over-heated, for example) for preservative purposes. Owing on the one hand to the partial and irregular distribution of vitamines in the natural products, or on the other hand to their instability, it may happen that by these processes they are removed or destroyed. Safety in this connexion is evidently greater when a variety of foods is consumed. In countries where geographical conditions prevent such variety the danger is more imminent. It is a fact at any rate that the most striking evidence for the evil effects of the artificial treatment of food came from rice-eating districts of the East when they had been invaded by milling machinery from the West. Those who recognize beri-beri as a deficiency disease have good evidence both for the evil effects of fractionating natural foodstuffs such as the cereals, and for the nutritive importance of what we have agreed to call accessory factors. The evidence from disease would have led sooner to a conception of these food constituents and their functions but for a not unnatural bias in thought. It is difficult to implant the idea of disease as due to deficiency. Disease is so generally associated with positive agents the parasite, the toxin, the 'materies morbi that the thought of the pathologist turns naturally to such positive associations and seems to believe with difficulty in causation prefixed by a minus sign. Even in connexion with deficiencies arising within the body there is or was a similar tendency. When the importance of internal secretions was first recognized there seemed to be much hesitation in believing that symptoms might be frankly due to their failure. When each fresh internal secretion was described there was always an effort to show that its function was to ' neutralize ' some, always hypothetical, toxic substance. Symptoms, on this view, were due to the unmaslflng of a deleterious agent rather than to simple deficiency in a normal and necessary agent. To distinguish between these two possibilities was of course a scientific duty ; but, at any rate in the earlier literature of internal secretions, a bias against the simpler view interfered with the fair interpretation of experimental results. So in connexion with the newer conception of disease as due to dietetic deficiencies. Even when Eijkman, through his admirable studies, had clearly established more than twenty years ago that beri-beri arose during the consumption of decorticated and not of whole rice (making it clear, therefore, that something in the cortex was necessary to normal nutrition) he was led to suggest, not the simple view that the cortical substance was of direct use to the body but rather that it was necessary to neutralize the otherwise deleterious effect of a diet over-rich in starch. Save for the mental bias just referred to it is very difficult to see why so roundabout an explanation should have been thought necessary. Its effect in every case has certainly been to delay the recognition of deficiency diseases and indirectly also to delay the realization of the functions of what, for lack of a better term, have been called vitamines. Fortunately in connexion with deficiency diseases it has proved possible by the choice of suitable animals to supplement clinical and sociological observation by experiments. As usual experimental work has made our knowledge more quantitative. The discovery, for instance, of avian polyneuritis and its relation to beri-beri by Eijkman and th6 demonstration by Axel Hoist that the guinea-pig easily develops the symptoms of scurvy and at the same time responds readily to anti-scorbutic treatment have made possible experimental work upon sound and illuminating lines. In this country the work done in connexion with beri-beri and scurvy at the Lister Institute has shown the value of experiments essentially quantitative in kind. Work on rickets now being carried out at Cambridge has further shown the value of experiment as a supplement to clinical observation when dietetics are concerned. This investigation seems to leave no doubt that a deficiency in the food, almost certainly involving a factor of the vitamine type, plays a fundamental part in the etiology of the disease. If, however, the evidence for the .existence and importance of vitamines had arisen entirely from the study of deficiency diseases our views might have remained too limited, too much confined to the standpoint of pathology and therapeutics. But in the very years during which the etiology of beri-beri was being cleared up and the suggestion established that a quantitative food deficiency may be responsible for striking symptoms, experiments were in progress on quite independent lines to show that something other than a supply of energy and protein is required to maintain so fundamental a physiological phenomenon as growth, and to demon- strate that normal metabolism as a whole is not possible without the influence of food constituents which because of their minute amount could justifiably be spoken of as ' accessory ' factors in nutrition. From such experiments then the conception of the vitamine also arose, and at the present time the evidence from all kinds of feeding experiments, whether carried out from the physiological or pathological standpoint, is being consolidated. In this country valuable contributions to the research are being made at Cambridge, the Lister Institute, the Cancer Hospital, and elsewhere. The work of McCollum and of Osborne and Mendel in the United States has made it clear that at least two substances of the vitamine type are concerned in growth, one of them being probably identical with the antineuritic substance discovered in connexion with beri-beri, B 2 while abundant evidence shows that the antiscorbutic factor is distinct from either of these. There is no occasion for surprise in the circumstance that students of normal metabolism have, like the pathologist, failed until recently to recognize the existence of such substances in the food. Nearly all the classical experiments have been made upon diets containing natural foods, the content of protein, fat, and carbohydrate being calculated from analysis. Not until these constituents had been thoroughly purified and animals fed upon ' synthetic ' dietaries made up of such purified materials could the suggestion arise that the familiar basal constituents of food were not by themselves capable of maintaining life. A point of interest arises here which is sufficiently cognate to the subject of this monograph to justify some reference to it. The tissues contain, of course, many known organic constituents other than proteins, fats, and carbohydrates. It is clearly a question whether any of them, or the constituent groups of any of them, must be supplied in diet. At one time or another many observa- tions have been made to determine this point. The result of adding certain of these known constituents has sometimes seemed to be positive in the sense of increasing growth, or of improving meta- bolism but the evidence has never been striking or even unequivocal. Now feeding experiments, such as those which have brought to light the necessity for a vitamine supply, seem to have proved at the same time that all known tissue constituents can be synthesized from the amino-acids of protein with the other basal foodstuffs. For, with their vitamine supply secured, animals in great numbers have now been shown to develop normally upon a diet containing only pure protein, fats, carbohydrates, and salts. We must believe, therefore, that substances such as the nucleic acids, the phosphatides, and other complex phosphorus compounds, creatine and the other bases in muscle, as well as many other necessary tissue constituents, must be synthesized from the basal foodstuffs. The vitamines may of course be themselves indispensable tissue elements in the structural sense ; exceptional in that they cannot be synthesized in the animal. If so it would seem that they share that characteristic with certain of the amino-acids of the protein and with those alone. To some, on the other hand, it seems probable that the functions of vitamines are in essence dynamic and catalytic, though the point remains unproven. 1 The purpose of the present monograph is to present in a collected form the essential facts concerning these accessory diet factors so far as they are at present known. It is hoped that it will be found, in particular, to make readily available information which is already capable of practical application. The practical importance of the facts will not be understood unless it be recognized that a deficiency in food, which when complete or- extreme leads to actual disease, may, when only relative, be responsible for ill health of a vague but still important kind. 1 So far back as 1911, when Hopkins described his own experiments to a meeting of the Biochemical Society (then the Biochemical Club) the subsequent discussion ranged chiefly round this point. The opinion of those who took part in the discussion was almost entirely in favour of the view that the function of such accessory food factors (as he termed them) must be that of stimulants rather than structural units. CHAPTER 11 ACCESSOKY FACTOES AND GKOWTH. THE growth of a young animal is believed to be determined by two factors. One of these is what has been termed the growth impulse. This is inborn, and represents the power to grow possessed by individuals of the particular species. It has not yet been possible to resolve and identify the multiplicity of factors which operate to determine the magnitude of the growth impulse, but it may be safely said that the chief variations are those introduced by hereditary influences. The growth impulse may therefore be termed the internal factor which controls growth and development. The second factor, which may be designated the external factor, must also be regarded as multiple, and is represented by the summation of those external conditions which influence growth. Of the many factors which together constitute this external influence the most important is, without question, the food supply. Neither of these fundamental factors can be regarded as inde- pendent of the other. The growth impulse, predetermined by evolutionary and hereditary influences, represents the power to grow inherent in the fertilized ovum, but this is in itself powerless to promote actual growth unless assisted by the co-operation of the external factor. On the other hand, the external factor, which for our purpose may be identified with the food supply, cannot stimulate growth beyond those limits which are predetermined by the growth impulse. It will, therefore, be realized what fundamental importance is to be attached to a full understanding of the food requirements of the animal body during the period of growth, since these represent the chief factors which are under voluntary control. For many years past it has been customary to estimate the nutritive requirements of the animal organism in terms of what have been for long regarded as the four fundamental food units, namely protein, carbohydrate, fat, and inorganic material, and to underestimate, if not entirely neglect, the possible significance of other less clearly defined dietary constituents. As a result of this narrow conception, we find that only within the last decade has research on animal nutrition advanced beyond the stage when primary importance was attached to the results yielded by elaborate studies of the nitrogen, energy, and mineral exchanges. Whilst acknowledging the great value of the many fundamental principles which have been deduced from such results, much that is misleading and inaccurate has undoubtedly followed from the attempt to allocate to every foodstuff a nutritive value estimated solely in terms of digestible protein, fat, carbohydrate, and salts. It is only necessary to consider the methods by which no inconsiderable proportion of this valuation has been made in order 6 to appreciate how these errors have arisen ; thus, for example, * protein ' is still computed in many cases from determinations of total nitrogen, without any attention being paid to the character of the nitrogen-containing substances actually present in the foodstuffs ; whilst the true nature of the substances frequently estimated as ' fat ' is almost invariably disregarded. The sole justification for assuming that proteins, fats, carbo- hydrates, and salts are all that is necessary for the nutritive require- ments of the body can only come from a successful experiment in which an animal has shown a normal development and standard of nutrition throughout its life cycle upon a diet composed of these units in a pure condition. EARLY EXPERIMENTS WITH PURIFIED DIETS. Many attempts have been made to accomplish this, although it was not an experiment of this nature that led to the first suggestion being 'advanced that in addition to these recognized dietary units other substances existed, which were equally indispensable for life. This suggestion was made by Lunin as far back as 1881 (1). He was investigating at the time the significance of inorganic salts in the nutrition of the animal, and as a result of some of his experiments found that whereas adult mice could live for several months in good health on a diet of milk, they invariably died within a month if they received a ration composed of what he believed to be the essential ingredients of milk, namely, caseinogen, milk-fat, milk-sugar, and the ash of milk. Commenting upon his experiments, he remarked : * Mice can live quite well under these conditions when receiving suitable foods (e. g. milk), but as the above experiments demonstrate that they are unable to live on proteins, fats, carbohydrates, salts, and water, it follows that other substances indispensable for nutrition must be present in milk besides caseinogen, fat, lactose, and salts.' In spite of the fact that this conclusion was given considerable publicity in the widely-read pages of Professor Bunge's Textbook of Physiological and Pathological Chemistry, it does not appear to have attracted the attention it merited. A number of investigators since Lunin's time have attempted to supply the nutritive requirements of animals, usually rats and mice, by means of artificial diets composed of mixtures of proteins, fats, carbohydrates, and inorganic salts, but it was found exceedingly difficult even to keep the animals alive for any appreciable time upon such diets. In practically every case, no matter how carefully the composition of the diets had been planned so as to provide what was considered an adequate and well-balanced supply of the neces- sary food units, the animals showed a steady decline in weight and health throughout the course of the experiments, and seldom sur- vived for any appreciable length of time. A valuable review of these earlier experiments is given in the monograph by Osborne and Mendel (2), which should be consulted for fuller details. Many suggestions were advanced to explain the repeated failures. By some it was considered that the monotony of the diet was in itself sufficient to bring about ultimate nutritive 7 failure ; a view that was difficult to reconcile with the fact that animals have been successfully reared and maintained at a normal standard of nutrition for considerable periods upon equally mono- tonous diets of single foodstuffs such as milk and egg-yolk (3, 4). Another explanation frequently put forward was that the absence of flavouring agents from the artificial diets induced loss of appetite, with a consequent failure to partake of sufficient food to satisfy the nutritive requirements for maintenance (5). Attempts were there- fore made to increase the palatability of the rations by the addition of flavouring substances, but in spite of this, the experimental animals still failed to flourish. Eeference must, however, be made to the experiments of Kohmann, who has done much pioneer work in this field of research. This investigator claimed to have been able to satisfy the nutritive requirements of mice with rations preparedly mixing a number of isolated and * purified ' food components (6). His animals not only showed increases of body weight upon such diets, but were actually sufficiently well nourished to produce young. A full criticism of his results will be given later, but it may be stated here that there is every reason to believe that he had failed to use sufficient care in ensuring the purity of the components of his food mixtures. In the year 1911 Osborne and Mendel published in America (2) their classical monograph in which they described experiments on the nutrition of rats over considerable periods upon diets composed of isolated food substances. They used dietaries containing proteins of a high degree of purity, together with starch, sugar, lard, agar, and inorganic salts. The agar was included in order to form an indigestible intestinal ballast or ' roughage ',' whilst the inorganic salts were given either|as a mixture of the pure crystalline salts or in the form of a preparation known as ' protein-free milk '. The latter product was prepared by evaporating down milk after removal of the fat and proteins. It therefore contained the salts, lactose, and extractives of milk. With regard to these experiments, they say (2 II. 59) : ' Although these apparently successful experiments indicated that the com- binations of isolated food-stuffs employed satisfied the nutritive requirements of the rats, and consequently constituted a complete food for the maintenance of mature animals, a prolongation of the observations has led to a less favourable outcome. A continuation of the experiments over longer periods has shown that in every case, sooner or later, the animal declined ; and unless a change^in the diet was now instituted within a comparatively short time, the animals died.' Mention must now be made of the work of Stepp (7), who found that whereas mice lived satisfactorily for several months upon certain foods, such as wheat bread made with milk, they were unable to live longer than a month when fed upon the same diet after it had been subjected to prolonged extraction with alcohol and ether. That the extraction itself had only lowered the nutritive value of the foodstuffs by removing some essential component was demonstrated by restoring the extract to the extracted food, when it once again became adequate for the nutrition of the mice. 8 He made a number of experiments in order to ascertain the nature of the essential factor removed by the extraction process. The addition of the ash of the extract, or of certain neutral fats in a pure condition, did not remedy the deficiency, but he found that milk and egg-yolk did. This led him to suggest the existence of an un- identified indispensable dietary unit, which he appears to have regarded as a member of the lipoid class. He was unable to identify this substance with cholesterol, lecithin, kephalin, and cerebrone, since all members of that class of substance failed to restore the nutritive value of the extracted foodstuffs. He remarks in one of his papers : * It is not impossible that unknown substances indis- pensable for life go into solution with the lipoids, and that the latter thereby become what may be termed carriers for these sub- stances.' We must now turn our attention to the classical experiments carried out by Hopkins in this country. As early as 1906 he wrote as follows (8) : ' But further, no animal can live upon, a mixture of pure protein, fat, and carbohydrate, and even when the necessary inorganic material is carefully supplied, the animal still cannot flourish. The animal body is adjusted to live either upon plant tissues or other animals, and these contain countless substances other than the proteins, carbohydrates, and fats. Physiological evolution, I believe, has made some of these well nigh as essential as are the basal constituents of diet ; lecithin, for instance, has been repeatedly shown to have a marked influence upon nutrition, and this just happens to be something already familiar, and a sub- stance that happens to have been tried. The field is almost un- explored, only it is certain that there are many minor factors in all diets, of which the body takes account. In diseases, such as rickets, and particularly in scurvy, we have had for long years knowledge of a dietetic factor, but though we know how to benefit these conditions empirically, the real errors in the diet are to this day quite obscure. They are, however, certainly of the kind which comprises these minimal qualitative factors that I am considering. Scurvy and rickets are conditions so severe that they force themselves upon our attention, but many other nutritive errors affect the health of individuals to a degree most important to themselves, and some of them depend upon unsuspected dietetic factors.' The results of his experiments extending over several years were published in 1912 (9). He fed young rats upon an artificial food mixture containing caseinogen, starch, cane sugar, lard, and inorganic salts. When the animals were fed upon the diet composed of these constituents in the crude condition, they were able to live and to show a certain amount of growth. When, however, the components had been carefully purified, growth invariably ceased after a com- paratively short period, and the rats declined and died. A most important feature of his experiments was that he showed by estima- tions of the energy consumptions of the animals that this failure was not due to an insufficient food intake. It was found that they ceased to grow at a time when they were consuming food in more than sufficient quantity to maintain normal growth. Another series of animals received in addition to the basal ration FIG. 1. Lower curve, six rats on artificial diet alone. Upper curve, six similar animals receiving in addition 2 c.c. of milk each per diem. Abscissae, time in days ; ordinates, average weight in grm. (Reproduced by permission from the Journal of Physiology, 1912 44 432.) FIG. 2. Lower curve (up to eighteenth day), eight male rats upon pure dietary ; upper curve, eight similar rats taking 3 c.c. of milk each a day. On the eighteenth day, marked by vertical dotted line, the milk was transferred from one set to the other. Average weight in grms., vertical ; time, horizontal. (Reproduced by per- mission from the Journal of Physiology, 1912 44 433.) 10 of purified foodstuffs a very small daily allowance of milk. In all cases the nlilk addendum, although its total solids amounted to only 4 per cent, or less of the whole food eaten, induced normal and continual growth. The extraordinary effect of this apparently insignificant addition to the diet upon the growth of the experimental rats is well illustrated by the above two figures, which have been reproduced from the original paper. A similar growth-stimulating action was exerted by the addition of protein-free and salt-free extracts of milk-solids, or of yeast, to the basal diet. As the milk ration was administered separately and in advance of the administration of the main dietary, it could not have affected the palatability of the food or diminished its monotony. The importance of -these early experiments is so great that an extract from Hopkins' discussion of his results is reproduced : * It is possible that what is absent from artificial diets and supplied by such addenda as milk and tissue extracts is of the nature of an organic complex (or of complexes) which the animal body cannot synthesize. But the amount which seems sufficient to secure growth is so small that a catalytic or stimulative function seems more likely. ' It is probable that our conception of stimulating substances may have to be extended. The original vague conception of such subjects being condiments, chiefly affecting taste, gained in definite- ness by the work of the Pavlov School. But the place of specific diet constituents which stimulate the gastric secretory mechanism can be taken by the products of digestion itself, and in this con- nexion the stimulant in the diet is by no means indispensable. Most observers agree that the addition to normal dietaries of meat extracts capable of stimulating the gastric flow does not increase the actual absorption of food, though this point could be properly tested by adding them to an artificial dietary known to be free from analogous substances. As was emphasized above, the milk did not affect absorption in my experiments. But such undoubted stimulating effects due to diet constituents as those discovered by Pavlov may quite possibly be paralleled elsewhere in the body on more specific and indispensable lines. Stimulations of the internal secretions of the thyroid and pituitary glands which are believed on very suggestive evidence to play an important part in growth processes, can be legitimately thought of. On the other hand, the influence upon growing tissues may be direct. If the attachment of such indispensable functions to specific accessory constituents of diets is foreign to current views upon nutrition, so also is the experimental fact that young animals may fail to grow when they are absorbing daily a sufficiency of formative material and energy for the purpose of growth.' These experiments undoubtedly mark the beginning of a full appreciation of the importance of what Hopkins has termed the accessory factors of the diet. Additional evidence of the existence of such factors rapidly accumulated ; Osborne and Mendel (10) confirmed their previous finding that when they replaced ' protein-free milk ' as a source of carbohydrates and salts in their artificial food mixture by lactose 11 and a mixture of pure inorganic salts approximating to the com- position of the ash of milk, the animals were unable to thrive. Summing up they say, ' whether the deficiency of the purely artificial diet is to be attributed to improper proportions of its constituents, to improper combinations of these constituents, or to the lack of some essential element, is at present difficult to define.' That they did not regard their failure as being entirely due to the absence of accessory factors may be assumed from their statement that they had achieved a considerable degree of success in feeding in the absence of the hypothetical organic hormones (10). Hopkins and Neville (11) challenged this statement and attempted to repeat the experiments of the American workers. Using very carefully purified ingredients they were unable to obtain any appreciable growth on such diets, and the animals, in spite of a satisfactory consumption of food, were all dead before the expiration of forty days. In com- plete confirmation of the earlier experiments of Hopkins, it was found that when a small ration of milk, 2 c.c. per day was given, in addition to the basal diet, this failure was not shown. DIFFERENTIATION OF Two ACCESSORY FACTORS. In 1913 McCollum and Davis in America confirmed the fact that rats were unable to grow upon an artificial ration consisting of caseinogen, lactose, lard, and inorganic salts (12). They also stated their belief that cessation of growth upon such diets was 'due to the animal running out of some organic complex which was indispensable for further growth, but since their rats lived in fairly good health upon this mixture after growth had ceased, they concluded that maintenance could be satisfactory in the absence of that substance. They found that the ether-soluble fraction of butter and eggs sup- plied the missing factor, but that lard and olive oil were unable to do so. In concluding their paper, they remark : * Our observation that ether extracts from certain sources improve the condition of animals on such rations strongly supports the belief that there are certain accessory articles in certain foodstuffs which are essential for normal growth for extended periods.' Very shortly after the appearance of this paper, Osborne and Mendel reported a continuation of their experiments (13). The greater efficiency of natural milk as a food, as compared with artificial mixtures containing what were supposed to be the only constituents of milk, impressed them with the belief that there existed some unidentified substance in the natural product which was not only necessary for growth but also for prolonged maintenance. They found that young rats which fed upon a diet containing protein, starch, lard, and protein-free milk grew at a satisfactory rate for a short time, but that sooner or later growth invariably ceased and that the animals declined. This decline could, however, be arrested and a normal rate of growth resumed when butter was substituted for the lard in the diet. Later these investigators found that the active substance was concentrated in the butter-fat fraction of the butter (14). Since 12 the authors claimed that their butter fat was free from nitrogen and phosphorus and devoid of any ash-yielding or water-soluble components, it appeared probable that the growth factor was not a substance of the type to which the anti-beri-beri factor, then being carefully studied by Funk, apparently belonged. The latter worker, however, challenged their claim as to the purity of butter fat, and showed that appreciable traces of nitrogen-containing substances could be extracted from that product prepared by the method described by Osborne and Mendel (15). Numerous naturally occurring fats and oils were examined for the presence of the substance which apparently played so important a part in the nutrition of the animal body, and it was found in association with several animal fats, such as cod-liver oil and beef fat, but was absent from a number of oils of vegetable origin (16, 17). The distribution of this substance will, however, be more fully considered in a later section. For some time it was generally believed that the substance found in association with certain fats was the only accessory substance necessary to supplement the nutritive deficiency of a diet of purified proteins, fats, carbohydrates, and salts for growth. Several workers had, however, failed to secure the satisfactory nutrition of animals even when butter fat was added to the basal ration of purified food- stuffs. Thus Funk and Macallum in 1915 reported that young rats fed upon such a diet not only failed to grow but rapidly declined and died after many of them had shown symptoms which the authors believed to be analogous to those of avian beri-beri (18). On the basis of Funk's vitamine theory of beri-beri (see later) they added a small ration of yeast to the diets and found that this addition was immediately followed by growth. They failed, however, to appreciate the true significance of their own results, for they apparently disregarded the influence of any substance present in the butter fat, and expressed their opinion that * the growth-promoting factor is beyond question contained in the yeast '. The proof of the existence of a second essential accessory dietary factor of a type distinct from that found in association with fats was first given by McCollum and Davis (19). As the result of a careful investigation of the dietary deficiencies of rice they were forced to accept the conclusion ' that there are necessary for normal nutrition during growth two classes of unknown accessory sub- stances, one soluble in fats and accompanying them in the process of isolation from certain food-stuffs, and the other soluble in water but apparently not in fats '. They termed these two substances ' fat-soluble A ' and ' water-soluble B ' respectively ; preferring these provisional appellations to the much criticized word ' vitamine ', which had been introduced and used extensively by Funk. Their terminology has been widely adopted as being both convenient and non-committal. Amongst other interesting points they showed that the so-called water-soluble factor is present in milk, and that it is only removed from milk sugar by thorough crystallization, a fact confirmed by Drummond (20). This observation supplied the solution to the apparent discordancy of many of the experimental results which had been obtained by various investigators, of whom many had employed in one form or another insufficiently purified lactose as a component of their dietaries, thereby unwittingly providing a more or less adequate supply of the water-soluble accessory factor. Thus, Funk and Macallum, who had taken great pains to ensure the purity of the components of their food mixture, and who excluded lactose on the grounds that it was known to contain traces of nitrogenous products derived from milk, were unable to obtain growth in the presence of butter fat until they supplied yeast, now known to be one of the richest sources of the water-soluble factor. Practically all investigators in this field of research have now admitted their belief in the indispensability of the two accessory factors, ' fat-soluble A ' and ' water-soluble B ' for growth and nutrition of the animal organism, but a brief reference must be made to certain opinions which have been advanced in opposition to this view. Foremost amongst the very small minority who have not yet acknowledged the indispensability of these constituents is F. Eoh- mann of Breslau. As the majority of his views and criticisms are contained in a book published in Germany in 1916 (21), and as no copy of this publication has as yet been available for reference in this country, we are unfortunately confined mainly to an examination of the criticisms it has roused in America (22). Apparently Kohmann asserts his belief that ' accessory foodstuffs are not necessary for the continued maintenance of fully grown animals ', and that if the long familiar nutriments are suitable in quality and quantity, nothing further is essential to the ration. He remarks : ' The assumption that some unknown substances are indispensable for growth is a convenient device for explaining experiments that result in failure a device that becomes superfluous as soon as the experi- ment succeeds.' But we must first examine his own experiments which he claims have been successful in achieving this result and which have led him to express these views. Kohmann has for many years past carried out a large number of feeding experiments with artificial mixtures of foodstuffs, and has thereby obtained valuable results upon such questions as the relative nutritive values of individual proteins. It is impossible to say, however, that he has exercised sufficient care in controlling the purity of the ingredients of his food mixture. Many of his dietaries con- tained protein in a very impure form, such as crude dried egg albumin and ' Kalzose ', the latter a commercial preparation of calcium-casein, admittedly containing milk sugar, and hence without doubt other products present in milk. The composition of the margarine, which he frequently employed as a source of fat, is never stated. It may, if derived from animal fat, contain large amounts of fat-soluble A (24). That he has failed to appreciate the difficulty of removing traces of the accessory substances from the ingredients of his diets may be judged from the fact that, in order to meet the possible objection that his diets contained these substances in some cases, he only extracted the components with cold alcohol. More 14 astounding is his admission that a successful nutrition with certain diets was frequently induced by the addition of alcoholic extracts of yeast or by small quantities of a product termed by him * Filtra- teiweiss '. Of this latter substance, Osborne and Mendel remark : ' according to his (Kohmann's) account of the method of preparation, this product may have contained some or all of the other constituents of milk among which are those proved to be especially efficient in promoting growth.' It is scarcely necessary to expend further time in considering work so full of inconsistences, and we may conclude by quoting the trenchant criticism of the American workers (22). * The thesis that a successful, i. e. positive, experiment in nutrition is far more significant than a negative one is doubtless valid. On the other hand, in dealing with substances which, like the alleged vit amines, are potent in surprisingly small amounts, the burden of the proof with respect to the complete absence of effective substances so widely distributed among the natural foodstuffs falls on those who deny the need of them.' ARE FAT-SOLUBLE A AND WATER-SOLUBLE B THE ONLY ACCESSORY SUBSTANCES NECESSARY FOR GROWTH AND NUTRITION? A later section of this book is devoted to a consideration of a third accessory substance, which may be sharply differentiated from both the factors ' A ' and ' B ' by its properties, namely the antiscorbutic substance. It will be seen there that this factor is undoubtedly indispensable for the normal nutrition of certain species of animals. The question, therefore, arises as to whether we must regard this substance as a third accessory factor indispensable as a component of a normal dietary for growth. It is difficult to give an answer to this question in the light of our present knowledge of the subject, because whilst we as yet understand very little of the requirements of the various species for this substance, it is undoubtedly true that wide variations occur. Practically all the researches which have led up to the discovery of fat-soluble A and water-soluble B have been carried out on young mice and rats. Neither of these animals appears to show any susceptibility to scurvy, if we may judge from the fact that they have been nourished with apparent success over long periods upon diets which can have contained but the merest traces, if any, of the anti-scorbutic substances. It is, however, quite conceivable that these two species represent types whose low susceptibility to the disease calls for a very small requirement of the preventive sub- stance. Failure to supply an adequate amount of the factor in such cases might only bring about a comparatively slight lowering of the nutritive standard of the animal, and experimental evidence is accumulating that this is actually what occurs in the case of the rat (23, 23 a). Strictly speaking, we must recognize at least three distinct accessory factors indispensable for normal growth and nutrition, but as the anti-scorbutic substance is fully treated else- where, the other two substances, fat-soluble A and water-soluble B, will alone be considered here. 15 300 250 200 ILLUSTRATIONS OF THE INFLUENCE OF FAT-SOLUBLE A AND WATER- SOLUBLE B ON THE GROWTH AND NUTRITION OF EATS. In Fig. 3 are given a number of curves illustrating the changes in body weight of rats which were fed from an early age upon an artificial ration of purified protein, carbohydrate, fat and in- organic salts, supplemented with adequate amounts of the factors 'A'and'B'. 1 The actual dietary employed was constituted as follows : Purified caseinogen . . . .v ." '. ; . . 20 parts starch . . . . . '"^ 55 Mixture of crystalline inorganic salts . . . 5 Butter fat (as source both of fat and the factor ' A ') . 15 Yeast extract (as a source of factor ' B ') . . . 5 FIG. 3. Showing normal growth of rats over 4-5 months with production, and satisfactory growth, of young on above dietary. Curve 1 represents the growth of a male rat, and Curve 2 the growth of a female rat. At the points marked on the second curve litters of young were born. Average curves for the growth of the second generation reared on this diet are given in Curve 3 (males) and Curve 4 (females). In one experiment four generations were reared upon this dietary as a sole source of food, and in every case the animals were well up to, and in some cases were considerably superior to, the normal standard. 1 The curves illustrating this section are taken from records obtained during experi- ments carried out by Drummond. They confirm those previously obtained by McCollum and his co-workers. 16 The composition of the salt mixture employed is one recommended by McCollum and Davis and is as follows (25) : Nad 5-19 parts MgS0 4 7-98 NaH 2 P0 4 1041 K 2 HP0 4 28-62 CaH 4 (P0 4 ) 2 16-20 Ca lactate 39-00 ,, Ferric citrate . . . . . 8-54 Iodine ...... trace. In addition to the above the animals received occasional doses of orange juice as an antiscorbutic. That the basal ration of protein, starch, and salts was free from accessory substances is shown by complete failure of young animals to grow when fed upon it, illustrated by the curves given in Fig. 10 (p. 19). Throughout this experiment the rats were apparently in excellent health and showed a normal appearance. A rat from this group was photographed after having received the diet for over twelve months (Fig. 4) ; one of the second generation raised on this ration is also shown (Fig. 5). From the close approximation of the weight curves of these animals to the standard curve for similar rats fed upon a mixed dietary of natural foodstuffs it appears probable that their nutritive require- ments had been completely supplied by the components of the artificial dietary. This is supported both by the fact that the experimental animals showed normal breeding propensities, many highly satisfactory litters of young having been produced and reared by females receiving the artificial ration, and by the animals showing a normal standard of resistance to disease. INFLUENCE OF THE ABSENCE OF FAT-SOLUBLE A FROM THE DIET. It has already been remarked that certain fats are very deficient in the fat-soluble factor. Such a fat is lard. Fig. 6 'represents the changes in body weight of rats fed upon a ration similar to that of which the composition has been given, but in which lard replaced the butter fat. It will be seen from these curves that for a short time the young animals are able to grow when the fat-soluble factor is deficient in their diet. The explanation of this is probably supplied by the assump- tion that the animal organism normally contains reserve supplies of the factor ' A ' in its own body. If this hypothesis is correct it is reasonable to assume that these reserves are mobilized for use when a deficiency occurs in the diet, but as soon as they are exhausted growth is immediately inhibited. During the period of temporary growth, throughout which it has been assumed that the reserve supplies are being utilized, the animals show a normal appearance and there does not appear to be any decline in their health, but when the reserves are exhausted and the deficiency becomes felt, not only do OF FIG. 4. Male rat reared from the age of five weeks upon the complete artificial ration described on p. 15. The animal is in perfect condition and has shown normal development for over twelve months on the diet. FIG. 5. Young rat, one of a litter produced by a female rat which had received the complete artificial ration since she was five weeks old. The father of this rat is shown in Fig. 4. The young rat is in perfect health and is well above the average weight for its age (eight weeks). FIGURE 7. they cease to grow but they become highly susceptible to bacterial infection. In the case of rats this lowered resistance first becomes apparent in many cases by the appearance of a characteristic infection of the external eye, which has been provisionally classified as a xero- phthalmia. It usually begins with a swelling of the lids of one or both eyes, which is followed by an inflamed and catarrhal condition of the conjunctivae (Fig. 7). This rapidly becomes worse and the discharge which is at first haemorrhagic, frequently becomes purulent. If untreated, the cornea may be involved and total blindness result. This condition is regarded by some (e. g. McCollum) as an almost FIG. 6. Showing preliminary growth and eventual decline on diet deficient in Fat-soluble A. Also recovery on addition of a source of Fat-soluble A. These curves illustrate how the absence of the fat-soluble accessory substance ultimately inhibits the growth processes of young animals. The broken curves represent the changes in body weight of rats fed upon a diet freed as far as possi- ble from all traces of that accessory factor. The small amount of growth, which usually occurs at first, probably indicates the utilization of reserve stores of the indis- pensable unit, but failure ultimately sets in. At the points marked on the curves by (o) the deficiency of the fat-soluble was made good by the addition of butter fat. This change was at once followed by a resumption of growth. In the small figure are given the weight curves of young rats born from females receiving artificial diets of this nature. Curve 4 indicates the normal growth of young rats nursed by mothers re- ceiving an adequate artificial ration similar to that used in the experiments illustrated in Figure 3. Curve 5 shows the development of young nursed by females receiving a dietary deficient in the fat-soluble factor. These two curves represent the average of a considerable number. The young nursed by the females on the inadequate ration were for some time not much below the normal standard as regards body weight, although they were at all times markedly undernourished. specific result of a deficiency of the fat-soluble factor. This view is certainly supported by the observation that if animals showing these symptoms be given a diet containing an adequate amount of the fat-soluble substance, the conjunctivitis usually clears up within a few days and the animal is very soon able to resume a normal rate of growth. An example of this recovery in growth is illustrated in Fig. 6. Fully-grown animals are apparently able to live for periods of many months in the absence of the dietary essential. Sooner or 1596 C is zoo later, however, the deleterious influence of the deficiency is made apparent by the appearance of the external eye disease, or by a greatly lowered general state of health, resulting in an abnormally high death rate from acute infections particularly those affecting the lungs. INFLUENCE OF THE ABSENCE OF WATER-SOLUBLE B FROM THE DIET. Contrary to the case of the fat-soluble factor, the animal does not appear to possess any appreciable reserve store of the water-soluble accessory upon which to draw in the event of an emergency arising. Consequently the ill-effects of a deficiency are more quickly apparent (26). An immediate cessation of growth is shown by young animals, followed by a short period during which body weight is more or less satisfactorily maintained. Adult animals show a very gradual fall in body weight following cf 50 >3 FIG. 8. Showing failure to grow on diet deficient in water-soluble ' B ', and recovery on adding that factor. These curves illustrate how a deficiency of the water-soluble growth factor im- mediately causes a cessation of growth, and show ths equally prompt resumption when an extract containing the active substance is added to the dietary of the experi- mental animal. Curves 3, 4, and 5 demonstrate that the more mature the animal, the longer can it survive against the deleterious effects of a deficiency of water-soluble A. the restriction, but sooner or later both young and old subjects suffer a rapid decline which invariably terminates fatally. Body weight curves illustrating these observations are given in Fig. 8. A number of the rats dying as a result of this deficiency have been observed to show symptoms of muscular inco-ordination due to involvement of the nervous system (see p. 35). Immediate recovery is effected by administering the missing factor, provided the condition of the animal is not extreme (Fig. 8). Fig. 9 illustrates the condition of a raf deprived of * water-soluble B '. INFLUENCE OF THE ABSENCE OF BOTH FAT-SOLUBLE A AND WATER-SOLUBLE B FROM THE DIET. When both these important constituents are absent from a diet it is usual to find that the deficiency of the second named is that which first exerts its deleterious action upon the growth and health FIG. 9. This photograph represents the condition shown by a young rat which is suffering from a deficiency of water-soluble B. There is no co-ordination of the movements of the hind quarters and the animal is unable to use the hind legs, which may be seen stretched out in a helpless manner. This animal showed a rapid recovery from this condition and was able to walk about with ease twenty-four hours after a dose of yeast extract had been given by the mouth. [To face p. 18.] 19, of the animal. This may be satisfactorily explained on the assump- tion that the body normally contains an available store of fat- soluble A. A deficiency of the latter, therefore, does not arise until the available reserves have been utilized, by which time the inadequate supply of the factor ' B ' has induced a cessation of growth and a serious disturbance of the general health. Young rats fed upon a diet inadequate as regards both these factors immediately cease to grow (see Fig. 10), and usually die show- ing symptoms associated with an absence of the water-soluble com- ponent, seldom surviving sufficiently long to exhibit the characteristic symptoms associated with a deficiency of the fat-soluble substance. PHYSIOLOGICAL SIGNIFICANCE OF FAT-SOLUBLE A AND WATER- SOLUBLE B. 1. Fat-soluble A. This factor in all probability is synthesized by the plant, and is found to be present in green leaves and in the zoo I ^ /' 6 / " / ,> ''/ f i/ J t ">. 7 ______ Animal receiving <**t ateftci'ent //? factor "#" comft/ete af/er */oo ._. J ^-f month-* V* .""X y Am ma/ receiving diet deficianf ir, to?/, "A'andV Animal ' rece/v/nf c/// dffic/ent xxxxxxx w />? factor "A" FIG. 10. Growth curves of rats fed on diet deficient in both fat-soluble A and water-soluble B. The curves represented by (....) show the inability of young animals to grow when both factors, A and B, are absent from the diet. When A alone is added, as for instance by the inclusion of butter fat in the ration (curves marked - - -), matters are not improved, but an immediate resumption of growth follows the restoration of both the missing units (curves marked ). When water-soluble B alone is added there is occasionally slight growth (curves marked x x x x), probably as a result of the utilization of reserve stores of the factor A, but it seldom lasts for long and is eventually followed by failure. embryos of many seeds. In the latter locality it is perhaps present in the form of a loose type of combination with some cell-constituent other than fat. This conclusion has been arrived at from the observa- tion that the simple processes used to extract fats, such as pressure or extractions with solvents, do not remove the fat-soluble factor from seeds, and this accounts for the absence of the factor from the vegetable oils prepared for consumption by these methods (17). The combination, whatever its actual nature may be, is very readily broken up. Treatment of the finely divided seed embryos with alcohol will effect the rupture, and subsequent ether extraction will remove not only the fats but the fat-soluble accessory substance. We as yet know nothing definite of the part it plays in the nutrition of the body. There is every reason to , believe that considerable amounts may be stored, perhaps in association with the reserve fat C 2 20 supplies, but this has not been experimentally proved. The fat derived from many tissues also contains the factor, so that it would appear to be closely associated with fat during transport of the latter in the body. The suggestion was at one time advanced that the fat-soluble accessory substance was a characteristic accompaniment of fats, such as milk-fat and egg-fat, elaborated by active organs for the express purpose of providing for the nutrition of the young, and that it was absent from purely depot fats, such as lard. This theory, however attractive, became untenable after it had been shown that the depot fat of oxen contained the factor in considerable amounts. No entirely satisfactory explanation of the deficiency of lard has yet been advanced. Whether the factor is intimately concerned in the metabolism of fats, or whether it plays some more direct part in the nutrition of the cell is as yet uncertain, but there is a certain amount of indirect evidence in favour of the latter view. There is apparently little appreciable loss of body fat in animals showing the characteristic symptoms associated with a deficiency of the fat-soluble substance, and such cases do not appear to show any obvious signs of a dis- turbed fat-metabolism (27). Storage of the factor appears to be particularly marked in adult animals, and the requirements, after maturity is reached, are of a smaller order than those of the growing young. The fully-grown rat is able to live for periods of several months upon what may be regarded as an almost complete deficiency of the fat-soluble factor, without showing appreciable loss of body weight or serious symptoms of ill-health, but at the same time it may be inferred that such animals are being maintained at a sub- normal level of nutrition by their decreased powers of propagation and increased susceptibility to infection. The storage of the fat-soluble factor is of considerable importance to the pregnant and nursing mother, for she is thereby enabled to provide her young with a high concentration of this factor without immediate dependence upon an external supply. The importance of this cannot be over-estimated, and it has been suggested that the ill-effects which follow a deficiency of the fat-soluble substance are much more serious and far-reaching when they occur in early life, particularly during the period when the young animal is dependent upon its mother (28). 2. Water-soluble B. The plant kingdom also provides the primary source of the water-soluble substance. The power to synthesize this substance is possessed generally by plants, even by some which appear low down in the natural classification. Thus, for example, yeast constitutes one of the richest known sources of the substance. It apparently exists free in the plant cell, although it has been stated that extracts of certain natural foodstuffs are more active after treatment with dilute acids : confirmation of this observation would tend to indicate the existence of the factor in some com- bination from which the acid liberates it. Of its action following absorption from the digestive tract, little, if anything, is known. The type of nutritive failure and the pro- duction of symptoms of nervous inco-ordination, produced by 21 , a deficiency of this factor, have already been described. A study of the onset of these ill-effects does not indicate that the normal animal possesses any large reserve of the water-soluble factor in an available form. The requirements of the body for the factor are much greater during growth than during maturity. As in the case of the fat-soluble substance, the quality of the milk of the nursing mother will suffer if her diet becomes deficient in this dietary essential. The adverse effects of this deficiency are, however, much more rapidly apparent in this case, for she has little, if any, reserve supply available in her own tissues. This point will receive further attention in a later section (p. 70). DISTRIBUTION OP FAT-SOLUBLE A AND WATER-SOLUBLE B IN NATURAL FOODSTUFFS. 1. Fat-soluble A. The presence of the fat-soluble factor was first detected in butter and egg-yolk, and, as far as is at present known, these foodstuffs represent the richest sources of that substance. It has also been found present in many oils and fats derived from the animal kingdom, as for example, cod-liver oil, shark-liver oil, beef fat, the fats of kidneys, heart muscle and liver tissues, herring oil, cod oil, salmon oil, and whale oil. On the other hand, the following oils and fats, all with one excep- tion, derived from plant sources, contain only small amounts or are deficient in the accessory factor ; sunflower-seed oil, corn oil, olive oil, cotton-seed oil, almond oil, arachis or peanut oil, linseed oil, coco-nut oil, and lard. Since there is good reason to believe that the animal organism does not possess the power to synthesize either of the factors A or B, it must derive its supplies of these substances from outside sources. The primary sources of fat-soluble A are found in the green leaves of plants, and the embryos of certain seeds. The former appear to be the richer source, but very few quantitative data upon the distribution of the substance have yet been obtained. It is, therefore, difficult to attach a definite value to any individual foodstuff as a source of fat-soluble A. This is particularly true in the case of the foodstuffs of animal origin, as their value as sources of that factor is in all probability directly determined by the nature of the diet which the animal has pre- viously received (28). Thus, the milk yielded by a cow which has for some time past been receiving a diet deficient in fat-soluble A, will, without doubt, sooner or later show the same .deficiency. The following tabulation of the chief foodstuffs has been made with the object of illustrating the distribution of the fat-soluble A factor. In the absence of quantitative data it has been impossible to do more than to indicate the relative values of the foodstuffs as sources of the accessory factor by the rough method of positive and negative signs. An attempt to give some idea of relative values has been made by the employment of more than one such sign in certain cases. A zero indicates the absence of the factor. (See also the combined table in which the anti-neuritic (water-soluble) and anti-scorbutic factors are included, p. 50.) 22 TABLE I Class of foodstuff. Individual foodstuff. Fats Butter Cream Margarine prepared from animal fats other than lard. (Value is in pro- portion to quality and percentage of animal fat.) Margarine prepared from vegetable fats or lard Nut butter .Vegetable oils such as are commonly used for salad oils or frying oils (e. g. olive and cotton-seed oils) Hardened fats, such as are sold as lard substitutes Cocoa butter Coco-nut oil Linseed oil Lard Mutton and beef fat Cod-liver oil and other fish liver oils Herring oil, salmon, or cod oil Meat and fish Lean meat (beef or mutton) Liver Kidneys Heart Brain Blood Sweetbreads ' Lean ' fish, such as cod, haddock ' Fat ' fish, as herring, salmon Fish roe Cereals Wheat embryo endosperm ,, bran Maize embryo endosperm Rice embryo and bran (i.e. so-called rice polishings) Rice (polished) Wholemeal bread White bread Custard powders and egg substitutes Millet Linseed Linseed cake (after expulsion of oil) Legumes Peas (dried) Kidney beans Soy-beans Other vegetables and fruit Cabbage (fresh) (dried) Lettuce Spinach Carrots (fresh) (dried) Onions Tomatoes Potatoes Oranges Bananas Apples (green) Nuts (Walnuts) Miscellaneous Cheese (prepared from whole milk) > , ( prepared from skim milk ) Fat-soluble A. + + + 4 + Variable. 4 + Inconclusive result. 4- + 4 4 + 4 4 Inconclusive result. 4- 4- 4- 4 4- 4 Inconclusive result. 4 + f + 4 4 4 4 Inconclusive result. + 4 4 4 4- 4 4 4 4 4 4 4- 4 4 4 23 . t C lass of foodstuff . Individual foodstuff. Fat-soluble A. Miscellaneous Eggs (yolk) + 4 + (white) (dried) + + Yeast (dried) Very slight, if any. ,, (extract) (commercial) Meat extract (commercial) Malt extract 2. Water-soluble B. In view of the probable identity of this with the anti-neuritic factor, the distribution of the two is treated on pp. 27 and 35. PROPERTIES OF THE ACCESSORY SUBSTANCES, FAT-SOLUBLE A AND WATER-SOLUBLE B. 1. Fat-soluble A. (a) When associated with fats it is not extracted by water, but is soluble in solvents which dissolve fats, e. g. ether, ligroin, and alcohol. It cannot, however, be extracted by fat solvents from green leaves. (b) It was at one time generally believed that the fat-soluble accessory factor was comparatively stable to heat. Thus, Osborne and Mendel (30) recorded that butter fat treated with steam for two and one-half hours did not appear to have lost its value as a source of the factor. Indirect evidence which supported that belief was given by the observation of Drummond (31), who found that certain fish oils, such as crude whale oil, which are frequently pre- pared by processes involving the use of high pressure steam for several hours, contained considerable amounts of the factor. More recent work has, however, cast some doubt on the stability of this accessory toward heat (32). The results of this investigation, carried out in America by Steenbock, Boutwell, and Kent, indicate that the fat-soluble accessory substance is gradually destroyed at 100 C., and that four hours' exposure to that temperature is sufficient to render butter fat of little greater nutritive value from the stand- point of vitamine supply than an equivalent quantity of lard. This result has now been confirmed by experiments of a similar character carried out in this country (33). (c) It is completely destroyed during the ' hardening ' of oil by the action of hydrogen, a process now widely employed for the preparation of edible fats (33). (d) It has been stated to be stable to alkali under the conditions employed for hydrolysis of fats at room temperature in a non- aqueous solution (34). 2. Water-soluble B. The properties of this factor are, as has already been remarked, identical with those described for the anti- beri-beri substance (29), and are treated under the heading of the latter (p. 35). ATTEMPTS TO ISOLATE FAT-SOLUBLE A. Scarely any attempt has bee* made as yet to isolate and identify this factor. The statement has been made that it may be removed from butter-fat by prolonged extraction with water, but recent work 24 does not confirm this (35). Certain facts may be interpreted as indicating that this accessory substance is a lipoid, but it has not been identified with any of the common representatives of that class of substance or with the free fatty acids (85). KEFERENCES. (1) LUNIN, G., Ztschr.f. physiol Chem., 1881 5 31. (2) OSBOBNE, T. B., and MENDEL, L. B. Feeding experiments with isolated food substances. Publication of the Carnegie Institute of Washington, 1911, No. 156. Parts I and II. (3) SOCIN, Ztschr.f. physiol Chem., 1891 59 93. (4) FALTA and NOGGEBATH, Hofmeister's Beitrdge, 1905 7 320. (5) McCoLLUM, E. V., Am. J. Physiol., 1909 25 120. (6) ROHMANN, R, Allg. med. Centr.-Ztg., 1903 1 ; 1908 9. (7) STEPP, W., Biochem. Ztschr., 1911 57 135 ; 1912 62 405. (8) HOPKINS, F. G., Analyst, 1906 31 395. (9) HOPKINS, F. G., J. Physiol, 1912 44425. (10) OSBOBNE, T. B., and MENDEL, L. B., J. Biol. Chem., 1912 13 233. (11) HOPKINS, F. G., and NEVILLE, D., Biochem. J., 1913 7 96. (12) MCCOLLUM, E. V., and DAVIS, M., J. Biol. Chem., 1913 15 167. (13) OSBOBNE, T. B., and MENDEL, L. B., J. Biol. Chem., 1913 15 311. (14) OSBOBNE, T. B., and MENDEL, L. B., J. Biol. Chem., 1913 16 423. (15) FUNK, C., and MACALLUM, A. B., Ztschr.f. physiol. Chem., 1914 92 13. (16) OSBOBNE, T. B., and MENDEL, L. B., J. Biol. Chem., 1915 20 379. (17) MCCOLLUM, E. V., SIMMONDS, N., and Prrz, W.. Am. J. Physiol, 1916 41 '361. (18) FUNK, C., and MACALLUM, A. B., J. Biol Chem., 1915 23 413. (19) MCCOLLUM, E. V., and DAVES, M., J. Biol Chem.> 1915 23 181. (20) DBUMMOND, J. C., Biochem. J., 1916 10 89. (21) ROHMANN, F., Kunstliche Ernahrung und Vitamine, Berlin, 1916. (22) OSBOBNE, T. B., and MENDEL, L. B., J. Biol. Chem., 1917 31 144. (23) HABDEN, A., and ZILVA, S. S., Biochem. J., 1918 12 408. (23 a) DBUMMOND, J. C., Biochem. J., 1919 13 77. (24) HALLIBUBTON, W. D., and DBUMMOND, J. C., J. Physiol, 1917 51 235. (25) MCCOLLUM, E. V., and DAVIS, M., J. Biol Chem., 1915 20 161. (26) DBUMMOND, J. C., Biochem. J., 1918 12 25. (27) DBUMMOND, J. C., Biochem. J., 1919 13 95. (28) DBUMMOND, J. C., lancet, Lond., 1918, Oct. 12. (29) DBUMMOND, J. C., Biochem,. J., 1917 11 255. (30) OSBOBNE, T. B., and MENDEL, L. B., J. Biol Chem., 1915 20 379. (31) DBUMMOND, J. C., J. Physiol, 1918 52 95. (32) STEENBOCK, H., BOUTWELL, P. W., and KENT, H. E., J. Biol Chcm., 1918 35577. (33) HALLIBUBTON, W. D., PATON, N., DBUMMOND, J. C., and others, J. Physiol , 1919 52 325. (34) MCCOLLUM, E. V., and DAVIS, M., J. Biol. Chem., 1914 19 245, (35) DBUMMOND, J. C., Biochem. J., 1919 13 81. CHAPTER III BEKI-BERI AND SCUEVY. THERE is now a general consensus of opinion that beri-beri and scurvy are diseases of dietetic origin, caused by the absence from the diet of an essential accessory factor. Experimental work on animals and experience based upon human diet confirm each other in the view that the factors concerned in prevention of beri-beri and scurvy are distinct and have a different distribution among natural foodstuffs. In modern European life, under normal peace conditions, the risk of these deficiency diseases, although a real one where infants are concerned, may be regarded as non-existent for adults ; the great variety of food taken ensures that an adequate amount of the preventive vit amines is regularly consumed. This fact will be appreciated later when the distribution of the factors preventing these diseases has been considered in detail. It is where extremes of climate limit the variety of available foodstuffs, as in the tropical or arctic zones, that a restricted diet may lead to outbreaks of deficiency disease. Thus beri-beri has proved to be mainly confined to rice-eating peoples in tropical climates ; scurvy is to be feared alike in tropical deserts or in arctic regions. Under war conditions, both for civilians and soldiers, the situation is altered, even in civilized European countries. Owing to dislocation of transport and other causes, many articles of common consumption become scarce or absent and the diet becomes more and more restricted. Accurate knowledge as to the value of various articles of food in respect of the accessory factors contained in them becomes of the utmost importance. It is necessary to ensure that an amount of these be consumed in the diet sufficient to prevent the disturbance of metabolism which eventually leads to ' deficiency diseases '. BERI-BERI. Beri-beri is a disease characterized by severe nervous disorder occurring principally among rice-eating populations, e. g. in Japan, the Malay Peninsula, Dutch Indies, Philippine Islands, &c. It is a form of severe peripheral neuritis, and may appear in two well- marked forms. In one (dry type) there is great wasting, anaesthesia of the skin, and finally paralysis of the limbs ; in the other (wet type) the most marked symptom is excessive oedema which may affect the trunk, limbs, and extremities. In severe cases the heart is usually involved, and death may occur suddenly from heart failure. The mortality is high. This disease has, in the past, been regarded as a tropical disease confined to rice-eating populations. It is now recognized that this, though the best known example of endemic beri-beri, is only a special case of what will inevitably occur when the diet consists too exclusively of a cereal which has been impoverished by excessive milling. The researches of Eijkman, Grijns, and later of Braddon, 26 Eraser, and Stanton have shown that the disease does not occur among the East Indian and Malay rice-eaters when unmilled or home-milled rice is taken, and that it can be cured or prevented by restoring to * polished ' 1 rice the valuable constituents (germ or embryo and silverskin or pericarp) known as ' rice polishings ', removed in the milling; see Fig. 11. The discovery of avian polyneuritis was an important step towards the elucidation of the human disease. Eijkman (36), who in 1897 was medical officer to a prison in Java where beri-beri cases occurred, noticed that the poultry of the establishment showed paralytic symptoms strangely reminiscent of those of his patients, and died with extensive degeneration of the peripheral nerves. unkucked hulled. 00 FIG. II. 2 Showing the various stages in Milling of the Rice Grain. I. Rice grain in the natural condition, retaining the husk or enclosing glumes. II. After removal of the husk, but retaining the pericarp or ' silver-skin ', and the embryo, which is shaded. III. After milling and polishing ; both ' silver-skin ' and embryo are removed and the grains are then ' polished ' by rubbing with talc between sheepskins. The fact that these fowls were largely fed upon the rice refuse of the institution was strongly suggestive of a dietetic origin for the disorder of the human inmates, a theory which, for many years previously, had attracted adherents. A careful study of polyneuritis gallinarum, as the disease was called by Eijkman, was published by Grijns (87) in 1901, but it was a matter for debate as to how far these experimental results could be applied to the human disease. On further study, avian polyneuritis has been accepted as the physiological equivalent of human beri-beri, both in its etiology, 1 Polished rice is a term which should strictly be applied to steam -milled rice after it has been subsequently polished with talc between sheepskins. The ' polishing ' itself does not injure the rice dietetically, but the expression has been used loosely to indicate the whole process of preparation, and to include the milling in which cuticle and germ are removed. 2 Reproduced from Chick and Hume, Trans. Soc. Trop. Med. and Hyg., vol. x, 1917, p. 149, with the permission of that Society. 27 symptoms, and method of cure. By its discovery a method has been provided for determining experimentally which foodstuffs prevent beri-beri, and their relative value for this purpose. Experimental work with fowls and pigeons figures largely in the pioneer work by the above-mentioned observers, and in the later researches, among others of Schaumann, Vedder, Chamberlain, and Funk. (For com- plete bibliographies upon this subject see 38, 39, 39 a.) McCarrison (39 b) has studied the loss of weight of various organs in pigeons fed with a diet which produces polyneuritis. With the exception of the adrenals the loss of various organs occurs much as in inanition but the adrenals increase to two or three times the weight of those of control birds. The adrenaline content of the enlarged glands appears to be increased proportionately to the size of the organ. McCarrison makes out a relationship between the hypertrophy of the adrenals and the occurrence of hydropericardiumin his experimental birds. He regards the excess of liquid as the equi- valent of ' wet beri-beri ' in man, and develops a theory of the cause of oedema in beri-beri on the basis of excessive adrenaline production. DISTRIBUTION AMONG FOODSTUFFS OF THE ANTI-BEEI-BERI OR ANTI-NEURITIC FACTOR. The work of the early investigators in this field yielded much valuable information upon this point, but the distribution of the anti-beri-beri vitamine was first studied systematically by Cooper (40) working at the Lister Institute. In response to war-time needs this work has since been rendered more complete by other workers in the same Institute (Chick and Hume, 41, 46). The methods adopted were substantially the same throughout; pigeons were used as experimental animals, and the experiments were of two general types preventive and curative. In the preventive experiments estimate was made of the daily ration of the particular foodstuff necessary to prevent polyneuritis when added to a vitamine-free diet consisting of highly milled and polished rice. Feeding was artificial, and in the absence of an adequate supply of vitamine in the supplementary foodstuff the birds developed severe symptoms in from 15-25 days, with paralysis of legs and wings, leading to complete helplessness and death within 24-48 hours. Protection was reckoned to be successfully accomplished if the birds showed no symptoms after a period of 50-60 days. In curative experiments a series of birds was fed with polished rice until severe symptoms were observed. The foodstuff to be tested was then given by the mouth in varying doses to separate birds and determination made of the dose required to cure the symptoms. One of the most striking features of avian polyneuritis is the dramatic swiftness with which a bird will recover from complete helplessness. It is not unusual to see normal gait and flight restored within a few hours after the adequate amount of vitamine has been administered. It is sometimes necessary to concentrate the factor contained in the foodstuff in order to get the requisite amount absorbed in time. In such cases a weighed quantity of the air-dried material is shaken up with alcohol in the cold, the alcoholic extract evaporated s 5 s ^ EH M W <* ft, s S s O P4 I" 2 tl* o o A ?l II s a 9 '-? CM O CO III 5 |2 O ^ GN1 2oc8 11 III 4 11^1:! & -~ *** ' I -" M M H S.2^ O OOOM^OO 29 O* ^ c H H II I =66 s '6 |6Aflbp M I I I I IO ' fits III! I I Pit I 1 I JOII * # !> il'"i K l bc'C * Wfi S 30 Btl illl 53 M ,. i a s ^ ii St. 31 to dryness under reduced pressure and the residue taken up in a measured quantity of water. Various doses of this watery solution are then given to a series of birds with severe symptoms in order to determine the minimum amount necessary to effect a cure. There is a considerable mechanical loss of the anti-neuritic factor in the process of extraction, and in making any comparison of values it is, therefore, necessary to make sure that the curative materials, as em- ployed, are strictly comparable (see above, Table III ; in the case of dry peas the curative dose of natural material is 10 grm., whilst that of the alcohol extract is the equivalent of 40 grm. ; in the case of wheat germ the amounts were 1 to 2-5 grm. and 16 grm. re- spectively). The results of the experiments on the distribution of the anti- beri-beri factor by Cooper ancf Chick and Hume are summarized in Tables II and III. The results in Table II, based on preventive experiments, must be considered the more valuable. If, however, the researches had been limited to this type of experiment, their scope must of necessity have been very much curtailed. In Table IV is shown the approximate relative value of some of the more potent of the foodstuffs investigated. TABLE IV. Showing approximately relative values of the more impor- tant foodstuffs (weight for weight) in natural condition for prevention of beri-beri, compiled from results in Tables II and 111. (Value of wheat germ taken as equal to 100.) Substance. Value* Approximate ivater content. Per cent. Wheat germ 100 10 to 13 Wheat bran 25 10 to 13 Rice germ 200 10 to 13 Yeast (pressed) 60 70 Peas (dried) 40 12 Lentils 80 Egg yolk 50 70 Ox liver 50 70 Beef muscle 11 75 Potatoes 4-3 80 It will be seen that the anti-beri-beri or anti-neuritic factor is widespread ; it was detected in almost all the natural foodstuffs examined. Its presence in yeast, the only unicellular organism studied, is significant of the universality of its distribution. Its principal sources proved to be the seeds of plants and eggs of birds, thus following very closely the distribution of the water-soluble growth factor and suggesting strongly the probable identity of the two (see p. 35). Cereals. The most important practical result emerging from this work is the fact that one of the chief sources of anti-beri-beri vitamine should be found in the cereals and edible pulses. In case of the cereals an interesting differentiation has been established (Chick and Hume 41) between the different constituents of the grain, the largest deposit of the anti-neuritic factor being found in the embryo, or germ, the bran (pericarp and aleurone layer) coming next in order of importance (see Fig. 12 a). The endosperm, 32 FIG. 12 a. 1 Diagram of a longitudinal section through a grain of wheat, showing : B, Pericarp, forming the branny envelope. A, Aleurone layer of cells forming the outer- most layer of the endosperm removed with the pericarp during milling. E, Parenchy- matous cells of the endosperm. O, Embryo or germ. FIG. 12 6. 1 Cross-section through the branny envelope and outer portion of the endo- sperm of wheat grain, showing : P, the pericarp ; E, endosperm, consisting of a, layer of aleurone cells and p, parenchymatous cells. 1 Reproduced, with the permission of the Controller of H.M. Stationery Office, from Figs. 1 and 2 in Dr. J. M. HAMILL'S ' Report to the Local Government Board on the nutritive value of bread made from different varieties of wheat flour ', 1911. sa especially when deprived of the aleurone layer (as is customary in the preparation of white wheaten flour or ' polished ' rice), is deficient in the anti-neuritic factor, and if employed too exclusively as a diet will occasion polyneuritis in birds or beri-beri in man [see also Edie and Simpson (42)]. The experiments were made chiefly with rice and wheat, but there is confirmatory evidence in case of maize and rye, and no reasonable doubt exists that, as a rule among cereals, the anti-beri-beri factor is chiefly concentrated in the embryo. The potency of the cereal embryo in curing avian polyneuritis was found to be very great. Chick and Hume found that 2-5 grm. (and occasionally 1 grm.) wheat germ was sufficient to cure a pigeon showing fully developed symptoms ; in case of rice and maize embryo, cures were obtained with amounts varying from 0-5 to 1 grm. and from 1 to 3 grm. respectively. The prevailing opinion, based on observation of rice-eating populations consuming decorticated rice, has located the principal deposits of the substance preventing -ben-heri in the cuticle of the husked grain, in the outer layer (aleurone layer) of endosperm, which is removed during steam milling. This view was based upon the connexion between beri-beri and decorticated rice, and the prevention or cure of the disease effected when rice bran or its extracts were added to the diet. A truer explanation recognizes the richer source of the vitamine to be in the germ (embryo) of the cereal removed with the bran. In the case of wheat and rice the germ, weight for weight, was found to be respectively five times and ten times as potent as the bran in the cure of avian polyneuritis. Pulses. The foodstuffs next "in importance to the cereals in prevention of beri-beri are the pulses. Grijns (37) and others have demonstrated the value of certain beans (' katjang-idjo 'Phaseolus radiatus) in prevention and cure of avian polyneuritis or human beri-beri. Dry peas and lentils were found to occupy a high place in the series of foodstuffs collected in Table IV. Eggs are also valuable sources of the anti-beri-beri factor, and this property was found unimpaired in two samples of dried eggs examined. These large deposits of anti-neuritic factor in the seeds of plants and eggs of animals (birds and fishes) suggest a due provision made for the wants of the young offspring during the early period of life. Vedder (43, p. 61) mentions the unusual prevalence of beri-beri among pregnant women in countries where the disease is endemic. Yeast is rich in this accessory factor and is unharmed in this respect by autolysis and extraction. Yeast extract A in Tables II and III was a commercial preparation (marmite) largely used as a substitute for meat extract in preparation of soup cubes, &c. Meat is comparatively deficient in this factor, and large quantities were required for curing or preventing pigeon polyneuritis. This was specially true of muscle fibre which was less valuable than heart, liver, or brain. In case of preventive experiments with fish, milk, and cheese protection was not obtained with the comparatively large amounts administered daily (experiments by Cooper, Table II). 1596 D 34 RESISTANCE TO DRYING, HEAT, &c., AND OTHER PROPERTIES OF THE ANTI-BERI-BERI OR ANTI-NEURITIC FACTOR. The anti-beri-beri factor withstands desiccation for long periods of time, as may be appreciated from the fact that its principal sources are found in dry foodstuffs. Its resistance to heat is also considerable. Many isolated observa- tions upon this point have appeared in the literature during the last twenty years, and most observers have not detected destruction of the anti-beri-beri vitamine after exposure to -100 C. Grijns (37) found that 1-2 hours' exposure to 120 C. destroyed the anti-beri- beri properties of unmilled rice, katjang-idjo beans, and buffalo meat. Eijkman (44) did not succeed in destroying the protective value of horseflesh by heating for two hours at 120 C. Hoist (45) traced loss of anti-neuritic properties in beef heated to 110 C. for half an hour, but in the case of dried peas or unpeeled barley kept at 1 15 C. for thirty minutes, no loss of anti-neuritic power was detected. Eecently Chick and Hume (46) have made a series of systematic experiments upon this point, with (a) wheat germ (natural con- dition, 15 -per cent, moisture), and (b) yeast extract (containing 65 per cent, water) exposed to temperatures from 100 C. to 124 C. in a steam autoclave. The results obtained are summarized in Table V, from which it will be seen that destruction of anti-beri- beri vitamine takes place very slowly at 100 C., but is much more rapid in the neighbourhood of 120 C. TABLE V. Influence of Exposure to high Temperatures upon the Anti-Beri-Beri Vitamine contained (a) in Wheat Embryo and (b) in Yeast Extract. Minimum amount in grm. required to cure a pigeon (300 to 400 grm.) suffering from acute polyneuritis. 1-0 to 2-5 2-5 5-0 10-0 did not cure 1-5 to 2-0 2-0 to 3-0 2-5 to 3-0 about 5-0 The practical conclusion to be drawn from this result is that in the baking of bread or biscuit, during which process the interior of the material does not rise above 100 C., no serious diminution in anti-beri-beri vitamine need be apprehended. This proved to be true in the case of pigeons, which throve well upon a sole diet of wheaten biscuit made from wholemeal flour. In preserving and canning foodstuffs, however, the temperatures employed are fre- quently much higher than 100 C., and it is safe to regard tinned foods of all descriptions with but few exceptions as vitamine-free. This is well seen in Table III, where the curative dose of raw meat for pigeons is given as the equivalent of 30 grams dry weight, and where, in two instances of tinned meats, no cures were obtained with amounts equivalent to 106 and 112 grams dry weight respectively. Substance. Wheat embryo, water content 11 to 14 % Yeast extract A, water content 65% Temperature. Time. C. Min. Unheated Control 98 to 103 120 100 to 117 40 118 to 124 120 Unheated Control 100 60 122 60 122 120 35, The anti-neuritic accessory substance can be extracted by alcohol from most of the foodstuffs in which it is found ; it is also soluble in water. It is not, as a general rule, extracted by ether or other fat-solvents, although Cooper (40) was able to detect distinct anti- neuritic properties in the ether extract from egg-yolk. It is probable that, as in the case of the fat-soluble accessory factor, the ' solubility ' of this vitamine is variable, and depends on the charactei of the material in which it is deposited. The anti-neuritic accessory substance is readily adsorbed from solutions upon the surface of various adsorbents, e. g. animal charcoal (Chamberlain and Vedder, 47), fuller's earth (Seidell, 48), colloidal ferric hydroxide (Harden and Zilva, 49). The anti-neuritic factor may be almost completely removed by one or another of these adsorbents, and there is no doubt that this property offers an explanation of the mistaken belief that the factor itself had been successfully isolated (p. 37). PROBABLE IDENTITY OF THE WATER-SOLUBLE B AND ANTI- NEURITIC FACTORS. It is now very widely believed that the water-soluble B and anti- neuritic factors are identical (see Drummond, 65). This conclusion is based in the first place upon the fact that when the distribution and properties of the water-soluble B factor, marked out by the use of rats, are compared with those of the anti-neuritic factor, established by experiments on pigeons, the closest possible agree- ment is found to exist between the two sets of results (see Tables VI and VII). Additional evidence is afforded by a comparison of the symptoms which are developed by rats and pigeons suffering from deficiencies of these factors. In both cases these point to the presence of a peripheral neuritis, and this is confirmed by the histological examination of the nerves. TABLE VI. Table showing Identity of Distribution of the Water-soluble B and the Anti-neuritic Factor. Value as source of Anti- Value as source of Water- beri-beri or Anti-neuritic Soluble B Factor (as shown Factor (as shown by experi- by experiments on rats). ments on birds)* 4-4-4- 4-4-44- 4-44- 4-4-4- Foodstuff. Rice germ Wheat germ Linseed Lentils Yeast . Egg-yolk Ox liver Wheat bran Fresh peas Dried peas Fresh carrots Dried carrots Meat muscle Milk . Potatoes Meat extract Meat tinned White bread or flour Polished rice. + + 4- + D 2 444 444- 4-44 4- 4 Slight 4- v. slight 36 f 11 0} 2 2% H o g ^ 5 S & |3 II II It H 3 S -is" ** a I ! 1! ons roxi - .e o ii *+-* o 6tli S o "^ J3 -3 -2 |2S JJ j Q OJ03CC ! S 1*4 1 . & 2 Qi O O F " i t*~j O lis > > f_i t 11 g K-h i^d .^ ii S 03 M 37 ATTEMPTS TO ISOLATE THE WATER-SOLUBLE ACCESSORY SUBSTANCE OR ANTI-NEURITIC VITAMINE. A large number of attempts have been made to obtain this accessory substance in a pure condition, but, as they have all hitherto failed, it is only necessary to review the more important investigations. Cooper and Funk (50) found that the curative substance present in an alcoholic extract of rice polishings was precipitated by phospho- tungstic acid, and that this method of preparation yielded a fraction which was * extraordinarily potent ', and free from proteins, carbo- hydrates, and phosphorus. Funk carried the investigation further (51), and fractionated the substances precipitated by phospho- tungstic acid by means of precipitations with silver nitrate, following the usual method employed for separating certain nitrogenous bases. From the silver nitrate-baryta fraction he obtained a crystalline sub- stance melting at 233 C., to which he gave the formula C 17 H 20 7 N 2 . This substance was curative for pigeons. This process of separation was applied to yeast and certain other foodstuffs (Funk, 52), and led to the isolation of a substance possessing similar properties Funk considered that the chemical properties of the curative substance placed it in the class of pyrimidine bases. The substances isolated by Funk do not, however, appear to be the active substance in a pure condition. Bather does it seem that they are nitrogenous bases contaminated with traces of the accessory factor (Barger, 53 ; Drummond and Funk, 54). Another attempt to isolate the factor was that made by Susuki, Shinamura, and Odake (55), who claimed to have prepared the anti-neuritic substance, which they termed oryzanin, in the form of a crystalline picrate. Drummond and Funk (54) failed to confirm their results. Edie, Evans, Moore, Simpson, and Webster (56) obtained an active fraction from yeast, from which they precipitated a crystalline basic substance belonging to the pyrimidine group. This they termed torulin. The precipitation of the active substance by silver nitrate and baryta has been confirmed by Cooper (57) and by Vedder and Williams (78) and Williams and Saleeby (79). Further, Williams and Seidell (60) prepared a sample of adenine from yeast, and found that this had anti-neuritic properties, but lost them on keeping. They attributed this change to isomerisation, but their results have not been confirmed by other workers (Voegtlin and White, 61 ; Harden and Zilva, 62). Quite recently Abderhalden, who on more than one occasion has vigorously attacked the vitamine theory, has published a paper with Schaumann (63), in which they report attempts to isolate the accessory substance of yeast. This long and obscure paper marks little or no advance in this field ; no active substances were obtained in a pure condition, the previous work of many investigators is ignored, and the subject is still further confused by the introduction of new names (eutonin and nutramine) for the accessory substances. Hofmeister, in his recent review of the vitamines (64), states that he has isolated from a very active solution a substance belonging to the pyrimidine series. It yields a crystalline hydrochloride and double salt with gold chloride, and he has given it the formula 38 C-H U N0 2 . The details of his work do not appear to have been published as yet. Drummond (65) has made a study of the water- soluble accessory factor in yeast extract, and has suggested that many of the results obtained by previous investigators may be explained by the readiness with which this substance is carried down in the adsorbed condition by precipitates. SCURVY. Scurvy has for many centuries been regarded as a disease due to dietetic errors, and rightly so. It was common knowledge in olden times, especially among seafaring folk, that scurvy occurred after deprivation for long periods of fresh foodstuffs, and that it could be prevented and rapidly cured when fresh vegetables and fruits were available. Thus Bachstrom in 1734 (Observationes circa scorbutum ; eiusque indolem, causas, signa et curam) wrote as follows : ' From want of proper attention to the history of the scurvy, its causes have been generally, though wrongfully, supposed to be cold in northern climates, sea-air, the use of salt meats, etc., whereas this evil is solely owing to a total abstinence from fresh vegetable food and greens ; which is alone the true primary cause of the disease. And where persons, either through neglect or necessity, do refrain for a considerable time from eating the fresh fruits of the earth, and greens, no age, no climate or soil are exempted from its attack. Other secondary causes may likewise concur, but recent vegetables are found alone effectual to preserve the body from this malady ; and most speedily to cure it, even in a few days, when the case is not rendered desperate by the patients' being dropsical or con- sumptive ' (translation given by Lind, 66, p. 394). Other theories of the etiology of scurvy dating from more recent times include, firstly, that of bacterial origin frequently advanced from different sources, and secondly, the suggestion that the cause is chronic poisoning from putrefying meat or fish. Many adherents of the former theory (e. g. Coplans, 67 ; Jackson and Moody, 68) have brought forward some experimental support for their view, the explanation being doubtless that any animal in a scorbutic con- dition, due to dietetic deficiency, will be a ready prey to a secondary infection. The second view, first advanced by Torup (quoted by Jackson and Harley, 69) and supported by the work of Jackson and Harley (69), has received wide acceptance in recent times, especially among the leaders of some Arctic expeditions, with the result that the nature of the stores taken and diet arranged was modified accordingly. There is, however, little, if any, support for this view in the final experience of the expeditions, and the inter- pretation placed by Jackson and Harley upon their experimental results has not been accepted by their scientific colleagues (Hoist and Frolich, 70, p. 42). It is convenient in this place to allude to the most recent attempt to explain scurvy on lines other than that of a deficiency disease, although the theory refers to the experimental scurvy of guinea- pigs rather than to human scurvy. McCollum and his co-workers (71, 72, and 73) consider guinea-pig scurvy to be caused by chronic constipation brought on by an unsuitable, rather than a defective. 39 diet. Their view is based mainly upon the different results obtained in guinea-pigs and rats, respectively, by a diet of milk and grain ; the guinea-pigs were found frequently to die of ' scurvy ', while the rats grew in a normal manner. These workers, therefore, consider that this diet contains adequate nutritive constituents for both types, so that the failure on the part of the guinea-pig must be referred to a defect in the physical properties of the diet, i. e. to lack of 4 roughage '. In the light of the recent experimental work on milk detailed on p. 45, it is evident that the discrepancy can be explained by the low value of milk as an anti-scorbutic foodstuff, coupled with a difference in the requirements of these two, animals as regards the amount of anti-scorbutic vitamine necessary for the maintenance of health. McCollum and his colleagues confine their interpretation of scurvy to the experimental disease produced in guinea-pigs, but this dis- order has been accepted on convincing evidence as the physiological equivalent of human scurvy by all other workers in this field. It is evident, therefore, that the identity of the two disorders must be denied or that the theory of McCollum must be applied also to human scurvy. The evidence against such a view is overwhelming, and as regards the guinea-pig disease, McCollum's views have been combated both in England and America in a series of publications (74, 75, 76, 77, 78, 79, and 80) showing how the experimental results of McCollum and his colleagues can reasonably be interpreted to give full support to the vitamine hypothesis. 1 Systematic experimental work on scurvy dates from the early part of the present century, when Professor Axel Hoist and his colleagues at the University of Christiania made a careful study of guinea-pig scurvy, its etiology, symptoms, and methods of pre- vention and cure, and decided that it offered a complete analogy with the human disease. One or two centuries previously, wherj scurvy was an everyday occurrence and the common dread of all seamen, rough * experiments ' were from time to time made upon the human material so unfortunately provided. Thus Lind, in the middle of the eighteenth century, published the following account of an ' experiment ', which expresses, as the result of his own careful observation, what became the common knowledge of his own day. ' On the 20th May, 1 747, I took twelve patients in the scurvy, on board the "Salisbury" at sea. Their cases were as similar as I could have them. They all in general had putrid gums, the spots and lassitude, with weakness of their knees. They lay together in one place, being a proper apartment for the sick in the fore-hold ; and had one diet common to all, viz. water-gruel sweetened with sugar in the morning, fresh mutton-broth often times for dinner ; at other times light puddings ; boiled biscuit with sugar, etc., and for supper, barley and raisons, rice and currants, sago and wine, or the like. ' Two of these were ordered each a quart of cyder a day. Two others took twenty-five drops of elixir vitriol, three times a day, 1 According to a private communication received from Professor McCollum he has been convinced by these arguments and now regards guinea-pig scurvy as a deficiency disease. 40 upon an empty stomach ; using a gargle strongly acidulated with it for their mouths. Two others took two spoonfulls of vinegar three times a day upon an empty stomach ; having their gruels and their other food well acidulated with it, as also the gargle for their mouths. Two of the worst patients, with the tendons under the ham rigid (a symptom none of the rest had) were put under a course of sea- water. Of this they drank half a pint every day, and sometimes more or less, as it operated by way of gentle physic. Two others had each two oranges and one lemon given them every day. These they ate with greediness, at different times upon an empty stomach. They continued but six days under this course, having consumed the quantity that could be spared. The two remaining patients took the bigness of a nutmeg three times a day of an electary recommended by an hospital-surgeon, made of garlic, mustard-seed, rad. raphan, balsam of Peru, and gum myrrh ; using for common drink, barley-water well acidulated with tamarinds ; by a decoction of which, with the addition of cremor-tartar, they were gently purged three or four times during the course. ' The consequence was, that the most sudden and visible good effects were perceived from the use of the oranges and lemons ; one of those who had taken them being at the end of six days fit for duty. The spots were not indeed quite off his body, nor his gums sound ; but without any other medicine, than a gargarism of elixir vitriol, he became quite healthy before we came into Plymouth, which was on the 16th. June. The other was the best recovered of any in his condition ; and being now deemed pretty well, was appointed nurse to the rest of the sick. ' Next to the oranges, I thought the cyder had the best effects. It was indeed not very sound, being inclinable to be aigre or pricked. However, those who had taken it were in a fairer way of recovery than the others at the end of the fortnight, which was the length of the time all these different courses were continued, except the oranges. The putrefaction of their gums, but especially their lassitude and weakness, were somewhat abated, and their appetite increased by it. ' As to the elixir of vitriol, I observed that the mouths of those who had used it by way of gargarism, were in a much cleaner and better condition than many of the rest, especially those who used the vinegar ; but perceived otherwise no good effects from its internal use upon the other symptoms. I indeed never had a great opinion of the efficacy of this medicine in the scurvy, since our longest cruise in the " Salisbury" from the 10th. August to the 28th. October, 1746 ; when we had but one scorbutic case in the ship. The patient was a marine (one Walsh), who after recovering from a quotidian ague in the latter end of September, had taken the elixir vitriol, by way of a restorative for three weeks ; and yet at length con- tracted the disease, while under a course of medicine recommended for its prevention. 1 There was no remarkable alteration upon those who took the electary and tamarind decoction, the sea-water, or vinegar, upon comparing their condition, at the end of the fortnight, with others who had taken nothing but a little lenitive electary and cremor- 41, tartar, at times, in order to keep their belly open ; or a gentle pectoral in the evening, for relief of their breast ' (Lind, 66, p. 56). Hoist and Frolich (70, 81) showed that scurvy could be induced in guinea-pigs by removing the greenstuff from the ordinary diet of grain and cabbage leaves, and by giving a diet consisting of grain and water only. It proved immaterial what grain was used, and the experiments included uncooked maize, oats, barley, and rice, as well as wheat and rye in the form of bread. In all cases this diet caused severe scurvy from which the animals died in from twenty to forty days, showing the haemorrhages and bony changes character- istic of the disease. By supplementing the scurvy diet with fresh vegetables, fruits and fruit juices, these observers were able to maintain the animals in health. For example, scurvy was prevented by a daily ration of 30 grm. of fresh raw 7 cabbage, dandelion leaves, sorrel, carrot or cranberries. When these anti- scorbutic materials were heated, their value was reduced by an amount depending on the temperature and time of heating ; after heating at 100 C. for one hour (and in some cases for half an hour) the same ration was found inadequate to prevent scurvy. A similar result was obtained with dried materials ; rations equivalent to 30 grm. of raw carrot, dandelion leaves or cabbage leaves failed to prevent scurvy when in the dried condition, and equally dis- appointing results were obtained with dried potatoes. The anti-scurvy substance originally present in the expressed juices of vegetables suffered a similar fate rapidly on keeping, but the expressed juices of acid materials, e. g., lemons, raspberries or sorrel leaves were found to be more stable in this respect. A further result of first-class importance was obtained by Fiirst (82) also working in Professor Hoist's laboratory. He found that, whereas dry cereals or pulses, e. g., oats, barley, lentils, peas, beans, were unable to prevent scurvy in the dry condition, if soaked in water and allowed to begin germination for 2-3 days they acquired anti-scurvy properties. This result has recently been confirmed by Chick and Hume (88), and Chick and Delf (84) as regards pulses. In the case of cereals it has been called in question by Weil, Mouri- quand, and Peronnet (85). The published account of their experi- ments is not very detailed, and it does not seem impossible that they might be interpreted in the opposite sense to that adopted by the authors themselves. From the researches of Hoist and his colleagues we are enabled to make the following generalization. The anti-scorbutic accessory factor is found in nature associated with living tissues in which metabolic processes are still proceeding. When these active processes cease or are greatly reduced, as in seeds, or when the tissues are destroyed, as in drying or heating, the anti-scurvy ' vitamine ' also disappears. In the case of seeds it is created anew during germination. The distribution of this factor thus presents a marked contrast with that of the anti-beri-beri factor, of which one of the principal sources is found in dry seeds. The researches of Hoist and his co-workers have been continued during the last three years by a group of workers at the Lister Institute, with the aim of making a more complete survey of the commoner 42 foodstuffs and assigning to each some quantitative value as regards anti-scurvy properties. Although the investigation is still incomplete, many results have already been published owing to the urgent need FIG. 13. 1 Weight Charts of five typical experiments with guinea-pigs showing the anti-scorbutic value of orange juice and fresh cabbage leaves : CURVE A. Typical scurvy on a diet of oats, bran and water. CURVE B. Typical scurvy on a diet of oats, bran and sterilized milk (heated to 120 C. for one hour in the autoclave). CURVE C. Typical scurvy on a diet of oats, bran and water, cured by addition of orange juice and autoclaved milk to the diet on the twenty-second day, when the symptoms were well marked. CURVE D. Weight chart showing influence of 5 c.c. fresh orange juice daily added to the ' scurvy ' diet ; autoclaved milk added to the diet on the fifty-sixth day. CURVE E. Normal weight chart on a diet of oats, bran and cabbage leaves (30 grm. . daily). CURVE F. Weight chart on diet of oats, bran, autoclaved milk and 3 c.c. fresh orange juice daily ; specially favourable circumstances warm weather, &c. for such knowledge presented by war-time conditions (Chick and Hume, 83 ; Chick, Hume, Skelton, and Smith, 86 ; Chick and Ehodes, 87 ; Delf, 88 ; Delf and Skelton, 89). To these workers and to other members of the staff at present engaged in this research we 1 Reproduced from the figure in the Transactions of the Society of Tropical Medicine and Hygiene with the permission of that Society; Chick and Hume, vol x, 1917, p. 152. FIG. 14. [To face p. 43.] 43, are indebted for much unpublished information, and for permission to incorporate it in the following paragraphs. Young, growing guinea-pigs (350 grm. weight) have been employed for the work, the methods used being in essential those of Hoist and his colleagues. The principal modification has been the substitution of water in the scurvy diet of grain and water by a daily ration of about 60 c.c. strongly heated (autoclaved at 120 C. for one hour) milk. This addition of milk very greatly improves the general condition of the experimental animals, although the onset of scurvy is not thereby seriously influenced. Normal growth takes place for the first 15 to 20 daj^s of the experiment in spite of absence of anti-scorbutic material ; with onset of scurvy symptoms, about the twentieth day, the weight of the animal begins to decline, death from acute scurvy ensuing about the thirtieth to fortieth day. The general type of the weight curve on a scurvy diet with and without the addition of autoclaved milk is well seen in Fig. 13 by observing the details of curves A and B respectively. The main object of the work has been to determine the minimal amount of the various foodstuffs that must be added daily to the ' scurvy ' diet in order to prevent occurrence of scurvy. When this minimal amount (or more) is consumed, growth does- not cease about the twentieth day, but the animal continues to grow in good health without symptoms of scurvy (Curve F, Fig. 13). As a general rule the animals were kept under observation during three months. The symptoms of severe scurvy in guinea-pigs are briefly the following : tenderness and swelling of the joints is the earliest sign, and the animal will frequently adopt a position (scurvy position), in which it rests on its side, while the painful member is held twitching in the air. In other cases the animal lies down with the side of its face upon the floor of the cage ('scurvy face-ache position '), indicating that its gums and jaw have become painful (see Fig. 14). Later on teeth become loose, solid food is refused, and death may occur a few days later. In cases where the anti-scorbutic given is only adequate for partial protection, the animal will usually live on for a con- siderable time, exhibiting swollen and painful joints, but growing up and enjoying a measure of health and spirits depending on the degree of protection afforded. In case of death from scurvy the principal lesions at the post- mortem examination show close analogy with those characteristic of the human disease. Haemorrhages occur in any position, but are most frequent in the limbs. In cases where they occur in the intestinal tract blood is frequently passed in life, and death occurs suddenly. Karification of the long bones is almost invariably present, and these frequently show fracture in the neighbourhood of the junction of shaft and epiphysis. The ribs are swollen and often fractured at the juncture of bone with cartilage. Table VIII, which is drawn up by the staff working at the Lister Institute, shows the approximate anti-scorbutic value of a whole series of foodstuffs ; the values given are based upon the minimal protective doses as determined for guinea-pigs. As in some instances the experiments are still incomplete, it is not possible to attach exact numerical values in every case. The series of signs are, 44 however, arranged to express the relative value of the various materials with as much exactness as is possible at the present time. TABLE VIII. Protective Value of Various Foodstuffs against Scurvy. = no value detected. Foodstuff. "^ Cereals. Whole grain Germ Bran Endosperm, e.g. white wheaten flour, polished rice Pulses. Whole dry Germinated. (Lentils) Vegetables. Cabbage, raw cooked \ hr. at 100 C. Runner beans, green pods Carrot (juice) Beetroot (juice) Swede (juice) Potato, "cooked \ hr. at 100 C. Onion Desiccated Vegetables Pickled Vegetables Fruits. Lemon juice, fresh ,, preserved Orange juice, fresh Lime juice, fresh preserved Grapes Apples dried Tamarind, dried *Mango ,, ('Amchur') *Kokum Fresh, whites ,, yolk Desiccated Raw (juice) Tinned Cow's, raw dried Pressed, autolysed Extract, ' marmite ' Value against Scurvy. Meat. Milk. Yeast. Min. Daily Ration to prevent Scurvy in Guinea-pigs. + 4- + + toO 4- + + 4- 4- + + + + + + + + toO Less than + ?0 ?0 ?0 Less than 4- Less than + Less than raw ?0 ?0 5g. 20 c.c. More than 20 c.c. 2-5 c.c. 20 g. 60 g. expressed as equivalent in fresh cabbage. v 1-5 c.c. 5 c.c. 1-5 c.c. 10 c.c. More than 20 g. More than 20 c.c 100 to 150 c.c. * Dried fruits much estimated as anti-scorbutic by natives of India. Vegetables. Among the most potent anti-scorbutic materials 'are placed raw cabbage leaves, the raw juice of swedes, and the juices Jof the citrous fruits. Eaw cabbage leaves take the first place, 1-5 grm. daily sufficing to protect from scurvy, or, in exceptional cases, as little as 0-5 grm. (Delf, 88). Among root vegetables there are great differences, raw carrot juice and raw beetroot juice proving comparatively feeble, while raw swede juice is placed in the front rank. The potato, which was tested in the cooked condition (after half-an-hour's boiling), would appear to occupy a mean position. In this connexion it is interesting to note that the cabbage and swede 45 are nearly allied species and belong to the same natural order of plants, viz. the Cruciferae. The old legend teaching that plants with a cruciform arrangement of the flower possess special virtue in the service of mankind thus receives scientific support at this late date. Fresh fruit. Among the fresh fruits examined, the orange and lemon are easily the most valuable anti-scorbutic agents. It is therefore the more surprising to find that the juice of fresh limes is distinctly inferior, but such it has proved to be. The experiments on this point include tests with monkeys as well as guinea-pigs. Ordinary preserved lime juice was found to be almost devoid of anti- scorbutic properties, but preserved lemon juice appeared to be distinctly more satisfactory. Experiments are now in progress with the aim of discovering some method of preserving lemon juice which shall retain its "original anti-scurvy value. Dried vegetables and fruit. Dried vegetables, in confirmation of Hoist's work, have been found practically useless (89, 90). From Delf and Skelton's results, cabbage leaves within two weeks of drying were found to have lost about 93 per cent, of their original anti-scorbutic value, while three months after drying less than 5 per cent, was retained. The dried fruits examined were old specimens, probably six months old. Some degree of protection was detected in the small doses that could be administered. Milk and meat. Milk and meat come last in the order of merit. Large quantities of fresh raw cow's milk, 100 c.c. daily and over, are required if scurvy is to be prevented by its agency alone (Chick, Hume and Skelton, 74, 75). In case of raw meat juice a daily ration of 20 c.c. offered only a very slight protection ; a larger ration was not tolerated. Among the foodstuffs in which no definite anti- scurvy properties could be detected by the method employed are the following : eggs (both fresh and dried), cereals, malt (dried and kilned), preserved lime juice, autolysed yeast and yeast extract (marmite), pickled cabbage. KBSISTANCB OF THE ANTI-SCURVY FACTOR TO HEAT, DRYING, &c. The absence of the anti-scorbutic principle from dried foodstuffs and its disappearance from powerful anti-scorbutics when these are reduced to the dry condition, is sufficient proof of the sensintiveess of the anti-scorbutic vitamine to drying. It may be regarded as an axiom that dry or dried foodstuffs will not prevent scurvy, and in this respect the contrast between the anti-beri-beri and anti- scurvy factors is very marked. With regard to exposure to high temperatures, the anti-scorbutic factor is also much more sensitive than the anti-beri-beri factor, or the fat-soluble A factor. The experiments of -Hoist and Frolich upon this point have already been referred to, but more complete series of experiments upon this point have recently been carried out at the Lister Institute by Dr. Marion Delf (89). Working with 46 raw cabbage leaves the minimum daily ration required to prevent scurvy in guinea-pigs was determined to be less than 1-5 grm. and greater than 0-5 grm., i. e. about 1-0 grm. When the cabbage was heated in water at 60 C. for one hour, symptoms of severe scurvy were just prevented by a 5 grm. ration. When the tem- perature was 70, 80, 90, or 100 C. for the same period, scurvy was not satisfactorily avoided. If the time was reduced to twenty minutes, the same ration, 5 grm., prevented scurvy when the temperature was 90 C. and just failed to do so when the temperature was 100 C. The conclusion drawn from these results is that when cooked for one hour at temperatures ranging from 80 to 100 C., cabbage leaves lose about 90 per cent, of the anti-scorbutic value originally possessed (anti-scurvy value of 5 grm. ration reduced by cooking to the equivalent of about 0-5 grm. raw cabbage). In a similar manner the loss on heating in water either (a) for sixty minutes at 60 C., or (b) for twenty minutes at 90-100 C., was estimated at about 80 per cent, of the original (anti-scurvy value of 5 grm. reduced to the equivalent of about 1-0 grm. raw cabbage). An interesting point emerging from these results is that the destructive influence of heat is enhanced to a comparatively slight degree with rise of temperature. Upon the above estimates, the rate of destruction is accelerated only about threefold (time required for destruction of 80 per cent, of the original value reduced to one- third) when the temperature is raised from 60 C. to boiling point, i. e. 40 C. If the process of destruction by heat of the anti-scorbutic factor is assumed to be influenced by rise of temperature in an orderly manner, this result points to a temperature coefficient of about 1-5 for 10 C. rise of temperature. It is suggested by Delf that this low result may be of value in guiding speculation as to the character of the unknown anti-scorbutic factor, that it is opposed to the enzyme or protein-like theory of its nature and would argue a much simpler constitution. Destruction or denaturation of protein bodies by heat has been shown to possess a much higher temperature coefficient than the ordinary chemical reaction, which is accelerated 2 to 3-fold for 10 C. rise in temperature. The results obtained in a few typical instances are collected in Table IX. At the same time it must be remembered that in Delf's experiment the anti-scorbutic material was contained in a tissue of vegetable cells, and it is possible that the low temperature coefficient may have reference to some extraneous limiting factor. Much further light may be expected from experiments now in progress on the effect upon anti-scorbutic value of heating the expressed juices of fruits and vegetables. 47' TABLE IX. Coefficient of heat change for Substance. 10(7. rise in temperature. Observer. Emulsin .... 7-14 Tamman(91) Haemoglobin. . .V . . 13-8 Chick and Martin (92) Egg albumin .... 635 (92) Bacteria, disinfection by heat, B. anthracis spores . . 10 Ballner (93) Bacteria, disinfection by heat, B. typhosus ... 136 Chick (94) Vibriolysin .... 1,000 Famulener and Madsen (95) Hydrolysis of cane sugar . . 3-6 Spohr (96) The above experimental results confirm those of Hoist and Frolich (70) in showing the great sensitiveness of the anti-scurvy factor to temperatures of 100C. and below. They are also in marked contrast with the results obtained with the anti-beri-beri factor. In this case little significant destruction was detected on exposure to 100 C. for one hour or over (see above, p. 34). This greater instability of the anti-scurvy factor is a matter of the greatest importance in estimating the anti-scorbutic value of cooked vegetables and the relative merits of different methods of cooking, and will be referred to later under that heading, p. 64. The anti-scorbutic factor is soluble in water and in alcohol (Harden and Zilva, 49 ; Hess and linger, 97) ; it passes through dialysing parchment (Hoist and Frolich, 70, p. 109), or a porcelain filter (Harden and Zilva, 49), without appreciable loss. The latter observers have also shown its behaviour towards adsorbents to be different from that of the anti-beri-beri factor ; it is not adsorbed upon the surface of fine precipitates such as fuller's earth or colloidal iron. When a mixture of equal volumes of autolysed yeast and orange juice was treated with fuller's earth the anti-neuritic factor was removed while the anti-scorbutic remained unaltered. Hoist came to the conclusion that the anti-scurvy factor was more stable in acid than in neutral media. His opinion was based mainly upon the fact that acid fruit juices retained their anti-scorbutic properties much longer than vegetable juices. Kecently Harden and Zilva (98) have shown that the presence of alkalis, even when dilute (1/50 normal sodium hydrate) and at room temperature, has a rapidly destructive effect upon the anti-scorbutic vitamine. These authors have called attention to the danger involved in the practice of adding sodium carbonate when boiling green vegetables. It has been suggested that the loss in anti-scorbutic value suffered by vegetables during cooking might be lessened if the water in which they are heated were made slightly acid with citric acid. The suggestion was originally made in respect of germinated lentils in order to preserve the anti-scurvy value as far as possible where these may form the only source of anti-scorbutic material in a diet (99). It has, however, been shown experimentally by Delf (88) that, when 0-5 per cent, citric acid is added to the water in which germinated lentils are boiled, the loss in anti-scurvy properties is, if anything, greater than when no addition of acid is made. It follows, therefore, that in cooking vegetables there should be no addition either of acid or alkali to the water in which they are boiled. 48 EEFERBNCBS, (36) EIJKMAN, C., Arch. J. path. Anat. [etc.], 1897 149 197. (37) GBIJNS, G., Geneesk. Tijdsch. v. Ned. 2nd., 1901 1. (38) SCHAUMANN, H., Arch.f. Schiffs- u. Tropen-Hyg., 1910 14 Beiheft 8, 325. (39) FUNK, C., Ergebn. d. Physiol, Wiesb. (Asher and Spiro), 1913, Jahrg. 13. (39 a) BBADDON, W. L., Causes and Prevention of Beri-beri, London, 1907. (39 b) MCCABBISON, In. J. Med. Res., 1919 6 275. (40) COOPEB, E. A., J. Hyg., 1913 12 436 ; 1914 14 12. (41) CHICK, H., and HUME, E. M., Proc. Roy. Soc., 1917, B 90 44. (42) EDIE, E. S., and SIMPSON, G. C. E., Ann. Trop M. and Parasitol, 1911 5 313, (43) VEDDEB, E. B., Beri-beri, London, 1913. (44) EIJKMAN, C., Arch.f. Hyg., 1906 58 150. (45) HOLST, A., J. Hyg., 1907 7 619. (46) CHICK, H., and HUME, E. M., Proc. Roy. Soc., 1917, B 90 60. (47) CHAMBEBLAIN, W. P., and VEDDEB, E. B., Philippine J. Sc. (B), 1911 6 395. (48) SEIDELL, A., U.S.A. Public Health Reports, 1916 31 366. (49) HABDEN, A., and ZILVA, S. S., Biochem. J., 1918 12 93. (50) COOPEB, E. A., and FUNK, C., Lancet, London, 1911 2 1266. (51) FUNK, C., J. Physiol, 1911 43 395. (52) FUNK, C., ibid., 1912 45 75. (53) BABGEB, G., The 'Simpler Natural Bases (Longmans), 1914 112. (54) DBUMMOND, P. C., and FUNK, C., Biochem. J., 1914 8 598. (55) SUSUKI, V., SHINAMUBA, T., and ODAKE, S. Biochem. Ztschr., 1912 48 89. (56) EDIE, E. S., EVANS, W. H., MOOBE, B., SIMPSON, G. C. E., and WEBSTEB, A.. Biochem. J., 19126234. (57) COOPEB, E. A., ibid., 1913 7 368. (58) VEDDEE, E. B., and WILLIAMS, Philippine J. Sc. (B.), 1913 8 175. (59) WILLIAMS, R. R., and SALEEBY, ibid., 1915 11 99 (60) WILLIAMS, R. R., and SEIDELL, A., J. Biol. Chem., 1916 26 431. (61) VOEGTLIN, C., and WHITE, J. Pharmacol and Exper. Therap., 1916 9 155. (62) HABDEN, A., and ZILVA, S. S., Biochem. J., 1917 11 172. (63) ABDEBHALDEN, E., and SCHAUMANN, H., Arch.f. d. ges. Physiol, 1918 172 1. (64) HOFMEISTEB, Ergebn. d. Physiol, 1918 16510. (65) DBUMMOND, J. C., Biochem. J., 1917 11 255. (66) LIND, A Treatise on the Scurvy, London, 2nd ed., 1757. (67) COPLANS, M., Tr. Epidemiol SOB., 1904 23 1. (68) JACKSON, L., and MOODY, A. M., J. Infect. Dis., 1916 19 511. (69) JACKSON, F. G., and HABLEY, V., Proc. Roy. Soc., 1900 66 250; Lancet, Lond. 1900, April 28. (70) HOLST, A., and FBOLICH, T., Ztschr. f. Hyg. u. Infektionskrankh., 1912 72 1. (71) McCoLLUM, E. V., and PITZ, W., J. Biol Chem., 1917 31 229. (72) MCCOLLUM, E. V., J. Am. M. Ass., 1918 71 937. (73) Prrz, W., J. Biol Chem., 1918 33 471. (74) CHICK, H., HUME, E. M., and SKELTON, R. F., Lancet, Lond., 1918, Jan. 5. (75) CHICK, H., HUME, E. M., and SKELTON, R. F., Biochem. J., 1918 12 131. (76) COHEN, B., and MENDEL, L. B., J. Biol Chem., 1918 35 425. (77) COHEN, B., Proc. Soc. Exp. Biol and Med., 1918, April 17. . (78) COHEN, B., and MENDEL, L. B., ibid., 1918, May 15. (79) HESS, A. F., and UNGEB, L. J., J. Biol Chem., 1918 35 479. (80) HABDEN, A., and ZILVA, S. S., Biochem. J., 1918 12 270. (81) HOLST, A., and FBOLICH, T., J. Hyg. t 1907 7 634. (82) FUBST, V., Ztschr. f. Hyg. u. Infektionskrankh., 1912 72 121. (83) CHICK, H., and HUME, E. M., Tr. Soc. Trop. M. and Hyg., 1917 10 141. (84) CHICK, H., and DELF, E. M., Biochem. J., 1919 13 199. (85) WEIL, MOUBIQUAND, and PEBONNET, Compt. rend. Soc. de biol, 1918 81, June 8. (86) CHICK, H., HUME, E. M., SKELTON, R. F., and SMITH, A. H., Lancet, Lond., 1918, Nov. 30. (87) CHICK, H., and RHODES, M., ibid., 1918, Dec. 7. (88) DELF, E. M., Biochem. J., 1918 12 420. (89) DELF, E. M., and SKELTON, R. F., ibid., 1918 12 448. (90) GIVENS, M. H., and COHEN, B., J. Biol Chem., 1918 36 127. (91) TAMMANN, G., Ztschr. physikal Chem., 1895 18 426. (92) CHICK, H., and MABTIN, C. J., J. Physiol, 1910 40 404. (93) BALLNEB, Sitzungsb. d. k. Akad. d. Wissensch., 1902 111 97. (94) CHICK, H., J. Hyg., 1910 10 237. (95) FAMULENEB, L. W., and MADSEN, TH., Biochem. Ztschr., 1909 11 186. (96) SPOHB, Ztschr. physikal Chem., 1899 2 195. (97) HESS, A. F., and UNGEB, L. J., J. Biol Chem., 1918 35 487. (98) HABDEN, A., and ZILVA, S. S., Lancet, Lond., 1918, Sept. 7. (99) GEEIG, E. D. W. (Private communication, quoted by Delf, 88. CHAPTER IV APPLICATION OF EXPEEIMENTAL WOKK TO THE PKACTICAL PKOBLEMS OF HUMAN DIETS. THIS section of the subject must necessarily be the one which arouses most interest, and it is, therefore, a matter for regret that our limited knowledge of the accessory substances should restrict to some extent the consideration of practical issues. Certain facts are, however, clear, and the application of these may be discussed with profit. The objection may be raised that the many theories concerning the accessory factors have been based solely on the results of experiments with animals such as rats, mice, guinea-pigs, pigeons, and, to a small degree, monkeys. There is, however, very little, if any, evidence that man's dependence on these factors is not as great as that exhibited by the rat or guinea-pig. We may assume that beri-beri and infantile beri-beri represent in man the ultimate result of a deficiency of the water-soluble factor, whilst symptoms of nutritive failure analogous to those shown by rats have been observed in young children suffering from a deficiency of the fat- soluble factor, and human scurvy is well known to result from a deficiency in the diet similar to that causing scurvy in guinea-pigs and monkeys. Evidence is also accumulating that accessory factors play a very important role in the growth processes of the higher animals, and many recent advances in animal husbandry have been based on these laboratory experiments with the lower species. It is of the greatest importance to emphasize the fact that a de- ficiency of an accessory factor may be of a much smaller order than that necessary to produce the typical syndrome of the disease usually associated with that deficiency, but may nevertheless be sufficient to induce a distinct failure of nutrition and health. This is par- ticularly true in the case of young children. Fortunately, in this country our natural dietary is so varied even amongst the poorer classes as practically to preclude an absolute deficiency of any particular factor arising, so that there is little fear of the incidence of a typical deficiency disease such as beri-beri. On the other hand, there is a very real danger that the improperly balanced dietaries consumed in many cases may lead to a partial deficiency of one or more of the accessory substances, if not of other components as well. The influence of these partial deficiencies, even when relatively slight, may be extremely serious when they occur in very early life, and, if we may judge from the experiments on animals, an adequate supply of these indispensable dietary components in later life may completely fail to make good the damage caused by the deficiencies in youth. Table X shows the distribution of the three accessory factors in the commoner foodstuffs and will be found useful for reference in reading the following pages. 1596 E 50 TABLE X. Classes of foodstuff. Fats and oils. Butter Cream Cod-liver oil Mutton and beef fat or suet Lard Olive oil Cotton-seed oil Coco-nut oil Coco butter Linseed oil Fish oil, whale oil, herring oil, &c. Hardened fats, animal or veg. origin Margarine prepared from animal fat Margarine from vegetable fats or lard Nut butters Meat, fish, &c. Lean meat (beef, mutton, &c. ) Liver Kidneys Heart Brain Sweetbreads Fish, white fat (salmon, herring, &c.) roe Tinned meats Milk, cheese, though weak and without appetite, were allowed to be up and about. No change was made in the diet, except that 10 grm. cod-liver oil were given twice daily. In the course of a few days the xerophthalmia improved, and within eight days was cured. At the same time the children gained weight, and improved in general health. No other cases of xerophthalmia developed. The skimmed milk in the diet was afterwards partly replaced by full milk. The author considers this disease to be one due to lack of the specific fat-soluble growth factor, and not to the lack of fat as such, which in the above instance had been supplied as margarine. The diet was, however, greatly deficient in the fat-soluble factor, owing to the use of skimmed milk, and cures resulted rapidly when cod-liver oil was administered. In many of the other cases of xerophthalmia described by Bloch, and showing marked oedema, the children had been nourished on diets consisting too largely of carbohydrates and had been deprived of all fats. The author, therefore, makes the suggestion that lack of fat may itself be the cause of this and other oedemata arising from malnutrition. Bloch considers this eye disease to be identical with that of Japanese children, called ' Hikari ', described by Mori (146), and successfully treated by the administration of fish- and other livers. The severer manifestations leading to blindness he considers due to superimposed infections. Throughout the work he calls attention to the frequency with which other diseases, such as diarrhoea, bronchitis, pneumonia, pyuria. discharges from ears and nose, and catarrhs of all sorts were found associated in these patients with xerophthalmia, showing a lowered resistance to infection resulting from their malnutrition. At the same time, it should be noted, especially in the light of the results discussed in a subsequent section of this report, that Bloch makes no mention o'f any signs of rickets 78 in most of the cases described. The children in many cases were not older than ahout twelve months, and it is possible that rickets may be a manifestation of a similar dietetic deficiency, less severe and extending over a longer period. ANTI-SCORBUTIC VALUE OF MILK AND ITS KEFBRBNCE TO INFANT FEEDING. Experimental work has shown (Frolich, 150 ; Chick, Hume, and Skelton, 133) that if scurvy is to be prevented by the agency of milk alone, a quantity must be consumed which practically amounts to a complete milk diet. This result is in accord with the physiological role played in nature by mammalian milk, as a food specially adapted for the complete nourishment of the young during the early period of life. Upon this basis fresh cow's milk must be classed among the less valuable foodstuffs in respect of anti-scorbutic qualities, and there is considerable evidence confirming this view. For example, Curran (151) mentions eighty cases among inmates of the Dublin unions in the Irish epidemic of 1847, who had consumed at least one pint of fresh milk a day for at least six months prior to the outbreak, although the diet had been deficient in fresh meat and vegetables. There is a considerable amount of evidence that the diet of the mother has effect upon the vitamine content of her milk. It is possible therefore that human milk may be poorer in anti-scorbutic properties than cow's milk, although it must be admitted that there is no direct evidence upon the point. The breast-fed infant has the advantage, however, that its nourishment is taken in the raw condition. It is probable that infants brought up on cow's milk do not receive any great excess of anti-scurvy vitamine, and that this is further diminished when the milk is fed to the child after boiling or in the dried condition. Medical authorities are much divided upon this point, many maintaining that an infant will thrive upon boiled milk or dried milk, without the addition of any supplementary anti- scorbutic substance, others that such is desirable and even essential. For example, Barlow (152), in his * Bradshaw lecture on Infantile Scurvy ', 1894, states his opinion that ' the boiling of cow's milk and prolonged sterilization (especially at high temperatures) lessens in some degree its anti-scorbutic quality '. Neumann (153) attributed the many cases of infantile scurvy, encountered in his private practice in 1901-2, to the practice of pasteurizing milk for infant feeding, recently introduced as a safeguard against transmission of bacterial infections. He found, on inquiry, that most of these infants had been receiving milk from one dairy in which it was the custom to * pasteurize ' the milk at 90-95 C. before delivery, and that in their homes, the milk, as an extra precaution, had been heated a second time in a Soxhlet or other apparatus at or near 100 C. for ten to fifteen minutes. Heubner (154) has published similar experiences and, in common with Neumann, has expressed the opinion that the marked increase of infantile scurvy which he noticed in Berlin about the same period must be attributed to the fashion of pasteurizing milk before feeding to infants, a custom which had been recently 79 introduced and enthusiastically adopted even to the extent- of repeating the operation more than once. The best studied instance to be found in the literature is probably that recently recorded by Hess and Pish (155). These investigators have described an outbreak of very mild and sub-acute infantile scurvy in the Hebrew Infant Asylum, New York, among infants who had been fed for several months upon a diet of cow's milk previously heated to 63 C. (145 F.) for thirty minutes. It had always been the custom in this Institution to give orange juice as an extra anti-scorbutic to the babies fed upon pasteurized cow's milk, but it had been discontinued as a result of the pronouncement of the American Medical Milk Commission (1912) that for purposes of infant feeding heated milk might be considered the equivalent of raw milk. The result was that an outbreak of mild scurvy occurred two to four months later. The babies were not very ill, they were fretful, anaemic, had no appetite, and ceased to gain weigbrt or grow. They were all over six months old, and it would have been reasonable to attribute the condition to teething troubles. The scorbutic nature of the illness was proved, however, by the ease and rapidity with which the symptoms cleared up when orange juice or other anti- scorbutic was restored to the diet, or when raw milk was substituted for the pasteurized milk. These conclusions of Hess and Fish have received confirmation in very similar observations made by Miller (156). The whole question whether the nutritive value of boiled, is equal or inferior to that of raw, cow's milk is discussed fully by Lane Claypon (157), in whose book reference is made to many authorities who consider that there is no proof of any inferiority in case of heated or pasteurized milk. It is not always stated, however, whether an extra anti-scorbutic was or was not employed with the diet of heated milk. Apart from this point it is probable that the difference of opinion existing on the whole question may be partly accounted for by differences in the length of time during which the milk was heated and the tem- peratures to which it was raised, and also by differences in sus- ceptibility of different infants and in the periods for which they were maintained upon that diet without change. As regards dried milk there is an exactly analogous difference of opinion among medical authorities. Pritchard (158) believes that * when dried milk or other preserved foods are employed it is well to give some fresh fruit juice or other anti-scorbutic '. Naish, on the other hand (159), considers that ' the risk of scurvy upon such a diet is non-existent ', and adds that he is not in the ' habit of ordering any orange juice or any other anti-scorbutic '. His view is shared by Dr. Helen Campbell among others (see Coutts, 160, p. 90). In the present state of our knowledge upon these points it is our duty to give our infants the best possible chance, and the wise course is to omit no precaution that may ultimately prove to have been necessary. An additional anti-scorbutic should therefore be given to infants who are reared on any artificial food, other than raw cow's milk. Even in this case, and that of the breast-fed infants, such a course might also prove beneficial. 80 As regards the most suitable anti-scorbutic material to be adopted, orange juice is easily the best from many points of view ; it is a very potent anti-scorbutic, is convenient, needs no preparation, and is acceptable to the vast majority of infants. Under peace conditions oranges are cheap and plentiful, but during the war their scarcity and high price placed them out of reach of many poor mothers. The juice of raw swedes seems to be the best substitute for orange juice at the moment ; the anti-scurvy value of these vegetables was found by Chick and Ehodes (161) to be very great and far in excess of the other root vegetables examined. Swedes have also the merit of being abundant and cheap. The preparation of the juice is exceedingly simple. The clean cut surface of the raw swede is grated on an ordinary kitchen grater and the pjalp folded in a small piece of muslin and squeezed with the fingers, when the juice readily runs out. Its slight sweetish taste is not disliked by infants and it is already recommended in some infant welfare centres. Canned tomatoes have been found satisfactory by Hess and linger (155 a). Where the presence of starch is not a drawback, cooked potato is also recommended by some workers (Barlow, 152; Hess and Fish, 155). The cooked potato is shaken up in water and the resulting fluid used as a diluent of cow's milk in the same manner as barley water. For curative purposes potato cream, orange juice, or grape juice is usually employed. It has recently been shown, however (Harden, Zilva, and Still, 162), that lemon juice, from which the free citric acid has been removed by treatment with calcium carbonate and alcohol, is a valuable remedy inasmuch as, owing to the absence of acid, a very large amount can be administered without causing digestive disturbance. A very serious effect of the anti-scorbutic deficiency in the guinea- pig is the degenerative change which occurs at an early stage in the odontoblastic cells of the teeth (Zilva and Wells, 162 a). It has not yet been ascertained to what degree a similar change occurs in infants or whether the permanent teeth are affected by an anti- scorbutic deficiency in early childhood. EEFERENCES. (130) DRUMMOND, J. C., Lancet, Lond., 1918, Oct. 12. (131) LTJSK, Science of Nutrition, 1918, 3rd ed., 394. (132) OSBOBNE, T. B., and MENDEL, L. B., J. Biol. Chem., 1918 34 537. (133) CHICK, H., HUME, E. M., and SKELTON, R. F., Biochem. J., 1918 12 131. (134) McCoLLUM, E. V., SIMMONDS, N., and PITZ, W., J. Biol. Chem^ 1916 27 33. (135) Medical Research Committee, Special Report Series, No. 20. A study of social and economic factors in the causation of Rickets, 1918. (136) HALLIBURTON^ W. D., and DBUMMOND, J. C., J. Physiol, 1917 51 235, (137) Report to the Local Government Board. Food Reports No. 24, 1918. Upon an inquiry as to dried milks with special reference to their use in Infant Feeding. (138) PBITCHARD, E., Pediatrics, 1913 25 632. (139) MCCOLLUM, E. V., SIMMONDS.. N., and PITZ, W., Am. J. Physiol., 1916 41 361. (140) Reports to the Local Government Board, Food Reports No. 15, 1911. On an inquiry as to condensed milks with special reference to their use as Infants' Foods. (141) Reports to the Local Government Board, Food Reports, No. 20, 1914. On the use of proprietary Infants' Foods for Infant Feeding. (142) HUTCHINSON, R., Food and the Principles of Dietetics, p. 434. London, 1911. (143) CHEADLE, W. B., Artificial Feeding of Infants, 1906. (144) STILL, F. G., Practitioner, 1900 64 611 ; 1905 75 462. (145) KNAPP, Ztschr. f. exp. Path. u. Therap., 1909 5 147. (146) MOBI, Jahrb. f. Kinderh., 1904. 81 (147) BLOCK, C. E., Ugesk. f. Laeger, 1917 79 309; Rigshospitakts Boerneafdeling: Meddeklser, 1918 2 1, 17. (148) MONRAD, Ugesk. f. Laeger, 1917 79 1177. (149) BLOCK, C. E., Rigshospitakts Boerneafdeling Meddeklser, 1918 3 57. (150) FROUCH, TH., Ztschr.f. Hyg. u. Infektionskrankh., 1912 72 155. (151) CURRAN, Dublin J. M. Sc., 1847 7 83. (152) BARLOW, T., Brit. Med. J., Nov. 10, 1894. (153) NEUMANN, Deutsche med. Wchnschr., 1902 247. (154) HETTBNER, Berl klin. Wchnschr., 1903 285. (155) HESS, A. F., and FISH, Am. J. Dis. Child., 1914 8 385. (155 a) HESS and UNGER, Proc. Soc. Exp. Biol. Med. t 1918 16 1. (156) MILLER, Ckveland M. J., 1917, August. (157) LANE-CLAYPON, J. E., Milk and its Hygienic Relations, London, 1916. (158) PRITCHARD, E., Pediatrics, 1914 26 300. (159) NAISH, ibid., 1914 26 247. (160) COUTTS, F. J. H., Report to Local Government Board, No. 116, 1918. (161) CHICK, H., and RHODES, M., Lancet, Lond., 1918, Dec. 7. (162) HARDEN, A., ZILVA, S. S., and STILL, G. F., ibid., 1919, Jan. 4. (162 a) ZILVA ; S. S., and WELLS, F. M., Proc. Roy. Soc., 1919, B. 90 505. 1596 CHAPTER VI EICKETS AS A DEFICIENCY DISEASE. IN consequence of the association of scurvy and rickets sometimes found in children, and further because of the efficacy of cod-liver oil as a therapeutic agent in rickets, it has naturally occurred to some (Hopkins, 163 ; Funk, 164) that this latter is also a deficiency disease, its development depending upon some dietetic error involving accessory food factors. Experimental medicine has provided much positive knowledge concerning disease, but it has also shown how unreliable are many views on etiology when the basis is supposition and conjecture unconfirmed by scientific investigation. Keference to the literature of rickets (Findlay, 165) shows that it occupies a pre-eminent place among diseases whose etiology is surrounded by conjecture, and it is hardly an exaggeration to say that every specialist in children's diseases has his own hypothesis as to the causative factor of rickets. This portion of the monograph consists largely of a summary of an experimental investigation undertaken by E. Mellanby for the Medical Eesearch Committee upon rickets in puppies (see Mellanby, 166). Although incomplete, the results are sufficiently definite to place rickets among the deficiency diseases. This investigation carried out on puppies has consisted essentially in placing the animal on standard diets found to produce rickets, and then adding to the standard diets other substances in order to determine the effect of these on the development of the disease. As a result of the work it has been found possible to make a provisional arrangement of foodstuffs into those preventing and those not pre- venting rickets. The standard diets used in the course of the work were as follows : Diet I. Whole milk, 200 c.c. Porridge, Oatmeal Rice 2 gr. NaCl Diet 11. Whole milk, 175 c.c. White bread NaCl 1-2 gr. Diet III. Separated milk, 175 c.c. White bread (70 % wheaten) Linseed oil, 10 c.c. Yeast, 10 grm. NaCl 1-2 gr. Diet IV. Separated milk, (250-350 c.c.) White bread (70%) * Linseed oil, 10 c.c. Yeast, 5-10 grm. Orange juice, 3 c.c. NaCl 1-2 gr. The abstraction of the butter from milk and its substitution by linseed oil in Diets III and IV was carried out in consequence of results obtained with the earlier diets. Similarly yeast and orange juice were added because they were found to improve the general condition of the animal and to allow better growth. In work of this nature it is obviously desirable to have such a diet as will allow the best possible general health and rate of growth consistent with a rapid development of the disease. As rickets is a disease that does not itself kill the child affected, and FIG. 10. Rickets following a diet of 175 c.c. whole milk, white bread ad lib, and 10 c.c. linseed oil per diem. Time of experiment, 5| months. Increase in weight during period of experiment 2,670 grm. 83 in its development is most obvious in the well-grown child, it is evident that experimental work of this nature can only be satisfactory when good growth is obtained and the mortality is not abnormally high. It is true that puppies on Diets III and IV are very susceptible to extraneous diseases like distemper and mange, and these can only be excluded by taking great care of the animals. Under laboratory conditions a puppy taken from its mother at six weeks old and placed on Diet IV ought to develop well-marked rickets within six weeks. The development and symptoms of rickets in puppies may be shortly described as follows (see Fig. 16). The bones are defectively calcined and the legs bend, the ligaments are loose and emphasize the leg deformity. Swellings at the epiphyses of bones are obvious, a rosary develops at the costo-chondral junctions, with other chest deformities according to the intensity of the affection. As in the rachitic child, the animals are lethargic and usually show great inability to run some time before the bone changes would prevent their so doing. That is to say, there is loss of tone of the muscle often accompanied by a lack of desire to carry out physical exercise. After death the calcium content of the bones is low and the structure, as revealed by histological examination of the bones, is characteristic and pathognomonic, the irregularity of the epiphyseal cartilages and the presence of osteoid tissue in abundance being the most marked features. Skiagraphs of the wrists of two dogs of the same family, one on a rachitic and the other on an anti-rachitic diet, are shown in Fig. 17, A and B. The following lists indicate roughly the effects of the various substances in preventing and allowing rickets when added to the standard diets (Table XIII). It will be necessary, however, to consider some of the substances more closely because their action occasionally varies under different circumstances. TABLE XIII. Substances not preventing Rickets in Substances preventing Rickets to varying puppies. extents in puppies. Bread ad lib. Whole milk (500 c. c. a day). Oatmeal. Cod-liver oil. Rice. Butter. .Separated milk ad lib. Suet. Yeast, 10-20 gr. a day. Olive oil. Orange juice, 5 c.c. a day. Arachis oil. Linseed oil. Lard. Babassu oil. Cotton-seed oil. Hydrogenated fat. Meat. . Calcium phosphate. Meat extracts. Sodium chloride. Malt extract. Meat protein. Milk protein. The substances having some preventive action on the development of rickets can obviously be classified into two categories (1) the group of extractives and (2) the group of fats and it is necessasy to discuss these groups in turn. Meat and malt extracts. In the earlier experiments on Diet II the addition of meat and meat extract definitely prevented rickets 02 84 (Diet II contained 5-7 grm. of butter-fat). Malt extract delayed the onset of the condition, and with large doses, e. g. about 20 c.c., some degree of protection was observed. When, however, Diets III and IV were the standard, the addition of meat (5 to 50 grm. in different experiments) did not prevent the development of character- istic symptoms. In all cases the meat seemed, however, to have an inhibitory influence which varied according to the experiment. For instance, meat added to the diet of a dog whose initial weight is small and whose rate of growth is also slight, may have a large influence on the development of rickets. With a large dog growing rapidly the influence of the meat is less evident, the increased metabolism in such a case apparently demanding a large supply of the anti-rachitic factor. 1 The explanation of the different results obtained with meat, according as whether Diets II or IV were used, apparently depended on the fact that Diet II was not so powerfully rickets-producing because of the presence of 5-7 grm. of butter-fat in the whole milk. In itself the butter in the milk (175 c.c.) was not sufficient to prevent rickets, but its effectiveness was increased considerably by the addition of a small quantity (10 grm.) of meat per diem. In addition to the above results, fresh vegetables (50 grm. potatoes and turnips per diem) have definite inhibitory action when added to the standard diets. This action is, however, not great, and is almost negligible in cooked turnip when the liquid is poured away after cooking. Fats. It will be seen in the tables recording the rachitic and anti- rachitic substances that there is a wide variation in the effect of fats as regards rickets. In the first place, it can be definitely stated that the animal fats tested were more effective in preventing rickets than the vegetable fats, and the latter varied greatly among themselves. Linseed and babassu oils and a hydrogenated fat seemed to have no preventive action on the development of the disease. On the other hand, arachis and olive oils showed moderate preventive action, while oils like coco-nut and cotton-seed occupied an intermediate position. Such results demonstrate that the action of the oils does not depend on fats per se but rather on something these substances contain. If we call this something an anti-rachitic factor it is clear that cod-liver oil and butter contain much more anti-rachitic faqtor than linseed oil. THE NATURE OF THE ANTI-RACHITIC FACTOR, The results obtained show clearly that diet plays an important part in rickets, and a study of the diets and experimental results seems to allow a satisfactory explanation along the lines of accessory food factors. It is generally agreed that there are at present three accessory food factors : (1) Fat-soluble A ; (2) Water-soluble B ; (3) Anti- scorbutic. Two of these three factors can be immediately eliminated, as may be seen by reference to Diets III and IV. For instance, yeast is a rich source of the water-soluble B accessory, and since yeast in the diet 1 See the analogous cases of beri-beri and scurvy (pp. 56, 67). FIG. 17 A, B. SKIAGRAPHS OF WI A. Duration of feeding, 3 months. Diet, 200 c.c. separated milk. White wheaten bread ad lib. Yeast, 5 grm. Linseed oil, 10 c.c. Orange juice, 3 c.c. Meat, 10 grm. Initial weight, 1,360 grm. Increase in weight in three months, 1,560 grm. In these photographs B is normal, outlines of the epiphyses and diaphysia also the large amount of cartilage betwe< with B, this difference being more partic results obtained in experiment of E. M [To face p. 84.] 3 OF TWO BOGS OF SAME FAMILY. B. Duration of feeding, 3 months. Diet as A, except that 10 c.c. cod-liver oil was substituted for linseed oil. Initial weight, 1,845 grm. Increase in weight, 2,400 grm. s well-developed rickets. Note the sharp Is of the bones in B as compared with 'A ; B epiphyses and diaphyses of A as compared y seen in the case of the ulna of A. Above 85 is compatible with rickets and shows no inhibitory effect, it is certain that the water-soluble B accessory is not the anti-rachitic factor. Again, orange juice in the diet allows rickets to develop, and therefore the anti-scorbutic factor is probably not concerned. We are now left with the fat-soluble A factor, and although the identity of this with the supposed anti-rachitic factor is not completely proved, there is some probability that the substances are the same. It is known, for instance, that animal fats are a good source of the fat-soluble A factor and that vegetable fats are deficient. Similarly the anti- rachitic factor is more abundant in the animal fats than in the vegetable fats. On the other hand, as seen above, there is a well- marked grading of the anti-rachitic factor in the vegetable fats which was not traced in the distribution of the fat-soluble factor as studied in experiments with rats. It is possible that further work will show a more harmonious distribution of the fat-soluble A and anti-rachitic factors in the vegetable fats, for, in the main, the agree- ment is sufficiently striking to suggest that the differences may be a matter of technique. 1 Considering that the detection of fat-soluble A has depended on the feeding of young rats while the work on rickets has been carried out on puppies it is not surprising that such differences should appear. It is much easier to prevent rickets in a small and slowly-growing puppy than in those of a larger type which grow rapidly. This is what might be expected from the accepted clinical observation that rickets attacks the large well-growing infant, and is not found in marasmic babies. In other words, rickets is a disease accompanying growth, and it appears that the more rapidly the animal grows the more anti-rachitic factor is necessary to keep the growth normal. Since, however, it is recognized that both fat-soluble A and water- soluble B are necessary for growth per se, in so far at least as rat feeding experiments are concerned, it might, at first sight, seem strange that a disease of growth should have its explanation in a deficiency of a growth-accessory factor. The experimental results indicate, that, however necessary it may be to have a minimum of fat-soluble A in the diet to ensure growth, it is by no means the case that the amount of growth has any relation to the amount of fat- soluble A eaten. On the other hand, there is an apparent relation between the rate of growth and the amount of anti-rachitic factor necessary to keep the growth normal and prevent the development of the rachitic syndrome. If, therefore, the anti-rachitic factor is the same as fat-soluble A, it appears from these experiments that the function of the latter is not so much to stimulate growth as to ensure the efficient working and normal development of organs and tissues. Whether the undoubted anti-rachitic effect shown in some cases (on Diets I and II) by lean meat and meat extract is due to the comparatively small amount of the fat-soluble accessory contained in them, or whether the explanation of their effect must be sought along other lines, is a question requiring quantitative investigation. It will be seen, therefore, that, although there is considerable 1 Moreover, the influence of green leaves, a valuable source of the fat-soluble acces- sory, has not yet been examined in connexion with rickets. 86 evidence that fat-soluble A and the anti-rachitic factor are identical, further work is necessary, and the above difficulties must be cleared up before it can be stated definitely that they are the same. APPLICATION OF THE FOREGOING FACTS TO OTHER HYPOTHESES CONCERNING THE ETIOLOGY OF ElCKETS. The main hypotheses that have been put forward previously concerning rickets fall for the most part under two groups : (1) Dietetic ; (2) Hygienic. There are, of course, other explanations, such as the action of the thymus (Matti, 167 ; Basch, 168 ; Klose and Vogt, 169) and other ductless glands, and the infection theory advocated by Koch (170), which cannot be so classified. It is obvious that an hypothesis involving ductless glands is not in antagonism to a dietetic explanation, more especially one concerned with acces- sory food factors; for nothing is known about the mode of action of the latter and very little of the former. (1) DIETETIC HYPOTHESES. (a) Bickets as a Disease due to Deficiency of Fat. The work of Bland Button (171) on the lion cubs at the Zoological Gardens has left its impression on English thought as regards rickets, and, together with the acknowledged efficacious results that follow the treatment of rachitic children with cod-liver oil and other fats, has brought about a general acceptance of the view that rickets is due to a deficiency of fat in the diet. The results here recorded make it clear that the efficacy of the treatment curative and preventive does not depend on the fat per se, but on whether it contains an abundance of anti-rachitic factor, animal fats being very superior in this respect to vegetable fats. (b) Excess of Carbohydrate in the Diet. When a diet contains excess of carbohydrate it means that it is made up largely of cereals. Now cereals, and more particularly cereals like wheat and rice, which have undergone some manufacturing process resulting in the loss of the embryo, are most deficient in accessory food factors. A diet, therefore, of such substances is un- balanced and most effective in producing rickets. (c) Deficiency of Fat and Excess of Carbohydrate (Holt, 172 ; Cheadle, 173). This condition comprises the first two hypotheses, and what is said about these can be extended to this suggestion. Such a combination would most certainly involve a deficiency of the anti-rachitic factor. (d) Deficiency of Calcium in the Diet. An abundance of calcium in the diet either in the form found in separated milk or as calcium phosphate will not prevent rickets when the diet is deficient in anti-rachitic factor. Similarly it has been found by Stoeltzner (174) and others that a diet otherwise 87 adequate, but containing too little calcium, will not produce rickets, although soft bones deficient in calcium salts are found in the animals. It is, however, probable that a deficient calcium intake associated with deficient anti-rachitic factor intake will bring about more strongly marked bone and other deformities associated with rickets than would occur if the supply of calcium salts were adequate. Lack of these salts must to this extent be an adjuvant factor in the etiology of rickets. The above are some of the more commonly held dietetic explanations of rickets, and it will be seen that there is nothing in them which is contrary to the accessory food factor theory. In fact this suggestion in a general way focuses the above theories to a central point. (2) DEFECTIVE HYGIENIC CONDITIONS. The Von Hansemann (175) theory of domestication includes in a comprehensive way all the unhygienic conditions associated with life in civilized and especially in closely packed communities. The defects involved in civilized environment and more particularly met with in urban life are (a) dietetic, (b) involving confinement and lack of fresh air. The dietetic portion of this combination has been dealt with above and is considered to be the prime factor in the etiology of rickets. The second factor confinement and lack of exercise will now be briefly discussed. The effect of confinement on the development of rickets has been studied by Findlay (176), who has decided that it is the real cause of the disease. Findlay's experiments involved the use of fourteen puppies, and during the experimental period of three months, five of these died. One on a diet of oatmeal and water and another on rice and water, died of marasmus. The remaining twelve were fed on porridge and milk (amount not stated), and of the nine animals that were confined, three died, one of marasmus, two of broncho-pneumonia with rickets. The confined animals were rachitic. Now, in an experimental research, designed to show the influence of confinement on the development of rickets, it is necessary that the diet used should be adequate and allow healthy development. That the diet given was not healthy is suggested by the high death rate recorded, and the experimental results only show that exercise is a factor in the production of rickets, but cannot be regarded as proof that it is the primary factor. Before the acceptance of this hypothesis is possible it must be shown that confinement on an adequate diet, that is to say, one compatible with the general health of the animal, always brings about rickets. In E. Mellanby's ex- periments puppies developed no signs of rickets during confinement for three months when fed on adequate diets. 1 Eeference may here be made to the recent statistical account of an investigation made by Miss Ferguson (178) on rickets, more particu- larly in Glasgow for the Medical Kesearch Committee. The interpretation of the results of this work are regarded as being adverse to the hypothesis that rickets is a dietetic deficiency disease, 1 Findlay's work has recently been confirmed and extended by Paton, Findlay, and Watson (177). 88 and the general conclusion, although undetermined in a definite sense, is that the factors favouring the development of rickets are : 1. Insufficient space in houses. 2. Confinement in such houses. 8. Imperfect parental care. No support is given to the diet hypothesis. Some of the dietetic results obtained by Miss Ferguson have been criticized elsewhere (166), and it remains doubtful whether accurate conclusions as to the diet of individual children, particularly of infants below two years of age, can be drawn from a consideration of family diet budgets. PKOPHYLACTIC TREATMENT OF KICKETS WITH COD-LIVER OIL. The therapeutic treatment of rickets with cod-liver oil and malt Is generally regarded as efficacious, but the successful prophylactic trials made with cod-liver oil by Hess and Unger (179) gives additional weight to the view advanced in this monograph that rickets is a deficiency disease due to insufficient amount of anti- rachitic factor in the animals' diet. These workers arranged for the distribution of cod-liver oil among the children of negro families in the Columbus Hill District of New York. Eickets is rife in this district and is said to affect 90 per cent, of the whole number of infants. The oil was given to infants between four months and a year old, and the results obtained were so striking as to deserve reproduction here (Table XIV). TABLE XIV. Oil given. Duration of No. of Infants not Infants Per cent. Average total. Therapy. Infants. developing developing Non-Rachitic. Rickets. Rickets. 54 oz. 6 months 32 30 2 93 23 oz. 6 months 541 80 21 oz. 4 months 12 7 5 58 Not given 16 1 15 6 It will be observed that, whereas without cod-liver oil 94 per cent, of the children developed rickets, when cod-liver oil was given in the largest dose, only 7 per cent, of the children were so affected. The cod-liver oil was a more potent factor than breast feeding, for even when breast fed almost all these coloured babies developed rickets. The development of rickets in breast-fed babies is uncommon in England, but that it does occur is undeniable and demands the attention of research, for, in a normal way, an abundance of whole milk in the diet is incompatible with the development of rickets. Probably the explanation is to be found in the inability of the maternal organism to synthesize accessory food factors (see p. 70), so that a mother's milk is only adequate when she received a suffi- ciency of these substances in her own diet. It is interesting that Hess and Unger (180), as the result of a statistical investigation of the dietaries of negro women in New York, found a large proportion were eating too little fat ; also that the amount of milk they con- sumed was abnormally small, while vegetables and fruit only formed a, small part of their diet. 89 GENERAL CONSIDERATIONS OF EICKETS AS A DEFICIENCY DISEASE. It is but little realized how great and widespread is the part played by rickets in civilized communities. If the matter ended with bony deformities obvious to the eye it would be bad enough, but investigations have demonstrated that such deformities only repre- sent a small part of the cases affected. Schmorl's (181) histological investigations on children dying before the age of 4 years showed that 90 per cent, had had rickets. The relation between defective teeth and rickets has been placed on an experimental basis recently by the work of May Mellanby (182), and there can be little doubt that any remedy which would exclude the one would almost certainly improve and might eradicate the other (see Pig. 18). The rachitic child carries the stigma of the disease throughout life in the form of defective teeth. Nor is this the most serious part of the evil, for the reduced resistance to other diseases of the rachitic child and animal is so marked that the causative factor of rickets may be the secret of immunity and non-immunity to many of the diseases which result in the high infantile death-rate associated with urban conditions. It is a striking fact to remember that in the West of Ireland, where the infantile death-rate is only 30 per 1,000, rickets is very rare, whereas in poor urban districts of this country where rickets is rife, the infantile death-rate varies from 100 to 300 per 1,000. It is at least suggestive that there may be some relation between rickets and the enormous death-rate of infants in towns even although the disease in itself does not kill. The experimental work with puppies has shown that the rachitic condition need not be at all advanced before the animal's whole behaviour is transformed. It becomes lethargic and is far more liable to be affected by such diseases as distemper, broncho-pneumonia, and mange. The low resistance of the animal to infection developed under conditions which ultimately may lead to rickets is impressive. There is some danger in applying laboratory results to a clinical condition more especially when the results are new and for the most part uncontrolled by clinical observation. But some remarks are necessary in this connexion, for, if experimental research can point to the real cause of a disease, then not only is the curative treatment indicated, but, what is of much greater importance in the case of rickets, it ought to be possible to indicate why rickets is widespread and to direct action along preventive lines. It appears from this work that the foodstuffs of an infant ought to contain a maximum amount of anti-rachitic factor, and that 'the type and amount of fat eaten are important. Since the amount of fat a child can eat is limited, it is necessary to give children the best fat from the point of view under consideration. They should, therefore, not be given vegetable margarines or any other vegetable fat. The natural fat for a child is the fat of milk, and to give it a vegetable fat not only limits the amount of butter it can eat even if procurable, but also weighs down the diet in the rachitic direction. If additional fat is given, then cod-liver oil is the best. Milk should remain the staple article of diet not only until weaning but for some years after this time. Milk is undoubtedly better than 90 the corresponding amount of butter. Under normal circumstances the child would then be assured of a good supply of anti-rachitic factor throughout its early life. Not, however, under all circumstances is this certain, for the work of McCollum, Simmonds, and Pitz (183) has shown that before an abundance of fat-soluble A appears in the milk the mother must have a good supply of this substance in her food. So far as the water-soluble accessory is concerned Drummond (184) has confirmed the results of these workers. This means that the animal's power of synthesizing these accessory food factors is small or absent. Green leaves are a good source of the fat-soluble A factor for the cow, and a well-fed cow, from this point of view, will give good milk. The nursing mother drinks this milk and eats eggs, butter, green vegetables, &c., and the accessory food factors are passed on to her mammary glands, thereby allowing the breast-fed child to get an adequate supply. If therefore a mother's diet is deficient in the anti-rachitic factor, it is easy to understand how even the breast-fed child may develop rickets. It is probable that the same argument applies even if it should subsequently prove that the anti-rachitic factor and fat-soluble A are not .identical. These suggestions may also explain why in children brought up on cow's milk rickets develops more commonly in the winter months, when the cow's diet is more artificial and may contain less of the anti-rachitic factor. As for the action of other foodstuffs it has been pointed out that meat has an anti-rachitic effect to some extent, and even in small quantities (10 grm. a day to a puppy) will render a slightly rachitic diet safe. Vegetable juices seem also to have some inhibitory action on the development of rickets. Nowadays, when proprietary foodstuffs for children are much in evidence, the public should insist on knowing the relative value of all these preparations as regards their accessory food factors as well as their content of proteins, fats, carbohydrates, and salts. Syn- thetic milks, especially those containing vegetable oils, should not be given to infants and children unless they have been proved to contain an adequate amount of anti-rachitic factor. Similarly, dis- pensing vegetable oils to children instead of cod-liver oil ought to be discountenanced. Finally, it may be stated that, although the experimental work here summarized deals only with young animals, it is certain that the necessity of having abundant anti-rachitic factor in the diet is also important at a later stage. Eecently May Mellanby (182) has demonstrated the effect of diets deficient in an accessory food factor, probably fat-soluble A, on the calcification of puppies' teeth (see Fig. 18). Apparently the formation of calcified enamel and the ade- quate spacing of the teeth in puppies is dependent on an abundance of some such factor. In human beings calcification of the teeth is a much slower process and continues till the eighteenth year. In order to ensure perfect calcification of the teeth, therefore, it is neces- sary that the diet should contain adequate supplies of the accessory food factor up to this time, and a deficiency at any period will be reflected in a corresponding defect in the formation of the teeth. The teeth appear to be most susceptible to this deficiency and the FIG. 18. Teeth of retriever puppies of same family. Age at death five months three weeks. Diet. 175 c.c. separated milk ; white bread (70 per cent, wheaten) ad lib. ; linseed oil, 10 c.c. ; yeast, 10 grm. In addition (d) received 5 grm. meat per diem. (e) ,, 10 (/) 50 From seventh to fifteenth week of experimental period (e) received 10 grm. of butter in addition. Increase in weight during experimental period (15 weeks) (d) 3,330 grm. (e) 4,145 grm. (/) 5,135 grm The enamel on most of the teeth of these dogs is poor, especially in the case of (/) ; it is best in (c) (butter). The size of the jaw in the case of (/) is in reality greater than is the case in (d) and (e). The magnification of the photograph is less. The photographs show (1) the good effect of butter on the enamel (e), (2) the fastest growing puppy (/) has the worst teeth. (Reprinted from paper of May Mellanby (Lancet, December 7, 1918) by permission of the Editor of the Lancet.) [To face p. 90. 91 results suggest that the defective teeth of civilized man may depend on some such type of unbalanced diet as has been suggested as the cause of rickets. In conclusion, there is good evidence that rickets is a deficiency disease due to diets which are unbalanced in that they contain too little of those substances rich in anti-rachitic factor and too much of those substances deficient in this respect. The anti-rachitic has, in many respects, a similar distribution to the fat -soluble A factor, and is possibly identical with this substance. EEFEEENCES. (163) HOPKINS, F. G., Analyst, 1906 31 395. (164) FUNK, C., Die Vitamine, 1914. (165) FINDLAY, L., Medical Research Committee, Special Report Series, No. 20, 1918. (166) MELLANBY, E., J. PhysioL, Proc. 52 xi, and Dec. 1918 ; Lancet, Lond., 1919. (167) MATTI, Ergebn. d. inn. Med. u. Kinderh., 1913 10 1. (168) BASCH, Jahrb. f. Kinderh., 1906 64 289. (169) KLOSE and VOGT, Beitr. z. klin. Chir., 1910 79. (170) KOCH, Jos., Ztsckr.f. Hyg. u. Infektionskrankh., 1912 72 321. ( 171 ) BLAND SUTTON, quoted by Cheadle and Paynton, Allbutt's System of Medicine, 3 85. (172) HOLT, Diseases of Infancy and Childhood, 1907, 251. (173) CHEADLE, Artificial Feeding and Food Disorders of Infants, 1906, 190. (174) STOELTZNER, W., Jahrb. f. Kinderh., 1899 50 268. (175) HANSEMANN, D. v., Berl. klin. Wchnschr., 1906 629 670. (176) FINDLAY, L., Brit. M. J., 1908 ii 13 (July 4). (177) PATON, N., FINDLAY, L., and WATSON, A., ibid., 1918. (178) FERGTJSON, M., Medical Research Committee, Special Report Series. No. 20, 1918. (179) HESS, A. F., and UNGER, L. J., J. Am. M. Ass., 1917, 1583. (180) HESS, A. F., and UNGER, L. J., ibid., 1918 70 900. (181) SCHMORL, Verhandl. dentalpath. Gesellsch., 1909, 58. (182) MELLANBY, MAY, Lancet, Lond., 1918, Dec. 7. (183) McCoLLiTM, E. V., SIMMONDS, N., and PITZ, W., J. Biol Chem., 1916 27 33. (184) DRTJMMOND, J. C., Biochem. J., 1918 12 25. CHAPTER VII PELLAGKA. PELLAGRA is a disease which is endemic in certain parts of the world, of which the chief are Northern Italy, Koumania, and the Southern States of North America. The disease may assume more than one form (Koberts, 185), but the characteristic symptoms in practically all cases consist of severe gastro-intestinal disturbances, accompanied by a bilateral symmetrical dermatitis which is painful and followed by desquamation and pigmentation. Until recently a survey of the literature on pellagra left the reader in obscurity regarding the etiology of the disease. The very multiplicity of theories that have been advanced was sufficient evidence that the correct explanation had not been discovered. The majority of these theories may be divided into two groups, according as they regard the disease as one of infective origin, or as the result of a faulty diet. Of those investigators who lean towards the belief in an infective origin, prominence must be given to Sambon, who made a careful study of the disease in Italy (186). From an epidemiological study of the disease, he concluded that it was not connected with the consumption of maize, but that it was an infec- tion, probably of a protozoal nature, and that flies of the genus Simulium (sand flies) were agents in the transmission. Although this theory has received a certain amount of support (187, 188), it has failed to convince many investigators who could find no relationship between pellagra and the Simulium flies (189). In no case has the announcement of the isolation of a specific organism from cases of pellagra been substantiated by further and more careful investigation. The strongest evidence against the infectivity of the disease is, however, given by the failure which has followed all attempts to transmit it to animals and man (190 and 191). In spite of this, many still regard the disease as a specific infection, but no further progress has been made in proving the correctness of this view. Turning now to those theories which ascribe the malady to dietary causes, it is found that to a large extent they originated in the observation that pellagra is frequently localized amongst people who subsist largely on a maize diet. Naturally the suspicion fell on maize, and for some little time considerable popularity was accorded to the theory which ascribed the malady to the consumption of diseased or spoilt maize (192 and 193). Several authors described the isolation of toxic substances from spoilt maize, and it was suggested that these products, produced by the growth of moulds, when ingested into the body were responsible for the production of a chronic intoxication resulting in pellagra. This and closely similar theories found many adherents, particularly in Italy. The first suggestion that pellagra was a disease produced by a deficiency in the diet was made by Funk (194). Following the researches which he made upon the etiology of beri-beri, he attempted 93 to draw an analogy between that disease and pellagra. As has already been pointed out in an earlier section, the polishing of rice removes a certain substance, present in the embryo, which is indis- pensable for normal nutrition, and an absence of which from the diet eventually induces the train of symptoms associated with beri-beri. Funk suggested that in the milling of maize a similar substance was removed,* and that an absence of that factor from the dietary would produce pellagra. He advocated the consumption of whole-meal maize and of potatoes to combat the disease (194). He was unable to produce the disease experimentally, and his theory, attractive as it might appear, was based purely upon conjecture, Since that time evidence has not been forthcoming which would justify the acceptance of his view, and a more careful study of the diet consumed in pellagrous districts is in itself sufficient to disprove the theory. In 1914 Goldberger in America reported a very careful study of the dietaries of pellagrous communities, and his results clearly indicated a controlling influence of diet both in the causation and the prevention of the disease (195, 196, and 197). His work has led to the advancement of the most satisfactory theory yet put forward regarding the etiology of pellagra. The accuracy of his conclusions, drawn from the study just referred to, was de- monstrated in a striking manner by his experimental production of the typical disease in man *by feeding upon a restricted diet similar to that consumed in pellagra zones (198). The diet he employed consisted of dishes prepared from corn, patent flour, polished rice, pork fat, corn starch, syrup, sweet potatoes, cabbage and turnip greens. Vedder (199) has also expressed the belief that the disease may be the result of a deficient diet, and pointed out that it is localized in districts where the population subsist on low protein and high carbohydrate rations derived very largely from corn products. In 1917 Chittenden and Underhill (200) reported the production in dogs of a pathological condition closely resembling human pellagra by restricting the animals to a diet of boiled peas, ' cracker meal ', and salad oil (cotton-seed oil). By quantitative variation in the food intake, the condition could be produced in varying periods of time ranging from one to six or eight months. They found that the pathological condition usually began suddenly with a refusal to eat, After abstention from food for a day or two, the animals showed a characteristic condition of the mouth and tongue, which were covered with pustules. At the same time severe gastro-intestinal disturbances were indicated by a haemorrhagic diarrhoea. In many cases the condition could be cleared up and a normal nutrition re-established by the addition of meat to the diet, but the amount of meat had to be above a certain level, otherwise no improvement resulted. In fact, it was found possible to induce the pathological condition in animals fed upon a meat, cracker meal, and lard diet, when the intake of meat was kept sufficiently low. Chittenden and Underhill believe that the condition they have described, which they suggest may be analogous to pellagra in man, may be referred to a deficiency of some essential dietary constituent or constituents. 94 A systematic analysis of the deficiencies exhibited by the dietaries characteristic of those consumed in pellagrous districts has been made by McCollum (201). As a result of the extensive researches which he has made on the nutritive value of various foodstuffs, it has been proved that the seeds of any plant (wheat, oats, maize, rice, beans, peas, flax, or millet), when fed as the sole source of nutriment, are inadequate for growth or prolonged well-being. This has been traced to the fact that they all exhibit somewhat similar deficiencies. First, the quality of the proteins they contain is low. This is particularly true of maize, which contains considerable amounts of the protein zein, the molecule of which is deficient in the biologically important amino-acid tryptophan. Secondly, they are usually deficient in certain inorganic elements, particularly sodium, calcium, and chlorine; and thirdly, with the possible exception of flax and millet, they are inadequate sources of the indispensable food unit which is termed fat-soluble A (see p. 22). McCollum has pointed out the most interesting fact, that the leaf of a plant may make good certain of the deficiencies of the seed. He has shown that cabbage and clover leaves are rich in the fat- soluble A and the inorganic elements sodium, calcium, and chlorine, and that a well-balanced food may be obtained by suitable mixtures of leaf and seed (202, 203). Working on this basis, McCollum has shown that the diets consumed by the populations of pellagrous districts show grave inadequacies, in that they are usually composed very largely of foodstuffs derived from seeds. Thus, he has analysed the rations employed by Goldberger in the experimental production of pellagra in the human being (see p. 93), ar^d has shown that 95 per cent, of the energy intake on those diets is derived from seed products or pork fat. Such a dietary shows three serious inadequacies : 1. It is undoubtedly deficient in the indispensable factor known as fat-soluble A. 2. It is probably deficient in sodium, calcium, and chlorine. 3. The quantity of proteins is low, and their nutritive value is below that of the proteins derived from meat or milk. The following dietaries are typical of those consumed in the pellagrous districts of Italy (204), and represent the type of ration low in fat-soluble A and poor as regards protein supply : I II Maize Beans Rice Potatoes Vegetable Lard Olive oil Fish Poultry j 1,091 grm. g 60 67 67 250 21 33 67 : 27 daily. t Polenta Milk Rice Potatoes Vegetable Lard Olive oil Beans Cheese . s 1,500 grm. 100 100 100 100 20 10 100 50 daily. i The theory which regards pellagra as a result of prolonged main tenance on an ill-balanced dietary of this type has recently received additional support by the careful investigation recorded by Gold- berger, Wheeler, and Sydenstricker (205). 95 A very exhaustive examination of the food purchased by pellagrous and non-pellagrous households in South Carolina was made. They showed that although the calorific intake of the pellagrous households was slightly less than that of the non-pellagrous, nevertheless the dietaries consumed by both types of household possessed calorific values comparing favourably with the recognized standards. The non-pellagrous undoubtedly enjoyed a larger supply of butter, and the animal protein foods, lean meat, milk, cheese, and eggs. Varying supplies of these foodstuffs, particularly fresh meat and milk, were associated with a corresponding inverse variation in the incidence of pellagra. In their concluding remarks they state : * the indications afforded by this study would seem very clearly to suggest that the pellagra-producing dietary fault is the result of some one or, more probably, of a combination of two or more of the following factors : (1) a physiologically defective protein supply ; (2) a low or inadequate supply of the fat-soluble vitamine ; (3) a low or inadequate supply of the water-soluble vitamine ; and (4) a defective mineral supply.' ' The pellagra-producing dietary fault may be corrected and the disease prevented by including in the diet an adequate supply of the animal protein foods, particularly milk, including butter and lean meat.' An important series of observations have been made recently by W. H. Wilson (unpublished work) of Cairo, which throw new light upon the etiology of pellagra, and go to prove that the disease is to be attributed principally if not entirely to a defective protein supply in the diet. This conclusion emerged after a careful analysis of a large series of diets known to have produced pellagra, together with others proved to be preventive or curative. Special attention was paid to the protein provided, not only as regards the actual amount, but also as regards the ' biological value '. The figures expressing the latter are taken from the work of K. Thomas (206), who investigated the nutritive properties of protein derived from various foodstuffs, and estimated their relative value in maintaining nitrogenous equilibrium in the human subject. These values were found to vary within wide limits, the ' biological value ' of vegetable protein (and especially of the protein derived from the maize grain) being less than that of milk or meat proteins. Equilibrium was not attained with maize diet, but Thomas calculated from the data obtained that the amount of ' tissue repair ' effected by 30 grm. protein from meat or milk would require over 100 grm. of maize protein. Other foodstuffs, such as fish, rice, potatoes, occupied an intermediate position. The reason for this difference lies doubtless in the fact that animal proteins are more appropriately constituted for animal nutrition as regards the amount and variety of their constituent amino-acids. The inferiority of maize proteins would be explained by the large proportion contained of zein, a protein which is devoid both of tryptophan and lysine, two amino-acids which are known to be essential for animal nutrition. Unfortunately the results obtained from the different experiments with maize were not concordant, and further confirmation is necessary before Thomas's figure for the biological value of maize protein can be accepted as final. 96 TABLE XV. ' Biological values ' of various proteins, as measured by the relative ' tissue -repairing ' values of equal weights. Ox meat . Cow's milk Fish Rice Potato 104 100 95 88 79 Caseinogen . 70 Peas . . . 56 Wheat flour . 40 Maize meal 30 It was after analysis of the pellagrous and non-pellagrous -diets from this point of view that Wilson arrived at the conclusion that danger of pellagra occurred if the * biological value ' of the protein ration was below a certain level. This level, as might be expected, varied within certain limits according to individual need and idio- syncrasy, hard manual labour needing a more generous provision in this respect. This view is in accord with many of the known facts of the incidence of the disease. For example, upon this theory pellagra would be likely to develop with greatest frequency upon a diet consisting largely of maize, owing to the low ' biological value ' of the proteins of this cereal ; at the same time the disease might occur among wheat or rice eaters, in cases of an exceptionally low total consumption, but it could rarely. occur among populations taking meat or milk even in small quantities. The protein deficiency which leads to pellagra and is possibly concerned with a defective supply of some essential amino-acid or acids may, in Wilson's opinion, occur also upon an adequate diet if assimilation is defective as the result of gastro-intestinal disease. This theory would account for certain rare cases, occurring in England and elsewhere, of a disease with symptoms closely resembling those of pellagra. In these instances there has frequently been a history of chronic diarrhoea and gas tro intestinal disturbance of long standing. EEFERENCES. (185) ROBERTS, Pellagra (London), 1912. (186) SAMBON, L. W., Journ. Trop. Med. (&c.), 1910 13 271, 287, 305, 319. (187) ROBERTS, S. R., Journ. Am. M. Assoc., 1911 56 1713. (188) British Pellagra Commission, Journ. Trop. Med. [&c.], 1911 14 344. (189) Thompson-McFadden Pellagra Commission; Summary of the First Report, Journ. Am. M. Assoc., 1914 62 8. (190) LAVINDER, C. H., FRANCIS, E., GRIM, R. M., and LORENZ, W. F., Journ. Am. M. Assoc., 1914 62 1093. (191) GOLDBERGER, J., Pub. Health Hep., Washington, 1916 31 3159. (192) CENT, Beitr. z. path. Anat. u. z. allg. Path., Jena, 39 ; cited by Funk, Die Vitamine, 1914. (193) OTTO, Ztschr.f. Klin. Med., 59 2. (194) FUNK, C., Journ. Trop. Med. [&c.], 1913 6 166. (195) GOLDBERGER, J., Pub. Health Rep., Washington, 1914 29 1683. (196) GOLDBERGER, J., ibid., 1915 30 3117. (197) GOLDBERGER, J., ibid., 1915 30 3338. (198) GOLDBERGER, J., and WHEELER, G. A., ibid., 1915 30iii. (199) VEDDER, E., Arch. Int. Med. 1916 18 137. (200) CHITTENDEN, R. H., and UNDERBILL, F. P., Am. Journ. Physiol., 1917 44 13. (201) McCoLLUM, E.V., and SIMMONDS, N., Journ. Biol Chem., 1917 32 29, 171, 347 ; 33 303. (202) McCoLLUM, E. V., SIMMONDS, N., and Prrz, W., Am. Journ. Physiol, 1916 41 333, 361. (203) McCoLLUM, E. V., SIMMONDS, N., and Prrz, W., Journ. Biol Chem., 1917 30 13. (204) LOMBROSSO and CAMTTRRI, cited by Funk, Die Vitamine, 1914. (205) GOLDBERGER, J., WHEELER, G. A., and SYDENSTRICKER, E., Journ. Am. M. Assoc., 1918 71 944. (206) THOMAS, K., Archivf. Phys., 1909, 291. APPENDIX [The following memorandum was issued by the Committee on Accessory Food Factors in June 1919; it contains a short summary of the present state of our knowledge upon this subject and of the practieal applications that can be made.] THE IMPORTANCE OF ACCESSORY FACTORS IN THE FOOD. SOME FACTS CONCEENING NUTBITION, FOE THE GUIDANCE OF THOSE ENGAGED IN ADMINISTEATION OF FOOD EELIEF TO FAMINE-STEICKEN DISTEICTS. EBCENT research has shown that the requirements of the human organism as regards diet cannot be met entirely by an adequate supply of protein, fat, carbohydrate, inorganic salts, and water. It has therefore modified the common belief of ten or more years ago, when the attention of physiologists was focussed upon the calorie or energy value of the diet. It is now established that, in addition to these necessary constituents, certain unidentified principles, known as accessory food factors or ' vitamines ', must also be present in order to maintain health and prevent the occurrence of ' deficiency diseases '. These substances have not so far been isolated, little is known of their chemical or physical properties, and at the present time their presence can only be detected by experiments with animals. These accessory factors or vitamines are widely distributed among naturally occurring foodstuffs, and in time of peace, under normal conditions of food supply , the variety of food consumed by European nations protects them from risk of any deficiency in these essential substances. Under the conditions arising from the war a different state of things exists ; in addition to a general shortage of food there is also a great restriction in the variety available, and danger from ' deficiency diseases ' is to be feared. Of these diseases scurvy is the best known, and the belief that it is caused by some deficiency in the diet has long been strongly held. Eecent research .has added to the deficiency diseases beri-beri, rickets, and other less well-marked disorders of growth and de- partures from health. The following notes have been compiled by the Committee on Accessory Food Factors in the hope that they may afford practical help to those occupied in the administration of food relief to the famine districts of Eastern Europe. The advice given is based upon the present state of our knowledge of the distribution of accessory food factors (vitamines) in natural foodstuffs and of the role played by them in preventing disease and in promoting health and growth. The accessory food factors at present recognized are three in number : (1) Anti-neuritic or anti-beri-beri factor, identified with the water-soluble B growth factor of the American investigators. 1596 H 98 (2) Fat-soluble A growth factor or anti-rachitic factor. (3) Anti-scorbutic factor. As far as is known the accessory food factors cannot be produced by the animal organism, and all animals are dependent for their supply directly or indirectly upon the plant kingdom. DISTRIBUTION AND PROPERTIES OF THE ACCESSORY FACTORS. (1) Anti-neuritic or Anti-beri-beri Factor (' water-soluble B : growth factor of the Americans). This vitamine prevents the occurrence of beri-beri in man and analogous diseases in animals. It is also necessary to promote satisfactory growth in young animals. It is widespread, and is found to some extent in almost all natural foodstuffs. Its principal sources are the seeds of plants and the eggs of animals, where it is deposited, apparently, as a reserve for the nutrition of the young offspring. Highly cellular organs such as the liver and the brain contain con- siderable amounts of this vitamine ; flesh contains comparatively little. Yeast cells are a rich source, so also are yeast extracts, e.g. * marmite '. In the case of peas, beans, and other pulses, this vitamine is distributed throughout the seed, but with cereals it is concentrated in the germ (embryo) and in the peripheral layer of the seed which in milling is peeled off with the pericarp and forms the bran. Beri-beri is occasioned by a diet composed too exclusively of cereals from which germ and bran have been removed by milling, as in the case of polished rice or white wheat flour. The disease is common where polished rice is the staple article of diet to the almost entire exclusion of other foodstuffs. It is rare, though not unknown, where white wheat bread is eaten, because the consumption of this type of cereal food is usually accompanied by a sufficiency of other foodstuffs containing the essential principle. It is unknown where rye bread is the staple food, because in the milling of rye there is no separation of the germ. (2) The Fat-Soluble A Growth Factor or Anti-rachitic Factor, necessary to promote Growth and prevent Rickets in young Animals. This vitamine appears to be necessary also to maintain health in adults, and it has been suggested that war oedema may be due to a lack of this factor in the diet. The main sources of this factor are two in number : (1) certain fats of animal origin, (2) green leaves. The most notable deposits of this factor are in cream, butter, beef fat, fish oils (for example, cod-liver oil, whale oil), egg yolk. It is present in very small or negligible amount in lard (pig fat) and in vegetable oils, as, for example, linseed oil, olive oil, cotton-seed oil, coco-nut oil, palm oil ; pea-nut or arachis oil is reported to contain it in larger amount. It will be noticed that this factor is found chiefly in the more expensive fats. While green-leaf vegetables contain the fat-soluble factor, root 99 vegetables are deficient in it ; war oedema has been frequently reported under circumstances in which root vegetables have formed a large proportion of the diet. (3) Anti-scorbutic Factor. This vitamine is necessary in a diet for the prevention of scurvy, and is found in fresh vegetable tissues and (to a much less extent) in fresh animal tissues. Its richest sources are such vegetables as cabbage, swedes, turnips, lettuce, water- cress, and such fruits as lemons, oranges, raspberries, tomatoes. Inferior in value are potatoes, carrots, French beans, scarlet runners, beetroot, mangolds, and also (contrary to popular belief) lime juice. Potatoes, although classed among the less valuable vegetables as regards anti-scorbutic value, are probably responsible for the prevention of scurvy in northern countries during the winter, owing to the large quantities which are regularly consumed. Milk and meat possess a definite but low anti-scorbutic value. This vitamine suffers destruction when the fresh foodstuffs con- taining it are subjected to heat, drying, or other methods of preservation. All dry foodstuffs are deficient in anti-scorbutic properties ; such are cereals, pulses, dried vegetables, and dried milk. Tinned vegetables and tinned meat are also deficient in anti-scorbutic principle. In case of tinned fruits the acidity of the fruit increases the stability of the vitamine, and prevents, to some extent, the destruction which would otherwise occur during the sterilization by heat and the subsequent storage. A table giving a summary of our knowledge as to* the distribution of these three accessory factors among the commoner foodstuffs is appended, p. 102. PRACTICAL APPLICATION OF THE FOREGOING FACTS TO THE PREVENTION OF DISEASE. (1) Prevention of Beri-beri. It is unlikely that any danger of beri-beri will arise among the famine-threatened districts of Eastern Europe as long as wholemeal flour from rye, wheat, barley, maize, or peas, beans, and lentils are provided. Mere shortage of food does not cause beri-beri, and poverty ensures that the whole grain is consumed for purposes of economy. (2) Prevention and Cure of Rickets or Growth Failure in Children or War Oedema in Adults. Evidence is accumulating that rickets is caused by a shortage not of fat as such, but of the ' fat-soluble growth factor ' which is con- tained in certain fats. Xerophthalmia, a severe disease of the external eye, leading, if untreated, to blindness, has also been attributed to lack of this factor. Infants and young children must therefore be supplied with the right kind of fat. To prevent rickets (1) full cream milk should be secured for artificially fed infants when possible ; failing that, (2) full cream dried milk or (3) full cream unsweetened con- densed milk. (2) is preferred to (3), and, in case of ignorant or careless mothers, even to (1), in order to prevent spread of infection H 2 100 and intestinal disorders. In all cases where (2) or (3) are used, an extra anti-scorbutic should be given (see below). Sweetened condensed milk is undesirable for the reason that the degree of dilution required by the high sugar content renders the food, as prepared, deficient in the fat -soluble (anti-rachitic) factor as well as in fat and protein. Milk and butter are the best sources of the anti-rachitic (or fat- soluble) factor for young and growing children ; margarines made from animal fats are also valuable ; those made from vegetable oils are to be condemned. If there is a shortage of butter it should be reserved for children, but if totally lacking the deficiency can be replaced by cod-liver oil and other fish oils, or by eggs. If all animal fats are unavailable, pea-nut oil should be selected in preference to other vegetable oils for preparation of margarines, &c., and some effort should be made to utilize the fat-soluble vitamine contained in green leaves. Green leaves are a cheap and readily available source of the fat- soluble vitamine, and adults can probably maintain good health when animal fats are substituted by vegetable fats if green-leaf vegetables are consumed in fair quantity. In case of this vitamine, the loss involved in ordinary cooking is not serious. Unfortunately infants or very young children cannot take green vegetables in the ordinary way, but the juices expressed from cabbages and other green-leaf vegetables, raw or even after steaming (not immersing in boiling water) for a few minutes, might be given even to infants if all other sources of this most necessary vitamine have failed. Purees, carefully prepared from cookod spinach or lettuce, can be tolerated in small quantities (one teaspoonful daily) by many young infants, and the amount taken can be increased regularly with age. In cases where rickets or growth failure or xerophthalmia are already well established, a daily dose of cod-liver oil is essential in addition to all other procedure. Pregnant and nursing mothers should have as liberal a supply of the fat -soluble factor as is possible. Kickets is not confined to artificially fed children. Breast-fed children depend for an adequate supply of this factor on the milk, which in turn depends upon the diet of the mother. (3) Prevention of Scurvy. Use of germinated seeds. If fresh vegetables or fruit are scarce or absent an anti-scorbutic food can be prepared by moistening any available seeds (wheat, barley, rye, peas, beans, lentils) and allowing them to germinate. It is necessary, of course, that these should be in the natural whole condition, not milled or split. The seeds should be soaked in water for 24 hours, and kept moist with access of air for 1-3 days, by which time they will have sprouted. This sprouted material possesses an anti-scorbutic value equal to that of many fresh vegetables, and should be cooked in the ordinary way for as short a time as possible. In case of shortage it should be remembered that salads are of more value than cooked vegetables. The extent to which the anti- scorbutic factor is destroyed during cooking depends chiefly upon 101 the time employed. When supplies are limited vegetables should be cooked separately and for as short a time as possible ; they should not be cooked for long periods with meat in soups or stews. Preserved foods, with a few exceptions, may be regarded as devoid of the anti-scorbutic principle. Lemon juice retains some value in this respect ; canned tomatoes (and presumably other tinned acid fruits) have also anti-scorbutic value. Canned vegetables are useless for prevention of scurvy, as also are dried vegetables. Infantile scurvy must be considered separately as many of the above foodstuffs are unsuited to infants or young children. To avert danger all artificially nourished infants should receive an extra anti- scorbutic. Cow's milk, even when raw, is not rich in the anti-scorbutic vitamine ; when heated, dried, or preserved, the amount contained is still further reduced. The most suitable anti-scorbutic material to use is fresh orange juice, 1-3 or 4 teaspoonfuls (5-15 c.c.) daily, according to age. Kaw swede (or, if unavailable, turnip) juice is a potent anti-scorbutic, and an excellent substitute for orange juice ; to obtain the juice the clean-cut surface is grated on an ordinary kitchen grater and the pulp obtained is squeezed in muslin. Tomato juice, even from canned tomatoes, and grape juice can also be used ; the latter is, however, less potent than orange juice, and a larger dose should be given. Pregnant and nursing mothers. If babies are breast fed it is important that the pregnant and nursing mother should receive an adequate supply of anti-scorbutic food in her diet. The popular belief that green vegetables are harmful in such cases is often without foundation. Infantile scurvy is not unknown in breast-fed children. It is evident that many of the above deficiency diseases are rife among the populations of Central and Eastern Europe. It is essential, therefore, that the principles set forth in the preceding paragraphs should be fully understood by all persons engaged in administering relief to these districts. Signed on behalf of the Committee, F. G. HOPKINS, Chairman. HAKKIETTE CHICK, Secretary. June, 1919. (Correspondence should be addressed to The Secretary, The Committee on Accessory Food Factors, The Lister Institute, Chelsea, London, S,W. 1.) 102 The Distribution of the Three Accessory Factors in the Commoner Foodstuffs. Classes of foodstuff. Fats and oils. Butter Cream Cod-liver oil Mutton fat Beef fat or suet Pea -nut or arachis oil Lard Olive oil Cotton-seed oil Coco -nut oil Coco butter Linseed oil Fish oil, whale oil, herring oil, &c. Hardened fats, animal or veg. origin Margarine prepared from animal fat Margarine from vegetable fats or lard Nut butters Meat, fish, &c. Lean meat (beef, mutton, &c.) Liver Kidneys Heart Brain Sweetbreads Fish, white fat (salmon, herring, &c.) roe Tinned meats Milk, cheese, cfcc. Milk, cow's whole, raw skim dried whole boiled Condensed, sweetened Cheese, whole milk skim Fat- soluble A or anti- rachitic factor. f + + Value in proportion to amount of animal fat contained Water-solu- ble B or anti- neuritic (anti- beri-beri) factor. Anti- scorbutic factor. very slight, if any very slight less than + + Undeter- mined less than + Fresh Dried Cereals, pulses, &c. Wheat, maize, rice, whole grain germ bran White wheaten flour, pure cornflour, polished rice, &c. Custard powders, egg substitutes, pre- pared from cereal products Linseed, millet Dried peas, lentils, &c. Peaflour (kilned) Soy beans, haricot beans Germinated pulses or cereals ?0 ?0 103 Classes of foodstuff. Vegetables and fruits. Cabbage, fresh cooked ,, dried canned Swede, raw expressed juice Lettuce Spinach (dried) Carrots, fresh raw dried Beetroot, raw, expressed juice Potatoes, raw cooked Beans, fresh, scarlet runners, raw Onions, cooked Lemon juice, fresh ,, preserved Lime juice, fresh preserved Orange juice, fresh Raspberries Apples Bananas Tomatoes (canned) Nuts Miscellaneous. Yeast, dried extract and autolysed Meat extract Malt extract Beer Fat- soluble A or Anti- rachitic factor. Water-solu- ble B or anti- neuritic (anti- beri-beri) factor. very slight + in some specimens Anti- scorbutic factor. very slight -f less than -J- + (at least) very slight very slight INDEX Accessory food factors : and growth, 5. differentiation of two, 11. distribution of the three, among food- * stuffs, 50, 51. Rohmann's criticism of theory of, 18. Adenine, alleged anti-neuritic properties of, 37. Adults, accessory food factors in diet of, 51. anti-beri-beri (water-soluble B) factor in diet of, 52. anti-scorbutic factor in diet of, 57. fat-soluble A factor in diet of, 52. Anti-beri-beri factor : distribution among foodstuffs of, 27, 28, 29, 30, 31, 35, 50. probable identity of, with water-soluble B factor, 35, 36 see also Water-soluble B factor. Anti-neuritic factor : see Anti-beri-beri factor and Water- soluble B factor. Anti-rachitic factor : nature of, 84. relation of, to fat-soluble A factor, 85. Anti-scorbutic factor : associated with living tissues, 41. best source of, for infant feeding, 80. distribution among foodstuffs of, 44, 50. effect of alkali and acid on, 47. effect of drying on, 45. effect of lack of, on growth, 42. effect of temperature on, 45. in diet of adults, 57. in diet of infants, 78. properties of, 45. relation of, to adsorbents, 47. Arctic explorations : incidence of scurvy in, 58. Beer, absence of anti- scorbutic factor from, 61. Kaffir, anti-scorbutic value of, 61. Beri-beri, 25. due to lack of an accessory food factor, 25. due to too exclusive diet of over- milled cereal, 52. outbreaks of, instances of, 53, 54. period of development of, 66. relation between incidence of, and type of rice consumed, 53. wet and dry types of, 25, 27. Breast-fed infants, development of rickets in, 88. Breast-feeding, of infants, 70. Cabbage, anti- scorbutic value of, 44, 51, 59. Cereals, distribution of anti-beri-beri (water-soluble B) factor among, 28, 29, 31, 50. ^j distribution of anti-scorbutic factor among, 44, 50. distribution of fat-soluble A factor, among, 22, 50. germinated, anti -scorbutic value of, 41, 44, 50, 60. Cheese, distribution of fat-soluble A factor in, 22, 50. occurrence of anti-beri-beri (water- soluble B) factor in, 28, 33, 50. Cooking, effect of, on anti-scorbutic value of foodstuffs, 64. Cruciferae, anti- scorbutic value of plants belonging to order of, 59. Deficiency disease, rickets as a, 89. Deficiency diseases, 2, 25. Diet of adults, accessory food factors in, 51. Diet of infants, accessory food factors in, 69. Dietary, basal, for experiments with rats, 15. Diets rich and poor in anti-beri-beri (water-soluble B) factor, examples of, 56. Eggs, absence of anti- scorbutic factor from, 44, 45, 50. distribution of fat-soluble A factor in, 23, 50. dried, value of, as source of anti-beri- beri (water-soluble B) factor, 55. occurrence of anti-beri-beri (water- soluble B) factor in, 28, 29, 31, 33, 35, 50. Eutonin, 37. Fat-soluble A factor : differentiation of, 12. distribution of, in natural foodstuffs, 21 ; table of, 22, 23, 50, 51. effect of temperature on, 23. in diet of adults, 52. in diet of infants, 71. influence of absence of, from diet, 16,18. influence of, on growth and nutrition of rats, 15. isolation of, attempted, 23. mode of occurrence of, in seeds, 19. physiological significance of, 19. properties of, 23. relation of, to metabolism of fat, 20. storage of, in animal organism, 20. Fats, animal, anti-rachitic value of, 84. distribution of fat-soluble A factor in 21, 22, 50. vegetable, anti-rachitic value of, 84. 106 Fish, distribution of fat-soluble A factor in, 22, 50. occurrence of anti-beri-beri (water- soluble B) factor in, 28, 29, 33, 50. Foodstuffs, anti-rachitic value of, 83. distribution of anti-scorbutic factor among, 44, 50. distribution of fat-soluble factor among, 22, 23, 50, 51. distribution of water-soluble B (anti- beri-beri) factor among, 27-35, 50. Fruits, distribution of fat-soluble A factor among, 22, 51. anti-scorbutic value of, 44, 45, 51, 57. dried, anti- scorbutic value of, 63. dried, occurrence of anti-beri-beri (water-soluble B) factor in, 30. Germinated pulses and cereals, anti- scorbutic value of, 41, 44, 50, 60. Germination of cereals and pulses, production of anti-scorbutic factor during, 41. Guinea-pig, experimental scurvy in the, 39, 43. Hygienic conditions, defective, and rickets, 87. Infants, accessory factors in nutrition of, 69. anti-rachitic factor in diet of, 89. anti -scorbutic factor in diet of, 78. artificial feeding of, 71. breast-feeding of, 70. fat-soluble A factor in diet of, 71. water-soluble B factor in diet of, 71. Infants' foods, proprietary : classification of, 74. value of, for infant feeding, 74. Kalzose, 18. Legumes, distribution of fat-soluble A factor among, 22, 50. Lemon juice : anti-scorbutic value of, 44, 51, 57. freed from citric acid, as a curative agent for scurvy, 80. Lime juice : anti-scorbutic value of, 44, 51, 58. relative anti-scorbutic value of, com- pared with lemon juice, 58. substitution of, for lemon juice in the Navy, 58. Maize theory of production of pellagra, 92. Malt, use of, as an anti-scorbutic, 61. M.alt extract : absence of fat-soluble A factor from, 23. anti-rachitic value of, 83. occurrence of anti-beri-beri (water- soluble B) factor in, 29, 51. Marylebone cream, 72. Meat, anti-scorbutic value of, 44, 45, 50, 63. distribution of fat-soluble A factor in, 22, 50. Meat (continued) : frozen, anti-scorbutic value of, 64. occurrence of anti-beri-beri (water- soluble B) factor in, 28, 30, 31, 50. tinned, anti-scorbutic value of, 64. Meat extract : absence of anti-beri-beri (water-soluble B) factor from, 29, 30, 51. absence of fat-soluble A factor from, 23, 51. absence of anti-scorbutic factor from, 51. anti-rachitic value of, 83. Milk, cow's, and milk products, distribu- tion of fat-soluble A factor in, 50. anti-scorbutic value of, 44, 45, 50. anti-scorbutic value of, for infant feeding, 78. as source of accessory food factors in infants' diet, 71. boiled, anti-scorbutic value of, for infant feeding, 78. occurrence of anti-beri-beri (water- soluble B) factor in, 28, 33, 35, 50. permissible modifications of, for use in infants' diet, 72. value of, as source of anti-rachitic factor in infants' diet, 90. condensed, manufacture of, 74. value of, in infant feeding, 74. dried, anti-scorbutic value of, for infant feeding, 79. methods of manufacture of, 73. synthetic, 73. value of, for infant feeding, 72. protein-free, preparation of, 7. Muscular inco-ordination in rats, due to lack of water-soluble B factor, 18. Nursing mother, accessory factors in diet of, 70, 90. Nutramine, 37. Oils, distribution of fat-soluble A factor in, 21, 22, 50. vegetable, anti-rachitic value of, 84. Onions, anti- scorbutic value of, 44, 60. Oryzanin, 37. Pellagra, 92. deficiency theory of origin of, 92. experimental in dogs, 93. experimental in man, 93. nature of diets producing, 94, 95. Wilson's observations on, 95. Pigeon, experimental avian polyneuritis in the, 27. Polished rice, nature of, 26. Polyneuritis, avian, discovery of, by Eijkman, 26. experiments of McCarrison on, 27. relation of, to human beri-beri, 26. relation of time of onset of, to size of carbohydrate ration, 56. Potatoes, anti-scorbutic value of, 44, 51, 60. occurrence of anti-beri-beri (water- soluble B) factor in, 30, 31, 35, 51. Pregnancy, accessory factors in diet during, 70. 107 Protein-free milk, preparation of, 7. Pulses, anti- scorbutic value of, 44, 50. germinated, anti-scorbutic value of, 41, 44, 50, 60. occurrence of anti-beri-beri (water- soluble B) factor in, 28, 30, 31, 33, 35, 50. Puppy, experimental rickets in the, 82. Purified diets : Hopkins's experiments with, 8. Lunin's experiments with, 6. McCollum and Davis's experiments with, 11. Osbome and Mendel's experiments with, 7. Rohmann's experiments with, 7. Stepp's experiments with, 7. Rat, basal dietary for experiments with the, 18. effect of anti-scorbutic factor on nutri- tion of the, 14. growth of, influence of fat-soluble A and water-soluble B factors on, 15-19. muscular inco-ordination in the, 18. salt mixture for experiments with the, 18. xerophthalmia in the, 17. Rice, milling of, 26. polished, nature of, 26. Rickets, 82. as a deficiency disease, 89. diets preventing and not preventing onset of, 82. etiology of, hypotheses concerning, 86. in puppies, symptoms of, 83. Mellanby's experiments on, in puppies, 82. prophylactic treatment of, with cod- liver oil, 88. relation of, to defective hygienic condi- tions, 87. Salt mixture for dietary experiments with rats, 16. Scurvy, 38. curative treatment of, 80. due to lack of an accessory food factor, 25. etiology of, theories concerning, 38. guinea-pig, Hoist's observations on, 39, 41. guinea-pig, symptoms of, 43. Lind's observations on, 39. period of development of, 66. Scurvy grass, 59. Swede juice, anti- scorbutic value of, 44, 51, 80. Teeth, effect of anti-rachitic deficiency on calcification of, in puppies, 90. effect of anti- scorbutic deficiency on structure of, 80. Temperature, effect of : on anti-scorbutic factor, 45. on fat-soluble A factor, 23. on water-soluble B factor, 34. Torulin, 37. Vegetables, anti-scorbutic value of, 44, 51. distribution of fat-soluble A factor among, 22, 51. dried, anti-scorbutic value of, 44, 45, 51, 61. Vitamines : see Accessory food factors. Water-soluble B factor : differentiation of, 12. distribution among foodstuffs of, 27, 28, 29, 30, 31, 35, 50. effect of temperature on, 34. in diet of adults, 52. in diet of infants, 71. influence of absence of, from diet, 18. influence of, on growth and nutrition of rats, 15. isolation of, attempted, 37. physiological significance of, 20. probable identity of, with anti-beri-beri factor, 35, 36. properties of, 34. relation of, to adsorbents, 35. Wheat germ, presence of anti-beri-beri (water-soluble B) factor in, 28, 29, 31, 33, 35, 50. Wheat grain, structure of, 32. Xerophthalmia in children, 75. in rats, 17. Yeast, absence of anti-scorbutic factor from, 44, 45, 51. occurrence of anti-beri-beri (water- soluble B) factor in, 28, 30, 31, 33, 35, 51. Yeast and yeast extract : distribution of fat-soluble A factor in, 23, 51. value of, as source of water-soluble B (anti-beri-beri) factor, 55. 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