chapter VII

). The other marked functional alteration in scurvy is increased susceptibility to infection; but how a vitamine deficiency induces this vulnerability cannot be explained. Clinical tests show that the blood contains sufficient antitoxin (diphtheria) to afford protection. Harden and Zilva found that "guinea-pigs fed on an unrestricted mixed diet, on a quantitatively restricted mixed diet, and a scorbutic diet showed no differentiation in amboceptor and agglutinin titres, and in the complement activity of the blood." If this is to be interpreted as indicating that the protective substances of the body undergo but little alteration, we must consider whether susceptibility to infection, as well as tendency to hemorrhage, is not due largely to alteration in the cement substance of the endothelial and epithelial membranes.

=The Mode of Action of the Vitamine.=--One of the most interesting as well as puzzling questions in regard to the antiscorbutic vitamine concerns the manner in which it prevents or cures scurvy. It is a subject which at present is in a state of flux, hardly having emerged from the realm of hypothesis, so that detailed consideration will profit little. At first the _modus operandi_ was explained and accepted as enzyme action, but it was soon evident, in view of the thermostability of the vitamine, that it could not be classed as a ferment or enzyme in the generally-accepted sense of this term. In general, it may be stated that there are two main views: one that the vitamine acts directly, and the other that it acts indirectly through the function of the endocrine glands. _Direct action_, furthermore, may be accomplished in at least one of three ways. The vitamine may (1) serve as a source of nutriment for the tissues, (2) exert an antitoxic effect on toxic products, or (3) function as a catalyzer. The first interpretation is evidently the simplest and conforms to the long-established knowledge of caloric food factors. That such small amounts as 2 c.c. of orange juice daily should suffice to protect an animal from nutritional disaster runs counter, however, to former conceptions of food nutrition. Further than this there is little against this viewpoint. In its favor is the fact that, up to a certain point, antiscorbutics act in direct ratio to the amount given; for example, 2 c.c. of canned tomato juice is insufficient to prevent scurvy in guinea-pigs, 3 c.c. will protect some but not all of a series, whereas when the amount is increased to 4 c.c. daily all animals will be saved. As we are considering new food factors it is manifestly unwise to judge them by old standards, and to decide offhand that they cannot possess such a high degree of nutritive power. This question must be regarded as still open.

The antitoxic theory suffers from the fact that the toxic origin of scurvy cannot be established. Before this is possible, it is clear that it will be difficult to bring forward convincing evidence of a neutralizing substance. Against this theory is the fact that elimination therapy is of no avail in the treatment of infantile scurvy. Hess and Unger (1919) failed to alleviate the symptoms by means of catharsis, diuresis, sweating and repeated intravenous injections of normal salt solution. In its favor it may be advanced, in a general way, that the vitamine, in many characteristics, resembles an antitoxin--in its extreme lability, its destruction by heat, aging and alkalies. On the other hand, antitoxins also are readily destroyed by acid which, as has been shown, exerts a protective influence on the antiscorbutic factor. The rapidity of action of the vitamines, one of the most impressive phenomena, calls to mind the neutralizing action of an antitoxin, and probably has given rise to the analogy. Williams suggests that the vitamines have "a general, non-specific, antitoxic or eliminative

## action" on toxic substances resulting from the metabolic decomposition

of food.

The theory has been advanced that the action of the vitamines is catalytic. Although this viewpoint has been taken regarding the water-soluble rather than the antiscorbutic vitamine, it will be well to review briefly the work on which it is based. Among the first to suggest this hypothesis were Vedder and Clark, who noted a relationship between the amount of vitamine required by fowls and their carbohydrate intake. Funk in 1913 made a similar observation in regard to beriberi, and in the following year, with von Schoenborn, showed that a vitamine-free diet led to hyperglycæmia, with diminished amount of hepatic glycogen, and that the addition of water-soluble vitamine diminished the hyperglycæmia and increased the liver glycogen. The work of Burge and his co-workers on the catalase content of tissue led to a similar conclusion. Their results may be summarized by the statement that the oxidative processes are hampered and fail to balance the autolytic changes, and, furthermore, that a relationship exists between the catalase activity, acidosis and normal oxidative processes. This theory would presuppose that scurvy is due to the formation of toxins which are normally in process of continual destruction in the body. According to some, these catalyzed toxic substances are metabolic in character, originating from incompletely oxidized food; according to another interpretation, they are the product of autolyzed tissue cells (tissue toxins). The difficulty with this explanation is that scurvy cannot be prevented or cured by a diet containing food of high catalytic power. For example, wheat embryos which, according to recent investigations of Crocker and Harrington, have a high catalytic activity, were found of no therapeutic value in relation to infantile scurvy (Hess, 3).

This problem has been approached from quite a different angle. As is well known, certain bacteria require serum, blood, milk, etc., in order to grow satisfactorily on artificial culture media. It has been established recently, primarily by the work of Lloyd, that this peculiarity in the cultivation of microörganisms is due largely to their requirement of vitamine. She found a relationship of the inverse order between the amount of amino acid present in the culture medium and the amount of vitamine required to stimulate the growth of strains of meningococcus. Reasoning from this experience, she suggests that the

## action of the accessory growth factors is to increase the reaction

velocity of the proteolytic metabolism. Here we find the vitamines once more regarded as catalyzers. This author, however, associates their

## activity with proteolytic rather than with carbohydrate metabolism.

Interesting and suggestive work of similar nature has been carried out in relation to the growth of protozoa and of yeasts (Eddy). Investigations of this kind, dealing with unicellular organisms propagated on a simple food, have the advantage of greatly simplifying the problem.

The recent work of Dutcher falls under this caption, differing merely in the fact that he attributes to the vitamines an _indirect action_. He has demonstrated that the tissues of polyneuritic birds show a decrease in catalase activity to a point 56 per cent. below normal, and that this

## activity is largely restored when the birds are cured with vitamine.

According to this writer the vitamine functions as a metabolic stimulant, and its lack results in a depression of the body oxidations with an accompanying formation of toxic metabolic products, injurious to the nervous system. The action is regarded as coming about in an indirect manner, being accomplished through the hormone action of the vitamines on one or more glands of internal secretion.

The _endocrine hypothesis_, suggested by Funk in his monograph, is not without some corroborative evidence. In testing the pharmacologic action of the water-soluble vitamine, Uhlmann found that it stimulated the various glands of the digestive tract, in this respect acting like pilocarpine. Some years ago Albert expressed the opinion that the action of this vitamine was "vagotropic" like atropine, and recently Dutcher has reported definite relief and cessation of polyneuritic symptoms by means of pilocarpine (0.5 mg. subcutaneously). He claims equally good results from thyroxin, the hormone of the thyroid gland, from desiccated thyroid and from tethelin (pituitary). Voegtlin and Myers conclude, as the result of experiments with brewers' yeast, that the chemical and physical properties of secretin and vitamine are identical.

The early work of Funk and Douglas, which showed that various _glands of internal secretion_ diminish in size and undergo degenerative changes when the diet is vitamine-free, the newer work of McCarrison and of Dutcher to the same effect, clearly point to an intimate relationship between some of the endocrine glands and the vitamines. As regards scurvy, the only work is that of Rondoni, McCarrison and of LaMer and Campbell on the adrenal glands, which were found by all to be enlarged in guinea-pigs suffering from this disorder. These investigations must be regarded as tentative rather than conclusive until confirmed by similar necropsy reports in man. In this connection it should be noted that thyroid, parathyroid or suprarenal extract is of no avail in the treatment of scurvy. This failure may, however, be explained by the fact that the normal balance of glandular activity was not established. We must bear in mind, however, that although the vitamines may influence the secretion of the glands of internal secretion, this explanation does not satisfactorily account for the symptoms of the "deficiency diseases." These disorders do not in the slightest respect resemble the clinical pictures which we are accustomed to associate with a lack of

## activity of the glands of internal secretion. If the polyneuritis of

beriberi and the hemorrhages of scurvy are attributable to a diminished secretion of the endocrine glands, then it will be necessary to revise present conceptions of their physiologic functions.

=The Fate of the Vitamine in the Body.=--One of the most important questions in relation to the antiscorbutic vitamine, quite apart from its chemical nature, physiologic function, and its source, is its fate in the human body after it has reached the alimentary canal or been carried to the tissues. It can be readily appreciated that our knowledge of this aspect is very meagre. We shall endeavor, however, to detail what little is known of this subject, conscious of the fact that investigations of the next few years may contradict our present viewpoints.

Most of the constituents necessary for the construction of tissue or for carrying on its functions can be synthesized by the animal body from the basal foodstuffs. It has been ascertained within the past decade that certain constituents--for example, some amino-acids of the protein molecule--are building-stones which cannot be primarily elaborated by the cells, but must be supplied by the food. At present the vitamines--including the antiscorbutic vitamine--are included in this new and essential group of substances which the human organism cannot manufacture. Animal experiments seem to bear out this conception of the vitamine whether we regard them as dynamic or as indispensable tissue elements in the structural sense.

A closely-related but less fundamental question is that of _the ability of man to store vitamines_--whether the tissues can hoard an excess of these factors, or whether, in this respect, we are carrying on a precarious hand-to-mouth existence in regard to cellular nutrition. It is of course clear that at all times the various organs and tissues must contain a certain amount of the vitamines. This has been shown for the water-soluble or "antineuritic vitamine" by the fact that even the organs of birds which have died of polyneuritis contain an appreciable quantity of the specific vitamine, although an insufficiency of this very factor has led to their death. That such is the case is demonstrated for the antiscorbutic vitamine by the fact that muscle tissue contains sufficient antiscorbutic to protect individuals subsisting largely on a diet of which raw meat constitutes the sole antiscorbutic agent (Stefánsson). It is very probable that some organs contain more of the vitamines than others; this has been proved for the "antineuritic" factor, and seems to hold good for the antiscorbutic--the liver being particularly rich. No quantitative study has been carried out from this point of view regarding the antiscorbutic vitamine, and it would be well worth our while to ascertain the relative antiscorbutic potency of the various organs of the body. Some time ago we undertook experiments to determine whether the guinea-pig is capable of storing this vitamine. One series of guinea-pigs was fed daily 6 c.c. of orange juice for a period of two weeks, whereas another series, of about the same weight, was given, in addition to the basal ration, only 3 c.c. per capita (the minimal protective dose). After this preliminary period both series were placed on a diet containing practically no antiscorbutic. Both groups came down with scurvy after about the same interval, leading to the conclusion that there could have been little if any storing of the excess vitamine by those which received twice the "minimal protective dose." The experiments of Harden and Zilva, who fed a concentrated lemon juice, showed that this potent agent also was unable to provide against a subsequent period of antiscorbutic deficiency. It should be realized that the results of these tests on guinea-pigs cannot be applied to man without tests on other species.

It might be thought that _the blood_--the purveyor of the vitamines to the tissues--would be particularly rich in these essential factors. Such, however, was not our experience in respect to the antiscorbutic vitamine. The blood possibly varies greatly in this respect according to the diet of the individual, or even according to the interval elapsing after the ingestion of antiscorbutic food. Our opinion is based on the surprisingly poor therapeutic effect of blood transfusion in the treatment of scurvy. To illustrate: An infant weighing about fifteen pounds received six intravenous injections of citrated blood--one of 200 c.c., given by the direct method, and a month later five smaller transfusions with citrated blood, which aggregated 205 c.c.[25] In spite of this addition of blood, the hemorrhage and congestion of the gums did not disappear, nor the general condition improve, as would have happened had 50 or 75 c.c. of orange juice been given by mouth. It seems probable that small quantities of vitamine are being transmitted at all times by the blood and supplied to the cells, but that its normal content of this factor is not great. The antiscorbutic potency of blood may perhaps be compared to that of milk. Animal investigation may show that various vessels--for example, those supplying or draining certain glandular organs--differ in the antiscorbutic quality of the blood which they carry. It is evident, therefore, that many transitory factors may influence the vitamine content of the blood, and that--as in the case of milk and fruits and vegetables--we are not dealing with a constant and unvarying agent.

[25] The dates and quantities of the transfusions were as follows: March 26th, 200 c.c.; April 27th, 30 c.c.; April 28th, 35 c.c.; April 29th, 30 c.c.; May 2nd, 75 c.c.; May 3rd, 35 c.c.

Nothing whatsoever is known concerning the _excretion_ of the antiscorbutic vitamine. No attempts have been made to recover it from the urine, or to ascertain if, when large amounts are ingested, the excess is thrown off by the body. This suggests the question--a corollary of that raised in connection with the vitamine content of the blood--whether it is immaterial if the vitamine is taken frequently in small amounts, or is provided only occasionally and at longer intervals in larger amounts. Is it of no moment whether the infant receive its quota of antiscorbutic every few hours through the medium of the breast milk, or only once a day in the form of orange juice or tomato? If we turn to studies on the other vitamines for enlightenment as to the possibility of excretion, we find that Muckenfuss recovered the water-soluble factor from ox bile and from human urine.[26] In this article he proposes the interesting question of a possible variation in the vitamine output under pathological conditions, which may be responsible for the development of functional disturbances in children.

[26] This investigation was carried out by means of activating fuller's earth with these fluids. This method is inapplicable to the antiscorbutic vitamine, which is not adsorbed by this material. An attempt to feed concentrated human urine to guinea-pigs resulted in their death.

It would be of interest to know the _fate of the antiscorbutic vitamine in the gastro-intestinal tract_. How is it affected by a lack of the acid gastric juice, or by the alkaline intestinal secretions, or by the bacteria in the lumen of the gut? From which part of the intestine is it largely absorbed? May an appreciable amount undergo destruction before this is accomplished? None of these questions can be answered satisfactorily in the present state of our knowledge, but they suggest that the mere fact that an adequate quota of antiscorbutic vitamine is provided in the food does not necessarily insure an adequate supply for the tissues. If in addition to the question of intake we must take into account that the vitamines may suffer various vicissitudes, it may come to pass that pathological conditions at times destroy or render them

## partially inactive. In this way we may account for irregularities in the

clinical course of disorders associated with vitamine deficiency.

Of prime importance, however, is the effect of the vitamines _on the glands of the alimentary tract and on the digestive processes_. A diminution of gastric juice, or in some instances a total absence, has been observed in adult scurvy, and noted by us in two cases of infantile scurvy. As mentioned elsewhere, some consider the function of the water-soluble vitamine analogous to that of secretin. In applying this hypothesis to scurvy it must be borne in mind that the sequence may be reversed, that the lack of vitamine may not lead to the gastric achylia, but that the achylia may come about secondarily as the result of the malnutrition.

We have referred to _"irregularities" in the course of the "deficiency diseases."_ A careful perusal of the literature leaves one with the impression that the most experienced observers are not entirely satisfied with the exclusively etiologic relationship of the vitamine to its respective nutritional disorder. In regard to scurvy, more

## particularly, there are numerous scattered reports where the disorder

did not yield to antiscorbutic foods as might have been expected, or where, on the other hand, it suddenly and inexplicably retrogressed, although there had been no alteration in the dietary.[27] These instances are not common, but they occur from time to time, and their occurrence must be accounted for. In relation to beriberi and avian polyneuritis improvement of this kind has frequently been explained on the theory of a sudden mobilization of vitamines from the tissues. There is, however, no data on which to base such explanations, and it may be that a lack of parallelism between vitamine intake and the clinical course may be due at times to processes taking place in the alimentary tract.

[27] Neumann, for example, writes that he has seen at least four children whose condition was not improved, although in addition to the milk, they took asparagus, spinach, and other vegetables or apple sauce. Some years ago we had a similar experience. It has likewise happened that infantile scurvy did not recur, although the diet was the same as that which originally led to the disorder.

The fat-soluble vitamine has been termed by some the growth vitamine. All the vitamines, however, are closely associated with the function of growth, which their deficiency tends to inhibit. In the chapter on symptomatology, it will be pointed out that infants suffering from scurvy fail to grow normally both in length and in weight. To a certain extent this may be due to a loss of appetite, which is one of the characteristic phenomena accompanying the scorbutic condition. On the other hand, this anorexia may be secondary and not primary to the impairment of the growth impulse, which may lead to a dysfunction of various body processes.

It is of little value to look ahead and try to foresee what the next decade will bring forth in regard to the nature of the vitamines. Investigation has broadened remarkably during the past few years and now embraces the chemical field--chemical and adsorption methods, the large realm of biology, including studies in physiology and pharmacology; and recently pathology has once more been called upon to aid in the solution of the problem. Probably additional vitamines will be discovered. From time to time it has been suggested that a specific growth vitamine exists quite distinct from the three which are recognized, and recently Mellanby has suggested still another food factor--a specific "rachitic vitamine." When we reflect that the characteristic functions of the various organs--the kidneys, liver, etc.--must depend on essential differences in chemical structure, the complexity of the entire problem of unidentified factors becomes evident.[28]

[28] For recent and comprehensive reviews of the vitamines the reader is referred to papers by A. B. Macallum (Trans. Royal Canadian Institute, Toronto, 1919) and by W. H. Eddy (Abstracts of Bacteriol. 1919, Vol. iii, 313.)

##