The Clinical Significance of the Interrelation of Nutrient Factors

THE CLINICAL SIGNIFICANCE OF THE INTERRELATION OF NUTRIENT FACTORS

S.

o.

WAIFE,

M.D.*

The practicing clinician is chiefly interested in getting his patient well as quickly as possible. This is a complicated process that involves much more than specific therapy, if available. He knows that every illness leads to an inadequate intake of food and an increased bodily need for some if not all of the essential nutrients. The pigeon-holing of facts which inevitably develops as knowledge accumulates, has, in the case of nutrition, made the doctor think of proteins, fats, carbohydrates, calories, vitamins and minerals as independent building blocks in the total structure of man. He may not be aware of newer concepts of human metabolism, a large complex family where everyone is related to everyone else in some way. We know from recent work with isotopically tagged molecules! that all nutrients enter into a gigantic metabolic pool and there undergo simultaneous transformation and synthesis into a multitude of substances, constantly being broken down and rebuilt again at variable rates of speed and always changing with the needs of body in health and disease, at work and at rest. We intend to show that this intimate interrelation of dietary nutrients speaks against the "specificity" of clinical lesions and that the disturbance in availability of anyone vital nutrient affects the optimum function of the organism. VITAMIN A

Let us first consider the ramifications of vitamin A. The two physical signs most clearly associated with avitaminosis A are impaired adaptation to darkness and hyperkeratosis. However, Stewart2 reported that daily doses of 150 mg. of ascorbic acid produced as great an improvement in dark adaptation as did daily doses of 24,000 I.U. of vitamin A. Furthermore, good adaptation was invariably shown by subjects with a dietary history adequate for vitamins A and C. Poor adaptation was associated with a low intake of both of these substances. Vitamins A and C are also partially interrelated in that the blood and liver content of ascorbic acid was less than half of normal in experimental vitamin A deficiency, while scorbutic signs could be aggravated by increasing the protein content of the diet in avitaminosis A.3 Several reports have shown that hyperkeratotic lesion, similar to those From the Nutrition Project, Philadelphia General Hospital. The Nutrition Project is supported by grants-in-aid from Swift & Co. and National Livestock and Meat Institute. * Assistant Director in Charge of Medical Education, Philadelphia General Hospital; Instructor in Medicine, University of Pf'nnsylvania School of Medicine and Woman's Medical College of Pennsylvania.

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seen in avitaminosis A, may be found in scurvy and niacin deficiency. The skin apparently may also reflect a disturbance in fatty acids and pyridoxine. If, as has been suggested,4 pyridoxine is connected with the utilization of unsaturated fatty acids we can see how a single lesion may be the result of an inadequate amount of two or more unrelated nutrients. Vitamin A, furthermore, is partially dependent on vitamin E. Adequate hepatic stores of vitamin A require a sufficient intake of vitamin E. Curiously, however, cod liver and other fish oils (which contain vitamin A) can destroy vitamin E in the intestines, if fed within a few minutes of each other.5 Here riboflavin enters the picture, for it can counteract this deleterious effect of marine fatty acids under certain conditions. In addition, a number of investigators have shown that in the presence of vitamin A deficiency large doses of vitamin D produced toxic changes which did not occur if adequate amounts of vitamin A were given. Vitamin A also plays a role in protein metabolism. It is essential for the growth of tissue (protein) in young rats, but not for its maintenance in adulthood. It has been said that vitamin A is held in the liver in the form of a protein complex. Alcohol invariably hastened the depletion of hepatic vitamin A stores, probably by lowering the liver component to which the vitamin is attached. It also appears that in patients with cancer an adequate intake of choline is necessary for normal lipid metabolism before vitamin A can be utilized. 6 Thus we have seen that vitamin A is directly involved in th~ metabolism of choline and vitamins C, D and E. THE VITAMIN B GROUP

The literature on the activities of this heterogenous group of essential nutrients is tremendous. Only a few points of clinical interest will be mentioned to show the scope and complexity of the effect of one substance on another. Riboflavin deficiency undoubtedly results in cheilosis. However, this clinical sign is not "specific" for ariboflavinosis. Machella, 7 for example, found improvement in nine of thirteen cases of cheilosis on pyridoxine alone. The other four also failed to respond to riboflavin administration. In fact, vitamin C produced healing in two cases which had not responded to the entire B complex group. Corneal vascularization may be due to riboflavin deficiency. Kruse 8 believes this is specific. However, it has been shown that the lesion merely means that at one time a deficiency of riboflavin occurred and trauma such as wind or dust may reactivate its presence. This vascularization may be a nonspecific response to a general vitamin deficiency.Io Similar lesions have been described in experimental deficiencies of vitamin A, tryptophane, lysine, zinc and sodium.It That the same lesion may be produced by multiple deficiency (Table 1) was further shown by Sydenstricker and his associatesI2 in rats where corneal vascularization fol-

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lowed the lack of anyone of the ten essential amino acids as well as pyridoxine and pantothenic acid. Tongue changes have long been considered a specific B complex lesion. However, Bakwin and othersl3 reported that in a group of sixty children with glossitis and fissures of the tongue even prolonged (as much as two years!) and adequate nicotinamide therapy produced a very slow, irregular response if at all. Furthermore, the diets of these children did not differ significantly from those with normal tongues and they concluded that it has not been proved that these common tongue lesions are caused by aniacinosis. Othersl4 also caution against the concept of a typical pellagra glossitis or "riboflavin tongue." Riboflavin and niacin are essential in the assimilation of protein and in its resynthesis into tissue protein. The site of this interaction is probably in the liver, for the hepatic content of those substances was increased by feeding a high protein diet and not by excessive feeding of TABLE

1

SOME EXAMPLES OF MULTIPLE NUTRIENTS AFFECTING SINGLE TISSUES

(Adapted from Follis l l ) Tissue

Cornea ..... Epidermis ....... . Kidney ...... . Bone .......... . Peripheral nerve .....

May be affected by deficiencies of:

Sodium, zinc, tryptophane, lysine, histidine, riboflavin, vitamin A Magnesium, zinc, riboflavin, pantothenic acid, pyridoxine, biotin, vitamin A, linoleic acid Magnesium, potassium, chlorine, choline, linoleic acid Calcium, phosphorus, vitamins A and D Pyridoxine, pantothenic acid, riboflavin?, niacin?

the vitamins themselves. Thiamine did not join this group, for it varied directly with its dietary intake. 15 Niacin alone of the B group can alleviate the severity of choline deficiency in rats. IS Many other relationships between these members of the water-soluble group exist. Thus, in a deficiency of thiamine, riboflavin or vitamin A, but no pyridoxine, there is a reduction in the vitamin C content of tissue. 17 Another example is the findings that thiamine and pantothenic acid deficiencies interfere with riboflavin mobilization in the liver,1 8 At times, however, the administration of one member may produce a deficiency syndrome. This curious finding occurred when six members of the B complex group (thiamine, riboflavin, pyridoxine, niacin, choline and.' pantothenic acid) were fed with inositol. A syndrome developed which could only be prevented by para-aminobenzoic acid, but if paraaminobenzoic acid were added to these same six vitamins, inositol deficiency resulted. 19 The explanation apparently lies in the bacterial synthesis of bacteria which will be discussed below. Another finding of clinical importance is that in cases of thiamine deficiency there is a disturbance in riboflavin metabolism, although the

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reverse does not hold true. Thus, riboflavin deficiency may exist as a result of impaired utilization in the presence of insufficient thiamine as well as a result of an intake insufficient for the body's needs. 20 Pellagra, of course, is known to be a "specific" deficiency disease. For many years the role of corn in the production of pellagra was a puzzle and challenge. It was known that aniacinosis may exist in corn eaters even when their diets contained more niacin than in other pellagraproducing diets. It was more recently found that niacin and tryptophane are essentially interchangeable. Corn protein has a very low tryptophane content and it is believed that pellagra represents a combined niacin and tryptophane deficiency. It is now known that pyridoxine (vitamin B 6 ) is necessary for the conversion of tryptophane to niacin. Hence, it is likely that here, too, is an example of either the nonspecificity of nutritional "lesions" or better, the close interrelation of all vital foodstuffs. TABLE

2

VITAMINS AND THE ENZYME SYSTEMS IN WHICH THEY ACT

Thiamine .. Riboflavin. Nicotinic acid. Pyridoxine .... Pantothenic acid. Biotin ... Folic acid.

(Adapted from Knight 16 ) Pyruvate decarboxylation and other thiaminoprotein enzymes Oxidation of pyridine nucleotide coenzyme, i.e., cozymase; D-amino acid oxidase; oxidation of hypoxanthine and certain aldehydes Dehydrogenases, (di- and tri-phosphopyridine nucleotides) .. " Amino acid decarboxylase; transaminase, tryptophase Acetylation ? CO 2 fixation

The type of diet, of course, greatly affects the balance between vitamins. A high fat diet may increase the body's need for riboflavin and vitamin E but may spare some nicotinic acid. Pantothenic acid deficiency becomes more pronounced on a high· carbohydrate diet than on a high fat diet, while if fat is omitted, a pyridoxine deficiency becomes more pronounced. Furthermore, the requirements for all members of the B complex group depends on the carbohydrate intake (for their role is predominantly in carbohydrate intermediary metabolism) (Table 2). But not all carbohydrates act similarly. Generally speaking, starch, lactose, glucose and sucrose in that order favor intestinal synthesis (vide infra) but that order varies somewhat with specific vitamins. 21 Significantly, thiamine synthesis is depressed by rice, which itself is practically devoid of thiamine, and this may explain the prevalence of beriberi in rice-eating regions. VITAMIN C

Ascorbic acid plays a role of many facets in our metabolism. While gingivitis is frequently found with lack of this vitamin, it may also be present in deficiency of vitamins A, D and niacin. Furthermore, scurvy

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exists without gingivitis and gingivitis in otherwise healthy individuals may not respond to ascorbic acid therapy. Vitamin C also favors the absorption of iron by preventing the oxidation of the ferrous form to ferric which is less readily absorbed. 22 In addition, vitamin A is related to vitamin C in that the ability of cattle to synthesize vitamin C is reduced in the presence of vitamin A deficiency. The clinical value of an adequate vitamin C intake is apparent. Since vitamin C is necessary for the formation and maintenance of intercellular supporting tissues and vitamin D is essential for the calcification of such tissues as growing enamel dentin and alveolar bone, it may be worthwhile digressing to review what we know about that most common of all diseases-dental caries. Dental Caries.-In brief, the progress of dental decay is not influenced by the mineral content of the teeth except for fluoride. Careful studies have disproved the concept that caries can be produced or prevented by alteration in vitamin and mineral intake. At the most, disturbed calcification such as may occur in rickets can affect the rate of progress of caries, but it is not a factor in its formation. While one would like to think that nutritional deficiencies (therefore amenable to treatment) may cause caries, it is, however, frequently observed 28 that caries occurs in people subsisting on an "optimal" diet and is just as frequently absent in people on a nutritionally deficient diet. In fact, it has even been suggested that there is a reduced susceptibility to dental decay in malnourished children. 24 At any rate, it is more than likely that the physical nature of the diet is more important than the chemical content. MINERALS

We have emphasized the vitamin problems chiefly because they have commanded a disproportionate segment of the recent investigative spot-light. Similar interrelations occur with minerals as well and are well known. For example, calcium utilization is adversely affected by an excess of magnesium and oxalate in the diet. While both calcium and magnesium absorption is favored by a high phosphorus diet (as well as an acid reaction in the intestine), iron and manganese form insoluble phosphate compounds in the bowel and may therefore impair phosphorus absorption.24 Follisll has made the interesting observation that certain myocardial lesions may occur in experImental potassium deficiency, but if there should be a concomitant thiamine deficiency no such lesion appears. Here we see a curious interrelation of vitamin and element in which deficiency of one protects the body from the injurious effect of another nutritiona:! deficiency. While the exact significance of these experimental findings is unknown at present, the close interwoven effect of all nutrients is obvious. NUTRIENTS AND IMMUNITY

For at least thirty years, the professional and layman's opinion that nutritional factors are important in resistance to disease has been studied

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scientifically. Early work showed that vitamins A and D and vitamin "B complex" deficiencies did not impair agglutinin, precipitin or hemolysin formation. More recently Cannon and his group2D have demonstrated the importance of protein metabolism in antibody formation in the experimental animal. Our work 26 • 27 has elaborated further on this problem in man. We have found that protein deficiency (as indicated by hypoalbuminemia) in man is associated with poor antibody titer levels and low quantitative antibody-nitrogen levels, particularly in the diabetic. Protein supplementation in general enhanced the antibody response, but not to levels reached by normoproteinemic patients. No relation was noted between the blood sugar and antibody response in diabetics. Furthermore, the degree of nitrogen storage (protein repletion) was not reflected in the antibody producing capacity in fourteen subjects.3o One member of the B complex group may also play a role in antibody formation. Axelrod 28 and Stoerck29 and their co-workers found that pyridoxine deficient rats had an impaired antibody producing ability. Since antibody is a modified globulin, a protein, studies of the relationship of nutrients and immunity opens new roads to our understanding of many biochemical as well as clinical problems. AMINO ACIDS

Much work has been done on the interrelationships of the essential amino acids. For example, it is known that the plasma level of certain amino acids is affected by the feeding of other amino acids. 32 Furthermore, Beyer and associates 33 showed that the renal reabsorption of one amino acid is affected by the presence or absence of others. Thus, there appeared to be competition for reabsorption between arginine and lysine but not between arginine and glycine; competition between leucine and isoleucine but not between leucine and arginine. It is conceivable that excess administration of a single amino acid may have a deleterious effect on tubular reabsorption of other essential protein building blocks. Even the land d (optical rotary forms) amino acids seem to compete with each other "for the means by which cells concentrate the amino acids presented them by the extracellular fluid."34 The absence of a vitamin may affect the urinary excretion of amino acids. That is, the excretion of arginine, phenylalanine, tryptophane and histidine, for example, is increased by riboflavin deficiency hut unaffected by lack of niacin. 3D Protein Metabolism.-A most significant finding was reported by Geiger 31 and co-workers. They showed that all the essential amino acids must be fed simultaneousLY for maximal growth in animals. After protein depletion, the increase in weight on re-feeding was retarded if protein and other nutrients were not fed at the same time. Furthermore, cataracts which develop on a tryptophane-deficient diet can be prevented only by feeding that essential amino acid simultaneously with the other amino acids. These reports emphasize the vital necessity of an adequate diet,

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adequate in all respects and balanced so that at the same time all essential nutrients can enter the body and be used where they are needed. A house is composed of many things and many things must be available at the one time before it can be built. . NUTRIENTS AND HORMONES

Like everything else in the body, our amino acids are under the influence of the endocrine glands. Friedberg and Greenberg 3b found that plasma amino acid levels were increased by thyroxine and adrenal cortical extract and decreased by insulin, epinephrine, estrogen, thiouracil and hypophysectomy. While the exact significance of this observation is not clear at present, it ObVIously represents an important interrelation of nutrient and hormone which needs further study. Recently, the role of the B complex group of vitamins in estrogen metabolism has been studied. It was at first suggested that vitamin B complex deficiency impaired the liver's ability to inactivate estrogen. 37 Later it was shown that the concomitant inanition was the chief factor in this effect.a8 It now appears that folic acid enables the tissues to respond to estrogen. This also has been confirmed by the administration of folic acid antagonists. 39 A recent survey by Samuels 40 collects the wealth of material on the interrelation of nutrients and hormones. VITAMIN IMBALANCE

On the principle that if 1 mg. of thiamine is good, 100 mg. is 100 times better, many physicians are prescrihing excessive quantities of one or several vitamins. However, many years ago, it was noted that if thiamine alone were used in the treatment of multiple dietary deficiencies, the symptoms of an acute niacin deficiency may occur; while the administration of niacin alone to pellagrins aggrevated certain signs presumably due to other nutritional deficiencies. Recently it was shown 41 that when patients with pernicious anemia and sprue were treated with folic acid, various signs of vitamin B complex deficiencies appeared only to disappear after liver therapy. Here, again, we see the importance of balance in the administration of nutrients. ANTIVITAMINS J;~ssential nutrients are interrelated. However, there is a large group of substances called "antivitamins" which are closely related structurally and which may induce specific avitaminoses. These metabolic antagonistf$ without specific physiologic activity in themselves apparently interfere with normal metabolic processes by competitive inhibition. 42 Awareness of the importance of this concept began in 1940 when Woods and Fildes,43 and subsequently others, showed that para-aminobenzoic acid, a member of the B complex family, has antisulfonamide activity. The chemical structures of these compounds are very similar. It was later found that the folic acid molecule contains the para-amino-

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benzoic acid structure and that folic acid was essential to the growth of certain bacteria. Sulfonamide drugs compete with para-aminobenzoic acid for a place in the folic acid (pteroylglutamic acid) molecule and if they win out are bacteriostatic by preventing the utilization of folic acid (an essential metabolite for certain bacteria). Recent reviews of biological antagonism have appeared to which the interested reader is referred. 44 Antivitamins have been found for vitamins C, E, K and in the B complex group (thiamine, riboflavin, niacin, pantothenic acid, folic acid, pyridoxine, biotin and inositol). Most of these were determined in vitro or in animals. Naturally occurring antiniacin and antithiamin compounds have been isolated. Woolley 45 has found a nicotinic acid antagonist in corn and it is likely that the frequency of pellagra in certain corneating sections of the South may be explained by this factor, in addition to the tryptophane-niacin deficiencydiscu~ed before. One amino acid has been found to have an antimetabolite analogue, methionine and methoxinine. 46 Avidin is a unique protein found in egg white which combines with biotin to give a complex not broken down during digestion. Cooking the egg destroys the biotin-combining power of avidin, and eliminates this antivitamin. A metabolic antagonist of particular clinical importance is that of a coumarin derivative (isolated originally from spoiled sweet clover) which prolongs the prothrombin time by interfering with the function of vitamin K. Structurally, vitamin K is very similar to one-half of the symmetrical configuration of Dicumarol. * BIOLOGICAL SYNTHESIS OF ESSENTIAL NUTRIENTS

No discussion of nutritional interrelation would be complete without mention of the lowly intestinal bacteria. Their ever-present activities are of definite clinical importance. In essence, it appears that certain vitamins can be synthesized (in the presence of an appropriate environment) by the intestinal flora and that others can be destroyed. For example, Young and Ja'lnes 47 found that vitamin C was destroyed by E. coli and A. aerogenes under both aerobic and anerobic conditions, but in the presence of fermentable carbohydrate (i.e., glucose) that vitamin was protected from microbic decomposition. 48 From this one can expect that avitaminosis C will occur if insufficient carbohydrate is presented to an intestine harboring a luxuriant growth of these common organisms. On the other hand, folic acid, biotin and para-aminobenzoic acid are synthesized by bacteria as their fecal and urinary excretion often exceeds the intake. 49 Moreover, there is experimental evidence that bacterial synthesis of thiamine, riboflavin, niacin and pyridoxine may occur. In fact, the presence of relatively insoluble carbohydrates such as dextrin or starch may provide a substrate for added bacterial synthesis of these

* Dr. Thompson discusses

this in more detail in his paper in this iSBue.

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vitamins, for the vitamin requirements of animals was reduced by feeding this type of carbohydrate. Another example of the effect of the type of diet on bacterial synthesis of vitamins is the report that a high protein diet tends to suppress riboflavin synthesis, but thiamine synthesis in thp intestine is enhanced by dextrose. Even under identical conditions of diet and environment, however, there is a wide variation in the quantities which one individual can synthesize as compared with another. Indeed, intestinal bacterial synthesis may reach high levels. Thus, Najjar and his co-workers so reported that on a riboflavin-free diet for over three months no clinical or chemical signs of deficiency occurred in human subjects. It is assumed that bacterial synthesis supplied this essential nutrient. Now, it has been shown by many investigators that intestinal bacteriostasis (induced by such sulfonamides as sulfaguanidine and sulfasuxidine) affects bacterial synthesis of certain vitamins particularly of the B complex group; and an otherwise adequate dietary intake may be insufficient if sulfonamide inhibition of vitamin synthesis occurs. Thus, a case of dysentery treated with sulfaguanidine suddenly developed pellagra,51 Apparently this drug disturbed the equilibrium between organisms producing and destroying niacin. In animals, it has been found that sulfaguanidine led to hypoprothrombinemia and that this condition could be prevented by administration of vitamin K, para-aminobenzoic acid or folic acid. Similar findings were noted with sulfadiazine, sulfathiazole and sulfasuxidine. While we cannot translate these experimental findings to the bedside, the implications should be obvious. It would seem that the patient receiving prolonged sulfonamide therapy for intestinal infections should have his nutritional intake adequately supplemented, parenterally, if possible. PERSONALITY AND VITAMINS

A much quoted result of vitamin B deficiency is said to be such phenomena as irritability, fatigue and loss of pep, vim and vigor. Such findings have been reported in careful studies by Henderson and his co-workers62 on the B complex group and by Foltz63 and others for thiamine. Mild depression, lassitude and somnolence has been described in experimental biotin deficiency in man. M However, the specificity of certain neuropsychiatric symptoms has been questioned by Brills5 and others. Allen,56 for example, studied 300 consecutive cases with a chief complaint of weakness and fatigue. In 80 per' cent no physical disorder was found. In only one of the 300 cases was vitamin deficiency held responsible for weakness. The most frequent physical disorders associated with these symptoms were chronic infections, including tuberculosis, diabetes, heart disease, myasthenia gravis and anemia. Fatigability is seen early in the development of pellagra, but it is also seen in almost every illness; while it may be the result of t.he anorexia

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and subsequent nutritional deficiency accompanying a disease it has not yet been proved that it is a characteristic of vitamin B complex deficiency. It is certain, however, that thousands of persons in conflict with their mothers-in-law, foremen or spouses are having their irritability and depression treated with vitamins. SUMMARY

This brief review of the highlights of a massive literature on nutritional interrelationships emphasizes two concepts. (1) The results of experimental single nutritional deficiencies show widespread effects of metabolic disorders. This can readily be understood by remembering the function of vitamins, to mention only one group of nutrients (Table 2). These essential metabolites by virtue of their place in intermediary metabolism directly or indirectly influence every tissue and structure of the body. (2) Every nutrient affects and in return is affected by every other nutrient because our body is like a vast city in which the carpenter depends on the cobbler for shoes, and the cobbler on the carpenter for h:s bench and both on the baker for bread. Clinically, a pure single nutritional deficiency is a theoretical improbability or even impossibility, although such disorders may be predominantly of one type or another. The treatment of these deficiencies, whether due to decreased supply or increased demand, or both, involves not only replacement of the primary substance but also the administration of all interrelated nutrients, for each essential metabolite is its brother's keeper. We have seen that certain clinical lesions, formerly said to be "specific" for a nutrient insufficiency, may be caused by several avitaminoses. Most vitamins and amino acids are interrelated and one may substitute in part for the other. Minerals as well as vitamins are greatly affected by the type of diet consumed. NutritIOnal factors are affected by hormonal changes and in turn affect the endocrine system. Antimetabolites have been discovered and perhaps are important clinically in inducing or aggravating deficiencies. Furthermore, bacterial synthesis of vitamins at all times must be considered in the total nutritional picture. Because, for optimal effect, all essential nutrients must be available to the body at the same time, all therapeutic considerations must include the time element. Excessive and unbalanced administration of vitamins may precipitate other avitaminoses. CONCLUSIONS

Generally speaking, there are no specific lesions caused by single deficiencies and amenable to specific therapy. All dietary correction must include the simultaneous administration of adequate amounts of well balanced necessary substances. An excess of anyone component cannot correct and may worsen the value of the diet as a whole. Large doses of vitamins to a patient who is not eating enough calories, carbohydratefl

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or proteins is as useless in correcting the metabolic disorder as is a diet lacking in essential food factors. One can almost describe dietotherapy as "all or none." All nutritional factors in some way are interrelated and optimal results will be obtained if this is remembered. Or to paraphrase old John Donne,-"no nutrient is an island entire in itself. . . ." REFERENCES 1. Schoenheimer, R.: The Dynamic State of Body Constituents. Cambridge' Harvard University Press, 1942. 2. Stewart, C. P.: J. Physiol. 96: (Proc. 28) 1939. 3. Mayer, J. and Krehl, W. A.: J. Nutrition 35: 523, 1948. 4. Burr, G. 0.: Federation Proc. 1: 224, 1942. 5. Moore, T.: The Interrelation of Vitamins. In Vitamins and Hormones, New York, Academic Press, 1945, Vol. 3. 6. Abels, J. C., Gorham, A. T., Pack, G. T. and Rhoads, C. P.: J. Clin. Investigation 20: 749, 1941. 7. Machella, T. E.: Am. J. M. Sc. 203: 114, 1942. 8. Kruse, H. D.: Milbank Mem. Fund Quart. 27: 5, 1949. 9. McCreary, J. F., Nicholls, J. V. V. and Tisdall, F. F.: Canad. M. A ..f. 51: 106, 1944. 10. Boehrer, J. J., Stanford, C. E. and Ryan, E.: Am. J. M. Sc. 205: 544,1943. 11. Follis, R. H. Jr.: The Pathology of Nutritional Disease. Springfield, Ill., C. C Thomas, 1948. 12. Sydenstricker, V. P., Hall, W. K., Bowles, L. L. and Schmidt, H. L. Jr.: J. Nutrition 34: 48, 1947. Bowles, L. L., Hall, W. K., Sydenstricker, V. P. and Hoch, C. W.: J. Nutrition 37: 9, 1949. 13. Bakwin, H. and others: Am. J. Dis. Child. 74: 657, 1947. 14. Jeghers, H.: New England J. Med. 227: 221, 1942. 15. Sarett, H. P. and Perlzweig, W. A.: J. Nutrition 25: 173,1943. 16. Griffith, W. H. and MuIford, D. J.: J. Nutrition 21: 633, 1941. 17. Sure, B., Theis, R. M. and Harrelson, R. T.: J. BioI. Chem. 129: 245,1939. 18. Supplee, G. C., Jenson, O. G., Bender, R. C. and Kahlenberg, O. J.: J. Bio!. Chem. 144: 79, 1942. 19. Martin, G. J.: Am. J. Physio!. 136: 124, 1942. 20. Sure, B.: J. Nutrition 27: 447, 1948. 21. Dann, W. J. and Darby, W. J.: Physio!. Rev. 25: 326, 1945. 22. Editorial: Nutrition Rev. 1: 154, 1943. 23. Editorial: Ibid. 5: 178, 1947. 24. Editorial: Ibid. 6: 155, 1948. 24a. Elvehjem, C. A. and Krehl, W. H.: J. A. M. A. 135: 279,1947. 25. Cannon, P. R., Chase, W. E. and Wissler, R. W.: J. Immuno!. 47: 133, 1943. Cannon, P. R.: Some Pathologic Consequences of Protein and Amino Acid Deficiencies, Springfield, Il!., C. C Thomas, 1948. 26. Wohl, M. G., Reinhold, J. G. and Rose, S. B.: Antibody Response in Patients with Hypoproteinemia with Special Reference to the Effect of Protein Supplementation. Arch. Int. Med. 83: 402 (April) 1949. 27.'Wohl, M. G., Waife, S. 0., Green, S. and Clough, G.: Proc. Soc. Exper. Bio!. & Med. 70: 305, 1949. 28. Axelrod, A. E., Carter, B. B., McCoy, R. H. and Geisinger, R.: Proc. Soc. Exper. Bio!. & Med. 66: 137, 1947. 29. Stoerk, H. C., Eisen, H. N. and John, H. M.: J. Exper. Med. 85: 365,1947. 30. Waife, S. O. and Wohl, M. G.: Unpublished data. 31. Geiger, E.: Science 108: 42, 1948. Schaeffer, A. J. and Geiger, E.: Proc. Soc. Exper. Bio!. & Med. 66: 309, 1947.

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32. Heir, S. W.: J. BioI. Chem. 171: 813,1947. 33. Beyer, K. H. and others: Am. J. Physiol.151: 202,1947. 34. Christensen, H. N., Streicher, J. A. and Elbinger, R. L.: J. BioI. Chem. 172: 515, 1948. 35. Scheweigert, B. S.: Proc. Soc. Exper. BioI. & Med. 66: 315, 1947. 36. Friedberg, F. and Greenberg, D. M.: J. BioI. Chem. 168: 405,1947. 37. Biskind, M. S.: Nutritional Therapy of Endocrine Disturbances. In Vitamins and Hormones, New York, Academic Press, 1946, Vol. 4, p. 147. 38. Jailer, J. W.: Endocrinology 43: 78, 1948. 39. Hertz, R.: Recent Progress in Hormone Res. 2: 161, 1948. 40. Samuels, L. T.: Nutrition and Hormones, Springfield, C. C Thomas, 1948. 41. Davidson, L. S. P. and Girdwood, R. H.: Lancet 1: 360,1948. 42. Wright, L. D.: J. Am. Dietet. A. 23: 289, 1947. 43. Woods, D. D. and Fildes, P.: J. Soc. Chem. Indust. 59: 133, 1940. 44. Wooley, D. W.: Physiol. Rev. 27: 308, 1947. 45. Wooley, D. W.: J. BioI. Chem. 163: 773,1946. 46. Knight, B. C. J. G.: J. Mt. Sinai Hosp. 15: 28, 1949. 47. Young, R. M. and James, L. H.: J. Bact. 44: 75, 1942. 48. Young, R. M. and Rettger, L. F.: J. Bact. 46: 351, 1943. 49. Elvehjem, C. A.: J. Am. Dietet. A. 22: 959, 1946. 50. Najjar, V. A., Johns, G. 0., Medairy, G. C., Fleischman,G. and Holt, L. E.: J. A. M. A. 126: 357,1944. 51. Hardwick, S. W.: Lancet 1: 267, 1946. 52. Hendcrson, C. R.: Am. J. M. Sc. 213: 488, 1947; 53. Foltz, E. E., Barborka, C. J. and Ivy, A. C.: Gastroenterology 2: 323,1944. 54. Sydenstricker, V. P., Singal, S. A., Briggs, A. P., DeVaughn, N. M. and lsbell, H.: Science 95: 176, 1942. 55. Brill, N. Q.: Am. Pract.1: 353,1947. 56. Allan, F. N.: New England J. Med. 231: 414, 1944.