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Biological Significance of Nutrients*
The Medical Clinics of North America March, 1943
BIOLOGICAL SIGNIFICANCE OF NUTRIENTS·
c.
A. EL.VEHJEM, Ph.D.t
WORKERS in the field of nutrition have been so busy finding additional nutrients essential for health that relatively little attention has been given to the biological action of the individual factors. At the time of Hippocrates (460:-370 B.C.), there existed a belief in the occurrence of a specific universal nutrient in the various materials used for food. It is surprising that this view was accepted by Beaumont as late as 1833. Although only one factor was recognized, much thought was given to the best sources of this nutrient. During the middle of the last century, Magendie demonstrated that the materials which we recognize today as carbohydrates, fats and proteins had different nutritive effects. Since that time, the number of individual factors shown to be required for optimum nutrition has been increasing continuously, and the only limitation of progre.ss has been our inability to estimate the amount and number of these materials in foods. Thus we were calorie conscious when we learned how to determine the caloric value of biological material. Interest in proteins, minerals and vitamins followed as the analytical procedures were developed. The various nutrients have been considered to a large extent as independent entities, and from a therapeutic point of view greatest emphasis seems to have been placed on the most recently identified factors. Any discussion of nutrition today certainly centers around vitamins. Early clinicians observed the biological action of nutrients when they found that an abundance of liver was a remedy for night blindness, and that vegetables and fresh fruits cured scurvy. Modem clinicians treat scurvy with ascorbic acid,
• From the Department of Biochemistry, University of Wisconsin. t Professor of Biochemistry, University of 'Wisconsin (Madison); Member, Food and Nutrition Board, National Research Council. 277
278
C. A. ELVEHJEM
beriberi with thiamine, and pellagra with niacin, anemia with iron and copper, rickets with calcium and vitamin D; but evidence for the exact action of these factors or how they fit into the general scheme of metabolism has not been readily available. Many of the early ideas concerning the action· of nutrients, especially some of the vitamins, were related to their action in counteracting toxic effects. We must still consider such effects, but the toxic substance may be due to disturbed metabolism as well as to pathogenic organisms. Thus, Peters has recently stated: "Surely it is more possible to understand how pathologically the brain in its metabolism may not only be subjected to the action of toxins (the usual view) but occasionally fails owing to self poisoning with its own misguided machinery."l . INTERRELATIONSHIP OF NUTRIENTS
Today we are beginning to realize the interrelationship of all nutrients and the interdependence of one factor upon another. There are several reasons for this change. First, our knowledge of metabolism has increased greatly during the past few years. We know that during the release of energy from carbohydrate in the human body, phosphoric acid, coenzymes containing vitamins, enzymes containing iron, and other enzymes activated by magnesium and manganese are all needed to complete the process. The conversion of carbohydrates to fat requires thiamine, the conversion of protein to fat requires pyridoxine. The requirement for choline which is found in lipids depends upon the intake of certain amino acids. Second, it is now possible to demonstrate a decrease in certain enzyme systems in severe nutritional deficiencies. Third, the availability of synthetic vitamins and purified minerals has made it evident that many nutritional diseases will .not respond to a single factor and that more than one vitamin or mineral may be needed. The success with which many deficiency diseases are now being treated is discussed in succeeding clinics, but maximum results have not been obtained even during the so-called "vitamin era." Greater progress will be made when all nutrients are considered together.
BIOLOGICAL SIGNIFICANCE OF NUTRIENTS
279
ENZYME SYSTEMS
Nutrients may be classified according to their function as follows: as structural material for supporting tissues, as fuel, and as catalytic material. The nutrients in the first two categories are discussed in later clinics. Those acting as catalysts are followed with a little more difficulty. Since many of these nutrients take part in enzyme systems, we should perhaps say a few words about biocatalysts. Everyone today agrees that the major part of an enzyme is protein in nature, and that under most conditions the living cell has little difficulty in building these proteins. Certainly, a part of the protein ingested in the human diet must be used for building the multitude of enzymes present in the entire body, and in this case it may be difficult to differentiate between the protein used for supporting tissues and that used for catalytic material. However, in many cases the enzyme contains an additional group or element which is generally termed the prosthetic group if it is firmly attached to the protein. For example, certain oxidases contain copper; other enzymes contain iron in much the same manner as hemoglobin does. The activity of still other enzymes may be altered greatly by the presence of kina~es or co enzymes. Kinases are enzymes which alter the activity of specific enzymes, and coenzymes are noncolloidal compounds which, when loosely attached to the protein part (}f an enzyme, activate the enzyme. Enzymes are highly specific, and both the protein and prosthetic groups may contain definite chemical configurations which enable them to accelerate a specific chemical reaction. Since enzymes show such a high degree of spec ificity, several enzymes are needed to carry out any appreciable part of the total mt;tabolism. Thus, we generally deal with enzyme systems, and even a simple system, such as the succinoxidase system, is made up of succinic dehydrogenase, cytochrome, and cytochrome oxidase. It is apparent, then, that many of the trace elements and vitamins are merely components of these essential catalytic systems, which the body cannot synthesize without proper precursors. As we learn more about these systems, the function of vitamins and
280
c.
A. ELVEHJEM
trace elements will be better understood, and as we have more vitamins available in synthetic form, greater progress will be made in studies on metabolism. 2 • 8 ENZYME·VITAMIN RELATIONSHIPS
Thiamine
In any discussion of the biological action of thiamine we turn immediately to the classical observation of Lohmann and Schuster, who isolated cocarboxylase in crystalline form and showed it to be the pyrophosphoric acid ester of thiamine. Much work, of course, was carried out before this final conclusion could be made. As early as 1911, Neuberg reported the presence in yeast of carboxylase, an enzyme which catalyzed the decarboxylation of a-keto acids. Later this enzyme was split into a protein component and a thermostable cocarboxylase. Similar work with animal tissues had shown a decreased respiration in tissues taken from polyneuritic animals, when pyruvate was used as a substrate. The addition of thiamine stimulated oxygen uptake and pyruvate removal. Today, it is fairly simple to demonstrate a reduced cocarboxylase content in tissues from experimental animals deficient in thiamine. Carboxylase has been isolated from both top brewers' yeast and animal tissues and found to be diphosphothiamine-magnesium"protein. The best preparation contained 0.46 per cent diphosphothiamine and 0.13 per cent magnesium. The protein component in the animal preparation appeared to be different from that in the yeast preparation. There is ample evidence, therefore, that cocarboxylase is the active form of thiamine in the animal body and that thiamine is phosphorylated during glycolysis. However, the reactions in which cocarboxylase is concerned are much more complex in animal tissues than in yeast. The exact changes which the prosthetic group undergoes during the catalytic process are still obscure. Thiamine may be related also to acetylcholine synthesis. Riboflavin At the present time, evidence indicates that riboflavin is related directly to more enzymes than any of the known
BIOLOOICAL SIGNIFICANCE OF NUTRIENTS
281
vitamins. In 1932, Warburg and Christian isolated from yeast an enzyme which acted as a carrier link between coenzyme II and molecular oxygen. In acid solution, this flavoprotein was broken down into the protein component and riboflavin phosphate. In living tissues, these flavoproteins probably do not react directly with oxygen but are more likely to function in the transfer of hydrogen from the reduced coenzyme to cytochrome C. As far as I know, no one has measured the amount of these flavoproteins in tissues taken from animals with riboflavin deficiency. However, there are other flavoproteins which apparently catalyze the direct reaction between the substrate and molecular oxygen. One of these is d-amino acid oxidase and this enzyme is definitely reduced in the liver and kidney of rats with riboflavin deficiency. Another flavoprotein belonging to this group is xanthine oxidase. This enzyme is also reduced during riboflavin deficiency. Very recently it has been shown that there is a definite decrease in the succinic acid oxidase content of the liver and heart taken from rats with riboflavin deficiency. Up to the present time, no one has shown conclusively that succinic acid oxidase contains riboflavin but the above results indicate that some part of the system may contain riboflavin. It is evident that riboflavin plays an important role in the entire respiratory mechanism in the animal body. The recent report by Sure and Dichek that riboflavin produces a profound effect on the economy of food utilization is further evidence that metabolism proceeds more efficiently when there is sufficient riboflavin for the optimum activity of the various enzyme systems. Nicotinie Acid. (Niacin)
Nicotinic acid apparently is related to only two coenzymes which are very similar in composition but which enter into a great variety of different reactions. In fact, practically all of the dehydrogenases which activate the oxidation of the . original substrates require these two coenzymes. Although nicotinic acid, adenine and ribose are constituent parts, nicotinic acid appears to be the only compound which the animal body has difficulty in synthesizing. Very recently
282
C. A. ELVEH]EM
we have tried to demonstrate the essential nature of ribose in the diet of rats receiving limited amounts of riboflavin, but no significant differences have been observed between the nits with and without added ribose. Whether the animal can actually make ribose from other carbohydrates or whether the basal rations contain minute amounts of riboflavin cannot be decided at the present time. Although some difficulty has been encountered in determining the cozymase content of animal tissue, it is rather definitely established that a decrease in the liver and muscle TABULATION* ENmME-VITAMIN RELATIONSHIPS, INCLUDING HYPOTHECATED POSSIBILITIESt
Vitamin Thiamine Nicotinic acid
Riboflavin
Enzyme System
Coenzyme or Prosthetic Group
Substrate
Carboxylase (a-keto acid Cocarboxylase : diphos- Pyruvic acid (a-keto glutaric acid) oxidase) phothiamine Numerous specific pro- Coenzyme I: cozymase: Lactic acid, malic acid. diphosphopyridine nu,(:I-bydroxybutyric acid. teinsj i.e., dehydrogenalcohol, glyceraldehyde ases, for the substrates cleotide listed diphosphate, glucose,: glutamic acid~ Coenzyme II: Warburg's Glucose-6-phosphate. isocoenzyme : triphosphocitric acid, glucose,f pyridine nucleotide glutamic acid: Alloxazine adenine dinu- Xanthine, hypoxanthine Xanthine oxidase c1eotide Alloxazine adenine dinu- d-Amino-acids d,-Amino-acid oxidase c1eotide (Coenzyme Il-cyto(Riboflavin phosphate) Coenzyme II chrome reductase) Coenzyme I (Coenzyme I-cytochrome reductase) (Succinic dehydrogenase) Succinic add
• Taken from Potter.' t The hypothecated possihilities are indicated hy parentheses. : These substrates can be oxidized in the presence of either coenzyme I or
n.
can be observed in experimental animals with nicotinic acid deficiency, as well as in the muscles of persons with pellagra. The blood cozymase undergoes little change during nicotinic acid deficiency. The tabulation summarizes some of the relationships which have been discussed. The remaining members of the B complex have not been associated with any specific enzymes, although it is well known that pyridoxine, pantothenic acid and biotin are closely associated with proteins in the living cell. It is interesting that recent work indicates a faulty metabolism of pyruvic acid in
BIOLOGICAL SIGNIFICANCE OF NUTRIENTS
283
livers taken from rats with either pantothenic acid or biotin deficiency. Ascorbic Acid and Dehydroascorbic Acid
Vitamin C has been associated with enzymes for many years, but as far as· I know no one has proved conclusively that it is a constituent of anyone biocatalyst. However, both ascorbic acid and dehydroascorbic acid exert either stimulatory or inhibitory effects on many of the proteolytic and oxidizing enzymes. It is well known that the enzyme which oxidizes ascorbic acid is a copper protein compound and ~s ~onsiderably more active than an equivalent amount of lomc copper. Para-aminobenzoic Acid
Finally, we may mention very briefly the relation of enzymes and para-aminobenzoic acid which has been isolated from vitamin-rich materials such as yeast and liver. Ansbacher has classified this compound as a vitamin. Although it is difficult to demonstrate any effect of para-aminobenzoic acid on the rate of growth in experimental animals, it does function in the growth of certain micro-organisms. Wisansky, Martin, and Ansbacher have shown that para-aminobenzoic acid retards the oxidation of tyrosine and dopa by tyrosinase. Lipman has reported that para-aminobenzoic acid is oxidized by hydrogen dioxide and peroxidase and that this reaction is inhibited by sulfanilamide. It is also oxidized by phenol oxidase in the presence of catalytic amounts of catechol. This reaction is not inhibited by sulfanilamide. 1 THE FUNCTION OF MINERALS
Although the mineral elements, calcium and phosphorus, are generally considered as nutrients for the building of bone tissues, we know that phosphorus, especially, is exceedingly important in cellular metabolism. There is a large variety of compounds in the body which contain phosphoric acid in ester form. Any discussion of carbohydrate metabolism must, of necessity, include many of these phosphorus compounds. In a recent review by Kalckar, the significance of these phos-
284
c.
A. ELVEHJEM
phoric acid esters is summarized on the basis that these compounds are more readily bound to catalytically active enzymes and that the energy released during the oxidation process can be directed and utilized more efficiently in the body.4 When we speak of iron and copper. deficiency in human beings or in experimental animals, we usually think of nutritional anemia, or a deficiency of hemoglobin in the blood stream. There are, of course, several other iron compounds in the body besides hemoglobin, but apparently the hemoglobin content of the blood is the first to undergo change and it is the easiest to measure accurately. The other iron compounds include cytochrome, catalase and peroxidase. As far as is known, there is no decrease in the cytochrome content of the tissues during severe iron and copper deficiency. However, Schultze has shown that a severe copper deficiency may produce a reduction in both the cytochrome oxidase and catalase content of certain tissues, especially bone marrow and liver. We have mentioned that ascorbic acid oxidase is a copper compound. Other enzymes containing copper include monoand polyphenol oxidases. These copper enzymes are found largely in plant materials, but Mann and Keilin have isolated from both red blood corpuscles and liver, protein compounds which contain 0.34 per cent of copper. The physiological function of these two compounds is still unknown. 2 Although it is now definitely established that zinc is an essential element in animal nutrition, the specific function of zinc is not clear. The intestinal phosphatases are reduced during severe zinc deficiencies, but in this case zinc may act merely as an activator for the enzyme. Carbonic anhydrase is undoubtedly a zinc protein compound, but attempts to demonstrate a decrease in the carbonic anhydrase content of the blood of zinc-deficient rats have failed. Holmberg has recently shown that the enzyme, uricase, contains 0.13 per cent zinc, but again the concentration of uricase in the livers of zinc-deficient rats was found to be normal. 6 Manganese has been shown to be an essential nutrient, but up to the present time no specific enzyme containing manganese has been isolated. It is well known, however, that
BIOLOGICAL SIGNIFICANCE'-OF NUTRIENTS
285
manganese, as well as magnesium, activates a number of enzymes in both the glycolysis and respiration mechanisms. The fact that chicks with a manganese deficiency develop perosis and that a decreased bone phosphatase accompanies this condition indicates that the function of manganese is closely related to phosphorus metabolism and to the enzyme affecting phosphorus-containing compounds. The enzyme, enolase, when isolated in purified form will function only upon the addition of salts of zinc, manganese, or magnesium; but the enolase which is active in vivo is probably the magnesium compound. Cobalt has a number of interesting effects onethe animal body and cobalt deficiencies have been observed in cattle and sheep grazing on areas deficient in this element, but the exact function of cobalt is unknown. EFFECTS OF NUTRIENTS ON INTESTINAL FLORA
It is important to mention an additional biological effect of nutrients and that is their effect upon the bacterial flora of the intestinal tract. Many vitamins and probably other nutrients stimulate the growth of certain micro-organisms and these organisms in turn can synthesize other vitamins and ,nutrients. Evidence is available to show that bacteriostatic agents, such as sulfaguanidine, retard the growth of rats on synthetic diets. Liver, yeast and grass are rich sources of factors which overcome this retarded growth. Similarly, it has been shown that rats kept on diets very low in biotin can grow normally because the bacteria in the intestinal tract synthesize this vitamin in sufficient amounts to meet the needs of the animal Although only a very short paragraph is here devoted to this subject, important developments which may be of significance in human nutrition will take place in the next few years. CONCLUSIONS
In conclusion I think it is evident that, in order to have an efficient human body, many nutrients are needed and that each nutrient plays an important role in the complete machine. The deficiency of a nutrient which functions in the smallest link of the chain may produce havoc. Many of the nutrients
286
C. A. ELVEHJEM
function in several enzyme systems, and it is still difficult to relate specific symptoms to a decrease in anyone enzyme. In other cases, we have not learned to associate symptoms with metabolic changes. Nevertheless the biochemical lesion may be present. Many of these lesions may be eliminated by the intelligent use of our present knowledge, but a complete understanding of the relation of nutrients to metabolism will assure even greater success. BIBLIOGRAPHY
1. Evans, E. A., Jr.: The Biological Action of the Vitamins. The University of Chicago Press, Chicago, 1942. 2. Green, D. ~.: Enzymes and Trace Substances, Advances in Enzymology and Related Subjects, edited by F. F. Nord and C. H. Werkman. Interscience Publishers, Inc., New York, 1941. 3. Potter, V. R.: Why 'Ve Need Vitamins. J. Am. Diet. Assoc., 18:359 (June) 1942. 4. Kalckar, H. M.: The Function of Phosphate on Cellular Assimilation. Biological Reviews, 17:28, 1942. 5. Ball, E. G.: Biological Oxidations and Reductions. Ann. Rev. Biochem., Vo!. 11. Edited by J. M. Luck and J. H. C. Smith. Annual Review, Inc., Stanford Univ. P. 0., California, 1942. 6. Wachtel, L. W., Hove, E., Elvehjem, C. A. and Hart, E. B.: J. BioI. Chem., 138:361, 1941.
BIOLOGICAL SIGNIFICANCE OF NUTRIENTS·
c.
A. EL.VEHJEM, Ph.D.t
WORKERS in the field of nutrition have been so busy finding additional nutrients essential for health that relatively little attention has been given to the biological action of the individual factors. At the time of Hippocrates (460:-370 B.C.), there existed a belief in the occurrence of a specific universal nutrient in the various materials used for food. It is surprising that this view was accepted by Beaumont as late as 1833. Although only one factor was recognized, much thought was given to the best sources of this nutrient. During the middle of the last century, Magendie demonstrated that the materials which we recognize today as carbohydrates, fats and proteins had different nutritive effects. Since that time, the number of individual factors shown to be required for optimum nutrition has been increasing continuously, and the only limitation of progre.ss has been our inability to estimate the amount and number of these materials in foods. Thus we were calorie conscious when we learned how to determine the caloric value of biological material. Interest in proteins, minerals and vitamins followed as the analytical procedures were developed. The various nutrients have been considered to a large extent as independent entities, and from a therapeutic point of view greatest emphasis seems to have been placed on the most recently identified factors. Any discussion of nutrition today certainly centers around vitamins. Early clinicians observed the biological action of nutrients when they found that an abundance of liver was a remedy for night blindness, and that vegetables and fresh fruits cured scurvy. Modem clinicians treat scurvy with ascorbic acid,
• From the Department of Biochemistry, University of Wisconsin. t Professor of Biochemistry, University of 'Wisconsin (Madison); Member, Food and Nutrition Board, National Research Council. 277
278
C. A. ELVEHJEM
beriberi with thiamine, and pellagra with niacin, anemia with iron and copper, rickets with calcium and vitamin D; but evidence for the exact action of these factors or how they fit into the general scheme of metabolism has not been readily available. Many of the early ideas concerning the action· of nutrients, especially some of the vitamins, were related to their action in counteracting toxic effects. We must still consider such effects, but the toxic substance may be due to disturbed metabolism as well as to pathogenic organisms. Thus, Peters has recently stated: "Surely it is more possible to understand how pathologically the brain in its metabolism may not only be subjected to the action of toxins (the usual view) but occasionally fails owing to self poisoning with its own misguided machinery."l . INTERRELATIONSHIP OF NUTRIENTS
Today we are beginning to realize the interrelationship of all nutrients and the interdependence of one factor upon another. There are several reasons for this change. First, our knowledge of metabolism has increased greatly during the past few years. We know that during the release of energy from carbohydrate in the human body, phosphoric acid, coenzymes containing vitamins, enzymes containing iron, and other enzymes activated by magnesium and manganese are all needed to complete the process. The conversion of carbohydrates to fat requires thiamine, the conversion of protein to fat requires pyridoxine. The requirement for choline which is found in lipids depends upon the intake of certain amino acids. Second, it is now possible to demonstrate a decrease in certain enzyme systems in severe nutritional deficiencies. Third, the availability of synthetic vitamins and purified minerals has made it evident that many nutritional diseases will .not respond to a single factor and that more than one vitamin or mineral may be needed. The success with which many deficiency diseases are now being treated is discussed in succeeding clinics, but maximum results have not been obtained even during the so-called "vitamin era." Greater progress will be made when all nutrients are considered together.
BIOLOGICAL SIGNIFICANCE OF NUTRIENTS
279
ENZYME SYSTEMS
Nutrients may be classified according to their function as follows: as structural material for supporting tissues, as fuel, and as catalytic material. The nutrients in the first two categories are discussed in later clinics. Those acting as catalysts are followed with a little more difficulty. Since many of these nutrients take part in enzyme systems, we should perhaps say a few words about biocatalysts. Everyone today agrees that the major part of an enzyme is protein in nature, and that under most conditions the living cell has little difficulty in building these proteins. Certainly, a part of the protein ingested in the human diet must be used for building the multitude of enzymes present in the entire body, and in this case it may be difficult to differentiate between the protein used for supporting tissues and that used for catalytic material. However, in many cases the enzyme contains an additional group or element which is generally termed the prosthetic group if it is firmly attached to the protein. For example, certain oxidases contain copper; other enzymes contain iron in much the same manner as hemoglobin does. The activity of still other enzymes may be altered greatly by the presence of kina~es or co enzymes. Kinases are enzymes which alter the activity of specific enzymes, and coenzymes are noncolloidal compounds which, when loosely attached to the protein part (}f an enzyme, activate the enzyme. Enzymes are highly specific, and both the protein and prosthetic groups may contain definite chemical configurations which enable them to accelerate a specific chemical reaction. Since enzymes show such a high degree of spec ificity, several enzymes are needed to carry out any appreciable part of the total mt;tabolism. Thus, we generally deal with enzyme systems, and even a simple system, such as the succinoxidase system, is made up of succinic dehydrogenase, cytochrome, and cytochrome oxidase. It is apparent, then, that many of the trace elements and vitamins are merely components of these essential catalytic systems, which the body cannot synthesize without proper precursors. As we learn more about these systems, the function of vitamins and
280
c.
A. ELVEHJEM
trace elements will be better understood, and as we have more vitamins available in synthetic form, greater progress will be made in studies on metabolism. 2 • 8 ENZYME·VITAMIN RELATIONSHIPS
Thiamine
In any discussion of the biological action of thiamine we turn immediately to the classical observation of Lohmann and Schuster, who isolated cocarboxylase in crystalline form and showed it to be the pyrophosphoric acid ester of thiamine. Much work, of course, was carried out before this final conclusion could be made. As early as 1911, Neuberg reported the presence in yeast of carboxylase, an enzyme which catalyzed the decarboxylation of a-keto acids. Later this enzyme was split into a protein component and a thermostable cocarboxylase. Similar work with animal tissues had shown a decreased respiration in tissues taken from polyneuritic animals, when pyruvate was used as a substrate. The addition of thiamine stimulated oxygen uptake and pyruvate removal. Today, it is fairly simple to demonstrate a reduced cocarboxylase content in tissues from experimental animals deficient in thiamine. Carboxylase has been isolated from both top brewers' yeast and animal tissues and found to be diphosphothiamine-magnesium"protein. The best preparation contained 0.46 per cent diphosphothiamine and 0.13 per cent magnesium. The protein component in the animal preparation appeared to be different from that in the yeast preparation. There is ample evidence, therefore, that cocarboxylase is the active form of thiamine in the animal body and that thiamine is phosphorylated during glycolysis. However, the reactions in which cocarboxylase is concerned are much more complex in animal tissues than in yeast. The exact changes which the prosthetic group undergoes during the catalytic process are still obscure. Thiamine may be related also to acetylcholine synthesis. Riboflavin At the present time, evidence indicates that riboflavin is related directly to more enzymes than any of the known
BIOLOOICAL SIGNIFICANCE OF NUTRIENTS
281
vitamins. In 1932, Warburg and Christian isolated from yeast an enzyme which acted as a carrier link between coenzyme II and molecular oxygen. In acid solution, this flavoprotein was broken down into the protein component and riboflavin phosphate. In living tissues, these flavoproteins probably do not react directly with oxygen but are more likely to function in the transfer of hydrogen from the reduced coenzyme to cytochrome C. As far as I know, no one has measured the amount of these flavoproteins in tissues taken from animals with riboflavin deficiency. However, there are other flavoproteins which apparently catalyze the direct reaction between the substrate and molecular oxygen. One of these is d-amino acid oxidase and this enzyme is definitely reduced in the liver and kidney of rats with riboflavin deficiency. Another flavoprotein belonging to this group is xanthine oxidase. This enzyme is also reduced during riboflavin deficiency. Very recently it has been shown that there is a definite decrease in the succinic acid oxidase content of the liver and heart taken from rats with riboflavin deficiency. Up to the present time, no one has shown conclusively that succinic acid oxidase contains riboflavin but the above results indicate that some part of the system may contain riboflavin. It is evident that riboflavin plays an important role in the entire respiratory mechanism in the animal body. The recent report by Sure and Dichek that riboflavin produces a profound effect on the economy of food utilization is further evidence that metabolism proceeds more efficiently when there is sufficient riboflavin for the optimum activity of the various enzyme systems. Nicotinie Acid. (Niacin)
Nicotinic acid apparently is related to only two coenzymes which are very similar in composition but which enter into a great variety of different reactions. In fact, practically all of the dehydrogenases which activate the oxidation of the . original substrates require these two coenzymes. Although nicotinic acid, adenine and ribose are constituent parts, nicotinic acid appears to be the only compound which the animal body has difficulty in synthesizing. Very recently
282
C. A. ELVEH]EM
we have tried to demonstrate the essential nature of ribose in the diet of rats receiving limited amounts of riboflavin, but no significant differences have been observed between the nits with and without added ribose. Whether the animal can actually make ribose from other carbohydrates or whether the basal rations contain minute amounts of riboflavin cannot be decided at the present time. Although some difficulty has been encountered in determining the cozymase content of animal tissue, it is rather definitely established that a decrease in the liver and muscle TABULATION* ENmME-VITAMIN RELATIONSHIPS, INCLUDING HYPOTHECATED POSSIBILITIESt
Vitamin Thiamine Nicotinic acid
Riboflavin
Enzyme System
Coenzyme or Prosthetic Group
Substrate
Carboxylase (a-keto acid Cocarboxylase : diphos- Pyruvic acid (a-keto glutaric acid) oxidase) phothiamine Numerous specific pro- Coenzyme I: cozymase: Lactic acid, malic acid. diphosphopyridine nu,(:I-bydroxybutyric acid. teinsj i.e., dehydrogenalcohol, glyceraldehyde ases, for the substrates cleotide listed diphosphate, glucose,: glutamic acid~ Coenzyme II: Warburg's Glucose-6-phosphate. isocoenzyme : triphosphocitric acid, glucose,f pyridine nucleotide glutamic acid: Alloxazine adenine dinu- Xanthine, hypoxanthine Xanthine oxidase c1eotide Alloxazine adenine dinu- d-Amino-acids d,-Amino-acid oxidase c1eotide (Coenzyme Il-cyto(Riboflavin phosphate) Coenzyme II chrome reductase) Coenzyme I (Coenzyme I-cytochrome reductase) (Succinic dehydrogenase) Succinic add
• Taken from Potter.' t The hypothecated possihilities are indicated hy parentheses. : These substrates can be oxidized in the presence of either coenzyme I or
n.
can be observed in experimental animals with nicotinic acid deficiency, as well as in the muscles of persons with pellagra. The blood cozymase undergoes little change during nicotinic acid deficiency. The tabulation summarizes some of the relationships which have been discussed. The remaining members of the B complex have not been associated with any specific enzymes, although it is well known that pyridoxine, pantothenic acid and biotin are closely associated with proteins in the living cell. It is interesting that recent work indicates a faulty metabolism of pyruvic acid in
BIOLOGICAL SIGNIFICANCE OF NUTRIENTS
283
livers taken from rats with either pantothenic acid or biotin deficiency. Ascorbic Acid and Dehydroascorbic Acid
Vitamin C has been associated with enzymes for many years, but as far as· I know no one has proved conclusively that it is a constituent of anyone biocatalyst. However, both ascorbic acid and dehydroascorbic acid exert either stimulatory or inhibitory effects on many of the proteolytic and oxidizing enzymes. It is well known that the enzyme which oxidizes ascorbic acid is a copper protein compound and ~s ~onsiderably more active than an equivalent amount of lomc copper. Para-aminobenzoic Acid
Finally, we may mention very briefly the relation of enzymes and para-aminobenzoic acid which has been isolated from vitamin-rich materials such as yeast and liver. Ansbacher has classified this compound as a vitamin. Although it is difficult to demonstrate any effect of para-aminobenzoic acid on the rate of growth in experimental animals, it does function in the growth of certain micro-organisms. Wisansky, Martin, and Ansbacher have shown that para-aminobenzoic acid retards the oxidation of tyrosine and dopa by tyrosinase. Lipman has reported that para-aminobenzoic acid is oxidized by hydrogen dioxide and peroxidase and that this reaction is inhibited by sulfanilamide. It is also oxidized by phenol oxidase in the presence of catalytic amounts of catechol. This reaction is not inhibited by sulfanilamide. 1 THE FUNCTION OF MINERALS
Although the mineral elements, calcium and phosphorus, are generally considered as nutrients for the building of bone tissues, we know that phosphorus, especially, is exceedingly important in cellular metabolism. There is a large variety of compounds in the body which contain phosphoric acid in ester form. Any discussion of carbohydrate metabolism must, of necessity, include many of these phosphorus compounds. In a recent review by Kalckar, the significance of these phos-
284
c.
A. ELVEHJEM
phoric acid esters is summarized on the basis that these compounds are more readily bound to catalytically active enzymes and that the energy released during the oxidation process can be directed and utilized more efficiently in the body.4 When we speak of iron and copper. deficiency in human beings or in experimental animals, we usually think of nutritional anemia, or a deficiency of hemoglobin in the blood stream. There are, of course, several other iron compounds in the body besides hemoglobin, but apparently the hemoglobin content of the blood is the first to undergo change and it is the easiest to measure accurately. The other iron compounds include cytochrome, catalase and peroxidase. As far as is known, there is no decrease in the cytochrome content of the tissues during severe iron and copper deficiency. However, Schultze has shown that a severe copper deficiency may produce a reduction in both the cytochrome oxidase and catalase content of certain tissues, especially bone marrow and liver. We have mentioned that ascorbic acid oxidase is a copper compound. Other enzymes containing copper include monoand polyphenol oxidases. These copper enzymes are found largely in plant materials, but Mann and Keilin have isolated from both red blood corpuscles and liver, protein compounds which contain 0.34 per cent of copper. The physiological function of these two compounds is still unknown. 2 Although it is now definitely established that zinc is an essential element in animal nutrition, the specific function of zinc is not clear. The intestinal phosphatases are reduced during severe zinc deficiencies, but in this case zinc may act merely as an activator for the enzyme. Carbonic anhydrase is undoubtedly a zinc protein compound, but attempts to demonstrate a decrease in the carbonic anhydrase content of the blood of zinc-deficient rats have failed. Holmberg has recently shown that the enzyme, uricase, contains 0.13 per cent zinc, but again the concentration of uricase in the livers of zinc-deficient rats was found to be normal. 6 Manganese has been shown to be an essential nutrient, but up to the present time no specific enzyme containing manganese has been isolated. It is well known, however, that
BIOLOGICAL SIGNIFICANCE'-OF NUTRIENTS
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manganese, as well as magnesium, activates a number of enzymes in both the glycolysis and respiration mechanisms. The fact that chicks with a manganese deficiency develop perosis and that a decreased bone phosphatase accompanies this condition indicates that the function of manganese is closely related to phosphorus metabolism and to the enzyme affecting phosphorus-containing compounds. The enzyme, enolase, when isolated in purified form will function only upon the addition of salts of zinc, manganese, or magnesium; but the enolase which is active in vivo is probably the magnesium compound. Cobalt has a number of interesting effects onethe animal body and cobalt deficiencies have been observed in cattle and sheep grazing on areas deficient in this element, but the exact function of cobalt is unknown. EFFECTS OF NUTRIENTS ON INTESTINAL FLORA
It is important to mention an additional biological effect of nutrients and that is their effect upon the bacterial flora of the intestinal tract. Many vitamins and probably other nutrients stimulate the growth of certain micro-organisms and these organisms in turn can synthesize other vitamins and ,nutrients. Evidence is available to show that bacteriostatic agents, such as sulfaguanidine, retard the growth of rats on synthetic diets. Liver, yeast and grass are rich sources of factors which overcome this retarded growth. Similarly, it has been shown that rats kept on diets very low in biotin can grow normally because the bacteria in the intestinal tract synthesize this vitamin in sufficient amounts to meet the needs of the animal Although only a very short paragraph is here devoted to this subject, important developments which may be of significance in human nutrition will take place in the next few years. CONCLUSIONS
In conclusion I think it is evident that, in order to have an efficient human body, many nutrients are needed and that each nutrient plays an important role in the complete machine. The deficiency of a nutrient which functions in the smallest link of the chain may produce havoc. Many of the nutrients
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function in several enzyme systems, and it is still difficult to relate specific symptoms to a decrease in anyone enzyme. In other cases, we have not learned to associate symptoms with metabolic changes. Nevertheless the biochemical lesion may be present. Many of these lesions may be eliminated by the intelligent use of our present knowledge, but a complete understanding of the relation of nutrients to metabolism will assure even greater success. BIBLIOGRAPHY
1. Evans, E. A., Jr.: The Biological Action of the Vitamins. The University of Chicago Press, Chicago, 1942. 2. Green, D. ~.: Enzymes and Trace Substances, Advances in Enzymology and Related Subjects, edited by F. F. Nord and C. H. Werkman. Interscience Publishers, Inc., New York, 1941. 3. Potter, V. R.: Why 'Ve Need Vitamins. J. Am. Diet. Assoc., 18:359 (June) 1942. 4. Kalckar, H. M.: The Function of Phosphate on Cellular Assimilation. Biological Reviews, 17:28, 1942. 5. Ball, E. G.: Biological Oxidations and Reductions. Ann. Rev. Biochem., Vo!. 11. Edited by J. M. Luck and J. H. C. Smith. Annual Review, Inc., Stanford Univ. P. 0., California, 1942. 6. Wachtel, L. W., Hove, E., Elvehjem, C. A. and Hart, E. B.: J. BioI. Chem., 138:361, 1941.