Pellagra and Amino Acid Imbalance C. GOPALAN
AND
KAMALA S. JAYA RAO
National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India
. . . . . . . . . . . . . . . . A. Role of Nicotinic Acid Deficiency. . B. Role of Leucine . . . . . . . 111. Biochemical Changes . . . . . . 1. Introduction. 11. Etiology . .
IV.
V. VI.
VII. VIII
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
.
.
. . . . . . . . .
A. Urinary Excretion of Tryptophan and Nicotinic Acid Metabolites. B. Plasma Tryptophan Levels. . . . . . . . . . . . C. Nicotinamide Nucleotides in Erythrocytes . . . . . . . Effects of Excess Leucine . . . . . . . . . . . . . A. Production of Canine Blacktongue . . . . . . . . . B. Leucine Excess and Maize . . . . . . . . . . . . C. Effect of Leucine on the Enzymes in the Tryptophan Nicotinic Acid Pathway . . . . . . . . . . . . . . . . . . Mental Changes in Pellagra . . . . . . . . . . . . . A. Clinical Symptoms and Signs . . . . . . . . . . . . B. Biochemical Basis for Altered Mental Function. . . . . . . The Skin in Pellagra. . . . . . . . . . . . . . . . Copper Metabolism . . . . . . . . . . . . . . . . Leucine-Isoleucine Imbalance . . . . . . . . . . . . . Summary and Concluding Remarks . . . . . . . . . . . References . . . . . , . . . . . . . . . . , .
505 506 507 509 509 509 512 512 515 515 516
516 517 517 519 521 522 523 523 524
I. INTRODUCTION Pellagra is a classical nutritional deficiency disease which had a worldwide distribution until the beginning of this century. It still continues to be an endemic public health problem in some parts of the technologically developing world. Excellent descriptions of the classical clinical features, which include dermatitis, diarrhea, and dementia, can be found in numerous publications and in standard textbooks of medicine and nutrition. Notwithstanding the statement of Gillman and Gillman (1951) that the typical manifestations of a disease are not the ones to be invariably seen, the dermatitis of pellagra is highly characteristic and diagnostic of the disease. It is bilateral, symmetrical, and seen on those parts of the body that are constantly exposed to sunlight. Thus it is commonly 505
506
C. GOPALAN AND K. S. JAYA RAO
seen on the extensor surfaces of the extremities and on the face and neck. Pigmentation on the face is generally limited to the cheeks and the bridge of the nose, and this characteristic pattern goes by the picturesque name of “butterfly pigmentation.” The dermatitis extending over the scapuloclavicular area is called Casal’s necklace or Casal’s dermatitis, perpetuating the name of the man who first described this disease in the 18th century. The remaining two of the classical 3 D’s of pellagra, namely, diarrhea and dementia, are not invariable features of the disease. The mental changes, when present, may range from mild symptoms like insomnia and depression to marked emotional instability and mania. Glossitis and angular stomatitis are seen in most of the cases. Nasolabial dyssebacia is also frequently seen. However, these are generally attributed to associated riboflavin deficiency and are not considered to be a part of the clinical syndrome per se.
11. ETIOLOGY Pellagra has been classically recognized to be a disease of the maizeeating populations. The association was suggested by Casal, Frappoli, and other European physicians in the early 18th century soon after the introduction of the maize crop on the continent. Credit, however, goes to Goidberger, who experimentally confirmed the association between consumption of maize and pellagra, both in humans and in dogs (Goldberger and Wheeler, 1920, 1928). He demonstrated the preventable nature of the disease and, more important, stressed that the disease was attributable to a faulty diet, not to any specific foodstuff. The etiology was suggested to be the deficiency of what Goldberger termed a pellagra-preventive or P-P factor. The beautiful anthology of some of his classical contributions to this field (Terris, 1964) is a testimony to the patience and scientific acumen with which he conducted his studies. Pellagra was common in the southern states of the United States and in Europe, where maize was commonly consumed by the poorer sections of the community. The disease has now been completely eliminated there. However, i t is still encountered among the Bantus of South Africa (Potgeiter et al., 1966), in Egypt, and in the maize-eating populations of India (Shah and Singh, 1967). I n the rice belt of Asia the disease is practically unknown (Aykroyd and Swaminathan, 1940; Gopalan and Srikantia, 1960). However, in the rural areas of the rocky Deccan plateau of India the disease is common among the adult population (Gopalan and Srikantia, 1960). The disease
PELLAGRA AND AMINO ACID IMBALANCE
507
accounts for about 1% of all admissions to general hospitals and for nearly %lo% of admissions to mental institutions in the city of Hyclerabad. The staple diet of these people is a millet, jowar (Sorghum vulgare), and the diets are not supplemented by any animal foods. Careful dietary histories of cases admitted to hospitals revealed that in 6% of the cases jowar was the staple food and the diets did not include maize. The rest consumed jowar together with rice whereas in few cases a history of occasional consumption of maize in addition to jowar was obtained. The disease afflicts both sexes equally. It is usually seen only in adults. Seasonal exacerbations and remissions have been described, the incidence being highest in spring when sunlight is in plenty. In India, where ample sunl.ight is present all through the year, the seasonal incidence is related to the food consumption pattern. Jowar is eaten during the lean season starting from June-July and the peak incidence of the disease is seen duriing the months of November to February. A. ROLE OF NICOTINIC ACIDDEFICIENCY Cioldberger and Tanner (1925) attributed pellagra to a lack of a pellagra-preventive factor in maize. Elvehjem et al. (1937) identified this fac1,or as nicotonic acid by demonstrating the curative efficacy of this vitabmin in canine blacktongue. Soon after, the clinical efficacy of nicotinic acid was also demonstrated in human pellagra (Fouts et al., 1937; Spies et CIL,1938). After these conclusive studies, it had come to be generally accepted that pellagra is a disease of nicotinic acid deficiency. Although maize is poor in nicotinic acid, it is no worse in this respect than other cereals (Aykroyd and Swaminathan, 1940). Krehl et al. (1945a) found that the marked growth depressive effect of maize in rats could be counteracted by nicotinic acid or by raising the casein content of the diets to 20%. Rice containing less nicotinic acid produced no such growth depreasion. The same group of workers (Krehl et al., 1945b) subsequently obtained data attributing the growth-depressive action of maize to its low tryptophan content. Rice is richer in tryptophan than maize. That tryiptophan is a precursor of nicotinic acid was subsequently established in experimental animals (Rosen et al., 1946; Singal et al., 1946) and in humans (Perlzweig et al., 1947; Sarett and Goldsmith, 1947). It is generally agreed that approximately 60 mg of tryptophan is equivalent to 1 mg of nicotinic acid (Horwitt et al., 1956; Goldsmith et al., 1961). From the foregoing observations, it was justifiably assumed that the pellagragenic nature of maize is due to its low content of nicotinic acid and tryptophan. Early workers in Egypt had postulated that pellagra might be due to a lack of tryptophan (Barrett-Connor, 1967). Goldberger
508
C. GOPALAN AND K. S . JAYA RAO
was also of the opinion that the disease might be one of amino acid deficiency (Goldberger and Tanner, 1922).
Availability of h'icotinic Acid Among the poorer sections of the population in Central America, maize forms the staple cereal. Despite providing 80% of the total calories and nearly 70% of the total protein (Bressani et al., 1953), the traditional association between maize consumption and pellagra is belied here, the disease being very rare (Aguirre et al., 1953; Squibb et al., 1959). A possible explanation forwarded for the rarity of pellagra in Central America is that corn is consumed here in the form of tortillas prepared after preliminary lime treatment. Much of the nicotinic acid in maize and other cereals is believed to be present in a bound form (Kodicek, 1940) considered unavailable to animals like chicks (Krehl et al., 1944; Coates et al., 19521, pigs (Kodicek et al., 1956), and rats (Kodicek, 1960). It has been claimed that the bound form is rendered free by alkali hydrolysis (Kodicek, 1940; Kodicek et al., 1959). Thus the rarity of pellagra in Central America has been attributed to the liberation of nicotinic acid consequent on lime treatment (Braude et al., 1955; Kodicek et al., 1956). Goldsmith et al. (19561, however, successfully induced symptoms and signs of nicotinic acid deficiency in human volunteers fed lime-treated maize diets. Another factor mentioned in the context of absence of pellagra in Central America is the high rate of coffee consumption. Roasted coffee contains considerable quantities of nicotinic acid (Teply et al., 1945)) and this may make significant contributions to a diet otherwise deficient in tryptophan and nicotinic acid (Teply et al., 1945; Goldsmith et al., 1959). In view of the reported unavailability of nicotinic acid in maize, the possibility that the vitamin might be present in a bound form in jowar too had to be tested. Although a marked increase in the nicotonic acid content of jowar following alkaline hydrolysis has been reported (Ghosh et at., 1963), Belavady and Gopalan (1966) could not confirm these findings. Nicotinic acid content of jowar was found t o range from 2 to 3.5 mg/100 gm. In acid-methanol extracts, 80% of the vitamin was found to be in a free form, and in methanol extracts nearly 60% was found to be in a free form. The growth pattern of rats and pups maintained on diets containing either untreated or lime-treated jowar was found to he similar. These studies supported the data obtained by chemical analysis that much of the nicotinic acid in jowar in its natural state is present in a free and available form. Thus the possibility that pellagra in jowareaters may be due to nicotinic acid in the millet being bound and unavailable could be ruled out.
PELLAGRA AND AMINO ACID IMBALANCE
509
B. ROLEOF LEUCINE Analysis of jowar, however, showed that the nicotinic acid and tryptophan content of this millet was similar to that of rice (Gopalan and Srikrzntia, 1960). Further analysis revealed a high content of leucine, a feature that jowar shares with maize. Leucine constitutes 12-14% of the protein of these foods in contrast to 8% observed in rice (Table I).Excess of certain essential amino acids in the diet has been shown to have a growth-depressive effect in experimental animals (Harper, 1964; Harper et al., 1970). The toxic effect of the excess amino acid was generally found to be more when the diet was inadequate in other respects than when com:plete. Addition of 1% or more of L-leucine to a 9% casein diet was found to cause growth retardation in rats, but this effect was absent when the animals were fed 18% casein diet (Harper et al., 1955). Pellagrins subsisting on jowar diets consume daily around 2000 kcal and 45 gm of protein derived almost solely from jowar and sometimes other cereals. Thus they are seen to consume a 9% vegetable protein diet containing 1-1.50/0 leucine. This observation was the starting point of the investigaton of the possible role of excess leucine in the causation of pellagra. Woolley (1946) and Borrow et al. (1948) questioned the widely accepted theory that pellagra is due to the low tryptophan-nicotinic acid content of maize, and, though they postulated the presence of a toxic pellagragenic agent, they were unable to conclusively demonstrate its presence.
111. BIOCHEMICAL CHANGES A. 'URINARY EXCRETION OF TRYPTOPHAN AND NICOTINIC ACIDMETABOLITES Najjar and Wood in 1940 reported the excretion of a whitish blue fluorescent substance in urine following the administration of nicotinic acid. T h k compound, subsequently demonstrated to be "-methyl nicotinamide (N'MeN), is one of the major urinary metabolites of nicotinic acid (Huff and Perlzweig, 1943a,b). Search for a second important metabolite (Eliinger and Coulson, 1944; Perlzweig and Huff, 1945) led to the identific ation of the 6-pyridone of N'MeN (Knox and Grossman, 1946). The principal metabolites of nicotinic acid in urine are now established as N'RIeN and "-methyl 6-pyridone 3-carboxylamide (6-pyridone) . The major pathway of tryptophan metabolism is the kynurenine pathway (Fig. 1 ) . 3-Hydroxyanthranilic acid, a metabolite in this pathway, can either be diverted to the glutarate pathway or be converted to quino-
5 10
C. GOPALAN AND R. S. JAYA RAO
TABLE I THECONTENT-OF NICOTINIC ACIDA N D CI.:RT.AIN AMINOACIDSI N SOMEFOODSTUFFS Foods tuff Itice Wheat Maize Jowar (Sorghum vulgare) a
Nicotinic acid
Tryptophan
Leucine
Isoleucine
1.2
1.2 1.1 0.8 1.2
8.0 6.5 14.9 12.9
6.0 3.5 6.4 6.1
5 ..i 1.4
1.8
Grams per 100 gm of protein.
linic acid (Henderson, 1949; Henderson and Hirsch, 1949; Moline et al., 1959; Nishizuka and Hayaishi, 1963). Although it was believed that quinolinic acid could be directly converted to nicotinic acid (Henderson, 1949; Henderson et al., 1949; Sarrett, 1951 ; Hankes and Segal, 1957), it has now been conclusively shown that it is an immediate precursor of nicotinic acid ribonucleotide (Nishizuka and Hayaishi, 1963). Tryptophan
1w
(5)
5-Hydroxytryptophan
iV-Formylkynuren ne
(6)
SHydroxytryptamine
1
5-Hydroxyindoleacetic acid
1
Kynurenh
1
3-H ydroxyk ynurenine L.
3-Hydroxyanthranilir acid 112)
or-Amino-8-carboxymuconic semialdehyde l(3)
pathway
Quinolinic acid 1(4)
Nicotinic acid ribonucleotidc
1
Intermediate
J\ Glutarate
-----+
Picolinic acid
Nicotinic acid adenine dinucleotide
I
1
NAD
1
N'-Methyl nicotinamide
1
6-Pyridone N'Methyl nicotinamide
Fro. 1. Pathways of tryptophan catabolism: (1) tryptophan oxygenase, (2) 3-hydroxyanthranilate oxygenase, (3) picolinic carboxylase, (4) quinolinate phosphoribos~ltransferase, (5) tryptophan hydroxylase, (6) 5-hydroxytryptamine decarboxylase.
PELLAGRA AND AMINO ACID IMBALANCE
511
011 barely adequate protein diets providing marginal amounts of tryptophan, addition of other amino acids has been shown to cause a disturbance in tryptophan-nicotinic acid metabolism and to result in nicotinic acid deficiency (Rosen and Perlzweig, 1949; Lyman and Elvehjem, 1951; Koeppe and Henderson, 1955). It was inferred that, in a diet providing marginal amounts of all amino acids, tryptophan is spared for conversion to nicotinic acid, whereas when other amino acids are in excess and tryptophan becomes a limiting amino acid, this conversion is impaired (Henderson e t aZ., 1953; Koeppe and Henderson, 1955). Investigations were, therefore, undertaken to see whether excess leucine would also bring about such disturbances in the tryptophan-nicotinic acid interrelationship. Urinary excretion of N’MeN, 6-pyridone) tryptophan, and quinolinic acid was found to be low in pellagrins (Belavady e t al., 1963). Hankes e t cd. (1971) also observed low levels of N’MeN, nicotinic acid, and kynurenic acid in the urine of pellagrins. Normal subjects and pellagrins were maintained on diets providing approximately 2300 kcals and 50 gm of protein, derived solely from vegetable sources. The diets provided about 8% protein and were similar to those consumed by the population which suffers from pellagra. Each subject was given in addition every day 10 gm of L-leucine orally. Administration of leucine brought about a marked increase in the excretion of quinolinic acid and a significant decrease in that of 6-pyridone (Belavady e t cd., 1963). Withdrawal of leucine resulted in a reversal to the basal pattern. Although in a preliminary study there was suggested evidence thal; leucine administration may increase urinary excretion of N‘MeN (Gopalan and Srikantia, 1960), subsequent studies showed that there is no consistent alteration in the excretion of this metabolite (Belavady e t al., 1963). The foregoing observations suggested that there might be a block in the further metabolism of quinolinic acid following leucine administration. More detailed investigations were carried out in experimental rats. Weanling albino rats were fed a basal diet containing 9% casein but adequate in other respects. The animals were also given supplements of either L-leucine or L-tryptophan. Quinolinic acid excretion was markedly increased. Excretion of N‘MeN although raised was not significant (Raghuramulu e t al., 1965a). Moreover, quinolinic acid excretion was found to increase with increasing doses of leucine. The effect was specific to leucine and was not seen with lysine, glycine, methionine, or threonine. Quinolinic acid excretion was found to be higher in rats fed a jowar diet than in those fed a wheat diet. The excretion of nicotinic acid and N’MeN Wafs also high.
512
C. GOPALAN A N D K. S . JAYA
RAO
B. PLASMA TRYPTOPHAN LEVELS Plasma tryptophan levels are in the low range in pellagrins. Truswell
et al. (1968) were of the opinion that the plasma level of this amino acid may be a good biochemical index of pellagra. However, individual variations in the levels are so wide th at it would appear that the plasma tryptophan level may not be of much significance in the pathogenesis of the disease (Ghafoorunissa and Narasinga Rao, 1975). The low plasma tryptophan levels are more likely a reflection of the general undernutrition in the pellagrins, since nonpellagrins from low socioeconomic groups, in which pellagra is common, had levels similar to those in pellagrins. On the other hand, plasma tryptophan levels in well-nourished nonpellagrins were higher than those seen in either of the former two groups. Truswell et a2. (1968) also observed that plasma tryptophan levels in children suffering from pellagra and kwashiorkor were of similar order. A decrease in urinary excretion of tryptophan was observed after a leucine load (Belavady et al., 1963). Feeding a high-leucine diet to rats resulted in decreased levels of plasma valine and isoleucine (Rogers et nl., 1962), and a similar observation has also been made in human subjects after a leucine load (Swendseid et al., 1965). The studies of Hagihira et a2. (1960) suggest that these alterations might be due to a competition between the amino acids during intestinal absorption. However, such a mechanism does not appear to operate in the case of leucine and tryptophan. A leucine load did not bring about any alterations in plasma tryptophan levels in normal volunteers (Ghafoorunissa and Narasinga Rao, 1971.) Further, the increase in plasma tryptophan levels following simultaneous administration of tryptophan and leucine was found to be similar to that observed after a tryptophan load alone.
C. NICOTINAMIDE SIXLEDTIDES I N ERYTHROCYTES The functional forms of nicotinic acid in the body are the two nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). I n view of the altered nicotinic acid metabolism in pellagra, studies were directed toward investigating the profile of these constituents. The total nucleotide concentration in the erythrocytes of pellagrins was similar to that in normals (Raghuramulu et al., 1965b). This is in line with the observations of Axelrod et al. (1941). The rate of nucleotide synthesis was, however, significantly lower in the pellagrins. Supplementation with L-leucine for 5 days did not alter the nucleotide concentration either in pellagrins or in normal subjects, but its rate of synthesis was suppressed in both groups to nearly
PELLAGRA AND AMINO ACID IMBALANCE
513
60% of the initial values. A rapid reversal to basal conditions occurred following withdrawal of leucine. Leucine added in vitro a t high concentratilons also inhibited the nucleotide synthesis (Belavady et al., 1973). As mentioned earlier, leucine supplementation led to an increased excretion of quinolinic acid and, i t may be logically assumed, to an increased concentration in blood. Quinolinic acid was found to inhibit nucleotide syntihesis in vitro, the inhibition being progressive with increasing concentrations of the compound (Raghuramulu e t al., 1965b). A similar mechanism may also be operative in v i m . The depressed rate of synthesis may, therefore, be due to a direct action of leucine or of quinolinic acid. The paradox of a normal concentration of the nucleotide in the face of a depressed synthesizing capacity of the erythrocytes was resolved in subsequent studies. Fractionation of the nucleotides was carried out by paper chromatography using the solvent system 95% ethanol: 1 M ammonium acetate, pH 5 , in the ratio of 7:3, as described by Preiss and Handler (1958a). Much of the nucleotide in the normal subjects resolved into NAD and NADP. In pellagrins a third spot corresponding to nicotinamide mononucleotide (NMN) was identified. Levels of NAD and NADP were lower than in normals (Srikantia et al., 1968a). Treatment with nicotinic acid brought about a marked reduction in the NMN content of the erythrocytes of pellagrins. NMN is normally not found in human and dog erythrocytes. However, the monkey, guinea pig, and rat have significant concentrations of NMN in their erythrocytes (Table 11). In dogs which developed blacktongue after feeding of jowar or maize diets, the total nucleotide concentration of the erythrocytes was not altered-a finding similar to that observed in pellagrins and in normal volunteers administered L-leucine. Nevertheless, NMN was detected in considerable concentrations. Monkeys fed jowar diets, however, showed a fall in the total concentration of nucleotides in the erythrocytes but no alteration in the NMN concentration (Belavady et al., 1968). Similar observations were also made in monkeys fed leucine (Belavady and Rao, 1973). It appears that there may be species variations in the changes in the nucleotide pattern following consumption of jowar. In species that normally have NMN in erythrocytes, the concentration is not changed, whereas those which normally do not have detectable quantities of the nucleotide show an increase. I n the absence of an adequate explanation for the normally observed species variations, it is not wise to speculate further on these divergent observations. The pathway proposed by Preiss and Handler (1958b) for the biosynthesis of nicotinamide nucleotides from nicotinic acid is depicted in Fig. 2. 'The incorporation of I4C-labeled nicotinic acid into nicotinic acid ribonucleotide as well as into nicotinic acid adenine dinucleotide
514
C. GOPALAN AND K. S. JAYA RAO
SPECIICS
~)IFFEHENCES I N
TABLE I1 NICOTINAMIDE NUCIXOTIDPCONCENTH ITIONS OF
ERYTHROCYTES
Species
Tot a1 nucleo tides (mg/100 1x11 of erythrocytes)
Chick (3)$ Dog (13) Iluckling (7) Guinea pig (3) Monkey (21) Rat (6) Man (12)
9 . r, 7.0 13.3 10.2 12.4 12.1 5.2
NAU (yoof total) 74
74 87.5 64 58 80.5 65. .i
NAIjP (70 of total)
NMN ( % of total)
26 22 12.5 24.5 28 12 34.3
Trace
AND
KAMALA S. JAYA RAO
National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India
. . . . . . . . . . . . . . . . A. Role of Nicotinic Acid Deficiency. . B. Role of Leucine . . . . . . . 111. Biochemical Changes . . . . . . 1. Introduction. 11. Etiology . .
IV.
V. VI.
VII. VIII
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
.
.
. . . . . . . . .
A. Urinary Excretion of Tryptophan and Nicotinic Acid Metabolites. B. Plasma Tryptophan Levels. . . . . . . . . . . . C. Nicotinamide Nucleotides in Erythrocytes . . . . . . . Effects of Excess Leucine . . . . . . . . . . . . . A. Production of Canine Blacktongue . . . . . . . . . B. Leucine Excess and Maize . . . . . . . . . . . . C. Effect of Leucine on the Enzymes in the Tryptophan Nicotinic Acid Pathway . . . . . . . . . . . . . . . . . . Mental Changes in Pellagra . . . . . . . . . . . . . A. Clinical Symptoms and Signs . . . . . . . . . . . . B. Biochemical Basis for Altered Mental Function. . . . . . . The Skin in Pellagra. . . . . . . . . . . . . . . . Copper Metabolism . . . . . . . . . . . . . . . . Leucine-Isoleucine Imbalance . . . . . . . . . . . . . Summary and Concluding Remarks . . . . . . . . . . . References . . . . . , . . . . . . . . . . , .
505 506 507 509 509 509 512 512 515 515 516
516 517 517 519 521 522 523 523 524
I. INTRODUCTION Pellagra is a classical nutritional deficiency disease which had a worldwide distribution until the beginning of this century. It still continues to be an endemic public health problem in some parts of the technologically developing world. Excellent descriptions of the classical clinical features, which include dermatitis, diarrhea, and dementia, can be found in numerous publications and in standard textbooks of medicine and nutrition. Notwithstanding the statement of Gillman and Gillman (1951) that the typical manifestations of a disease are not the ones to be invariably seen, the dermatitis of pellagra is highly characteristic and diagnostic of the disease. It is bilateral, symmetrical, and seen on those parts of the body that are constantly exposed to sunlight. Thus it is commonly 505
506
C. GOPALAN AND K. S. JAYA RAO
seen on the extensor surfaces of the extremities and on the face and neck. Pigmentation on the face is generally limited to the cheeks and the bridge of the nose, and this characteristic pattern goes by the picturesque name of “butterfly pigmentation.” The dermatitis extending over the scapuloclavicular area is called Casal’s necklace or Casal’s dermatitis, perpetuating the name of the man who first described this disease in the 18th century. The remaining two of the classical 3 D’s of pellagra, namely, diarrhea and dementia, are not invariable features of the disease. The mental changes, when present, may range from mild symptoms like insomnia and depression to marked emotional instability and mania. Glossitis and angular stomatitis are seen in most of the cases. Nasolabial dyssebacia is also frequently seen. However, these are generally attributed to associated riboflavin deficiency and are not considered to be a part of the clinical syndrome per se.
11. ETIOLOGY Pellagra has been classically recognized to be a disease of the maizeeating populations. The association was suggested by Casal, Frappoli, and other European physicians in the early 18th century soon after the introduction of the maize crop on the continent. Credit, however, goes to Goidberger, who experimentally confirmed the association between consumption of maize and pellagra, both in humans and in dogs (Goldberger and Wheeler, 1920, 1928). He demonstrated the preventable nature of the disease and, more important, stressed that the disease was attributable to a faulty diet, not to any specific foodstuff. The etiology was suggested to be the deficiency of what Goldberger termed a pellagra-preventive or P-P factor. The beautiful anthology of some of his classical contributions to this field (Terris, 1964) is a testimony to the patience and scientific acumen with which he conducted his studies. Pellagra was common in the southern states of the United States and in Europe, where maize was commonly consumed by the poorer sections of the community. The disease has now been completely eliminated there. However, i t is still encountered among the Bantus of South Africa (Potgeiter et al., 1966), in Egypt, and in the maize-eating populations of India (Shah and Singh, 1967). I n the rice belt of Asia the disease is practically unknown (Aykroyd and Swaminathan, 1940; Gopalan and Srikantia, 1960). However, in the rural areas of the rocky Deccan plateau of India the disease is common among the adult population (Gopalan and Srikantia, 1960). The disease
PELLAGRA AND AMINO ACID IMBALANCE
507
accounts for about 1% of all admissions to general hospitals and for nearly %lo% of admissions to mental institutions in the city of Hyclerabad. The staple diet of these people is a millet, jowar (Sorghum vulgare), and the diets are not supplemented by any animal foods. Careful dietary histories of cases admitted to hospitals revealed that in 6% of the cases jowar was the staple food and the diets did not include maize. The rest consumed jowar together with rice whereas in few cases a history of occasional consumption of maize in addition to jowar was obtained. The disease afflicts both sexes equally. It is usually seen only in adults. Seasonal exacerbations and remissions have been described, the incidence being highest in spring when sunlight is in plenty. In India, where ample sunl.ight is present all through the year, the seasonal incidence is related to the food consumption pattern. Jowar is eaten during the lean season starting from June-July and the peak incidence of the disease is seen duriing the months of November to February. A. ROLE OF NICOTINIC ACIDDEFICIENCY Cioldberger and Tanner (1925) attributed pellagra to a lack of a pellagra-preventive factor in maize. Elvehjem et al. (1937) identified this fac1,or as nicotonic acid by demonstrating the curative efficacy of this vitabmin in canine blacktongue. Soon after, the clinical efficacy of nicotinic acid was also demonstrated in human pellagra (Fouts et al., 1937; Spies et CIL,1938). After these conclusive studies, it had come to be generally accepted that pellagra is a disease of nicotinic acid deficiency. Although maize is poor in nicotinic acid, it is no worse in this respect than other cereals (Aykroyd and Swaminathan, 1940). Krehl et al. (1945a) found that the marked growth depressive effect of maize in rats could be counteracted by nicotinic acid or by raising the casein content of the diets to 20%. Rice containing less nicotinic acid produced no such growth depreasion. The same group of workers (Krehl et al., 1945b) subsequently obtained data attributing the growth-depressive action of maize to its low tryptophan content. Rice is richer in tryptophan than maize. That tryiptophan is a precursor of nicotinic acid was subsequently established in experimental animals (Rosen et al., 1946; Singal et al., 1946) and in humans (Perlzweig et al., 1947; Sarett and Goldsmith, 1947). It is generally agreed that approximately 60 mg of tryptophan is equivalent to 1 mg of nicotinic acid (Horwitt et al., 1956; Goldsmith et al., 1961). From the foregoing observations, it was justifiably assumed that the pellagragenic nature of maize is due to its low content of nicotinic acid and tryptophan. Early workers in Egypt had postulated that pellagra might be due to a lack of tryptophan (Barrett-Connor, 1967). Goldberger
508
C. GOPALAN AND K. S . JAYA RAO
was also of the opinion that the disease might be one of amino acid deficiency (Goldberger and Tanner, 1922).
Availability of h'icotinic Acid Among the poorer sections of the population in Central America, maize forms the staple cereal. Despite providing 80% of the total calories and nearly 70% of the total protein (Bressani et al., 1953), the traditional association between maize consumption and pellagra is belied here, the disease being very rare (Aguirre et al., 1953; Squibb et al., 1959). A possible explanation forwarded for the rarity of pellagra in Central America is that corn is consumed here in the form of tortillas prepared after preliminary lime treatment. Much of the nicotinic acid in maize and other cereals is believed to be present in a bound form (Kodicek, 1940) considered unavailable to animals like chicks (Krehl et al., 1944; Coates et al., 19521, pigs (Kodicek et al., 1956), and rats (Kodicek, 1960). It has been claimed that the bound form is rendered free by alkali hydrolysis (Kodicek, 1940; Kodicek et al., 1959). Thus the rarity of pellagra in Central America has been attributed to the liberation of nicotinic acid consequent on lime treatment (Braude et al., 1955; Kodicek et al., 1956). Goldsmith et al. (19561, however, successfully induced symptoms and signs of nicotinic acid deficiency in human volunteers fed lime-treated maize diets. Another factor mentioned in the context of absence of pellagra in Central America is the high rate of coffee consumption. Roasted coffee contains considerable quantities of nicotinic acid (Teply et al., 1945)) and this may make significant contributions to a diet otherwise deficient in tryptophan and nicotinic acid (Teply et al., 1945; Goldsmith et al., 1959). In view of the reported unavailability of nicotinic acid in maize, the possibility that the vitamin might be present in a bound form in jowar too had to be tested. Although a marked increase in the nicotonic acid content of jowar following alkaline hydrolysis has been reported (Ghosh et at., 1963), Belavady and Gopalan (1966) could not confirm these findings. Nicotinic acid content of jowar was found t o range from 2 to 3.5 mg/100 gm. In acid-methanol extracts, 80% of the vitamin was found to be in a free form, and in methanol extracts nearly 60% was found to be in a free form. The growth pattern of rats and pups maintained on diets containing either untreated or lime-treated jowar was found to he similar. These studies supported the data obtained by chemical analysis that much of the nicotinic acid in jowar in its natural state is present in a free and available form. Thus the possibility that pellagra in jowareaters may be due to nicotinic acid in the millet being bound and unavailable could be ruled out.
PELLAGRA AND AMINO ACID IMBALANCE
509
B. ROLEOF LEUCINE Analysis of jowar, however, showed that the nicotinic acid and tryptophan content of this millet was similar to that of rice (Gopalan and Srikrzntia, 1960). Further analysis revealed a high content of leucine, a feature that jowar shares with maize. Leucine constitutes 12-14% of the protein of these foods in contrast to 8% observed in rice (Table I).Excess of certain essential amino acids in the diet has been shown to have a growth-depressive effect in experimental animals (Harper, 1964; Harper et al., 1970). The toxic effect of the excess amino acid was generally found to be more when the diet was inadequate in other respects than when com:plete. Addition of 1% or more of L-leucine to a 9% casein diet was found to cause growth retardation in rats, but this effect was absent when the animals were fed 18% casein diet (Harper et al., 1955). Pellagrins subsisting on jowar diets consume daily around 2000 kcal and 45 gm of protein derived almost solely from jowar and sometimes other cereals. Thus they are seen to consume a 9% vegetable protein diet containing 1-1.50/0 leucine. This observation was the starting point of the investigaton of the possible role of excess leucine in the causation of pellagra. Woolley (1946) and Borrow et al. (1948) questioned the widely accepted theory that pellagra is due to the low tryptophan-nicotinic acid content of maize, and, though they postulated the presence of a toxic pellagragenic agent, they were unable to conclusively demonstrate its presence.
111. BIOCHEMICAL CHANGES A. 'URINARY EXCRETION OF TRYPTOPHAN AND NICOTINIC ACIDMETABOLITES Najjar and Wood in 1940 reported the excretion of a whitish blue fluorescent substance in urine following the administration of nicotinic acid. T h k compound, subsequently demonstrated to be "-methyl nicotinamide (N'MeN), is one of the major urinary metabolites of nicotinic acid (Huff and Perlzweig, 1943a,b). Search for a second important metabolite (Eliinger and Coulson, 1944; Perlzweig and Huff, 1945) led to the identific ation of the 6-pyridone of N'MeN (Knox and Grossman, 1946). The principal metabolites of nicotinic acid in urine are now established as N'RIeN and "-methyl 6-pyridone 3-carboxylamide (6-pyridone) . The major pathway of tryptophan metabolism is the kynurenine pathway (Fig. 1 ) . 3-Hydroxyanthranilic acid, a metabolite in this pathway, can either be diverted to the glutarate pathway or be converted to quino-
5 10
C. GOPALAN AND R. S. JAYA RAO
TABLE I THECONTENT-OF NICOTINIC ACIDA N D CI.:RT.AIN AMINOACIDSI N SOMEFOODSTUFFS Foods tuff Itice Wheat Maize Jowar (Sorghum vulgare) a
Nicotinic acid
Tryptophan
Leucine
Isoleucine
1.2
1.2 1.1 0.8 1.2
8.0 6.5 14.9 12.9
6.0 3.5 6.4 6.1
5 ..i 1.4
1.8
Grams per 100 gm of protein.
linic acid (Henderson, 1949; Henderson and Hirsch, 1949; Moline et al., 1959; Nishizuka and Hayaishi, 1963). Although it was believed that quinolinic acid could be directly converted to nicotinic acid (Henderson, 1949; Henderson et al., 1949; Sarrett, 1951 ; Hankes and Segal, 1957), it has now been conclusively shown that it is an immediate precursor of nicotinic acid ribonucleotide (Nishizuka and Hayaishi, 1963). Tryptophan
1w
(5)
5-Hydroxytryptophan
iV-Formylkynuren ne
(6)
SHydroxytryptamine
1
5-Hydroxyindoleacetic acid
1
Kynurenh
1
3-H ydroxyk ynurenine L.
3-Hydroxyanthranilir acid 112)
or-Amino-8-carboxymuconic semialdehyde l(3)
pathway
Quinolinic acid 1(4)
Nicotinic acid ribonucleotidc
1
Intermediate
J\ Glutarate
-----+
Picolinic acid
Nicotinic acid adenine dinucleotide
I
1
NAD
1
N'-Methyl nicotinamide
1
6-Pyridone N'Methyl nicotinamide
Fro. 1. Pathways of tryptophan catabolism: (1) tryptophan oxygenase, (2) 3-hydroxyanthranilate oxygenase, (3) picolinic carboxylase, (4) quinolinate phosphoribos~ltransferase, (5) tryptophan hydroxylase, (6) 5-hydroxytryptamine decarboxylase.
PELLAGRA AND AMINO ACID IMBALANCE
511
011 barely adequate protein diets providing marginal amounts of tryptophan, addition of other amino acids has been shown to cause a disturbance in tryptophan-nicotinic acid metabolism and to result in nicotinic acid deficiency (Rosen and Perlzweig, 1949; Lyman and Elvehjem, 1951; Koeppe and Henderson, 1955). It was inferred that, in a diet providing marginal amounts of all amino acids, tryptophan is spared for conversion to nicotinic acid, whereas when other amino acids are in excess and tryptophan becomes a limiting amino acid, this conversion is impaired (Henderson e t aZ., 1953; Koeppe and Henderson, 1955). Investigations were, therefore, undertaken to see whether excess leucine would also bring about such disturbances in the tryptophan-nicotinic acid interrelationship. Urinary excretion of N’MeN, 6-pyridone) tryptophan, and quinolinic acid was found to be low in pellagrins (Belavady e t al., 1963). Hankes e t cd. (1971) also observed low levels of N’MeN, nicotinic acid, and kynurenic acid in the urine of pellagrins. Normal subjects and pellagrins were maintained on diets providing approximately 2300 kcals and 50 gm of protein, derived solely from vegetable sources. The diets provided about 8% protein and were similar to those consumed by the population which suffers from pellagra. Each subject was given in addition every day 10 gm of L-leucine orally. Administration of leucine brought about a marked increase in the excretion of quinolinic acid and a significant decrease in that of 6-pyridone (Belavady e t cd., 1963). Withdrawal of leucine resulted in a reversal to the basal pattern. Although in a preliminary study there was suggested evidence thal; leucine administration may increase urinary excretion of N‘MeN (Gopalan and Srikantia, 1960), subsequent studies showed that there is no consistent alteration in the excretion of this metabolite (Belavady e t al., 1963). The foregoing observations suggested that there might be a block in the further metabolism of quinolinic acid following leucine administration. More detailed investigations were carried out in experimental rats. Weanling albino rats were fed a basal diet containing 9% casein but adequate in other respects. The animals were also given supplements of either L-leucine or L-tryptophan. Quinolinic acid excretion was markedly increased. Excretion of N‘MeN although raised was not significant (Raghuramulu e t al., 1965a). Moreover, quinolinic acid excretion was found to increase with increasing doses of leucine. The effect was specific to leucine and was not seen with lysine, glycine, methionine, or threonine. Quinolinic acid excretion was found to be higher in rats fed a jowar diet than in those fed a wheat diet. The excretion of nicotinic acid and N’MeN Wafs also high.
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C. GOPALAN A N D K. S . JAYA
RAO
B. PLASMA TRYPTOPHAN LEVELS Plasma tryptophan levels are in the low range in pellagrins. Truswell
et al. (1968) were of the opinion that the plasma level of this amino acid may be a good biochemical index of pellagra. However, individual variations in the levels are so wide th at it would appear that the plasma tryptophan level may not be of much significance in the pathogenesis of the disease (Ghafoorunissa and Narasinga Rao, 1975). The low plasma tryptophan levels are more likely a reflection of the general undernutrition in the pellagrins, since nonpellagrins from low socioeconomic groups, in which pellagra is common, had levels similar to those in pellagrins. On the other hand, plasma tryptophan levels in well-nourished nonpellagrins were higher than those seen in either of the former two groups. Truswell et a2. (1968) also observed that plasma tryptophan levels in children suffering from pellagra and kwashiorkor were of similar order. A decrease in urinary excretion of tryptophan was observed after a leucine load (Belavady et al., 1963). Feeding a high-leucine diet to rats resulted in decreased levels of plasma valine and isoleucine (Rogers et nl., 1962), and a similar observation has also been made in human subjects after a leucine load (Swendseid et al., 1965). The studies of Hagihira et a2. (1960) suggest that these alterations might be due to a competition between the amino acids during intestinal absorption. However, such a mechanism does not appear to operate in the case of leucine and tryptophan. A leucine load did not bring about any alterations in plasma tryptophan levels in normal volunteers (Ghafoorunissa and Narasinga Rao, 1971.) Further, the increase in plasma tryptophan levels following simultaneous administration of tryptophan and leucine was found to be similar to that observed after a tryptophan load alone.
C. NICOTINAMIDE SIXLEDTIDES I N ERYTHROCYTES The functional forms of nicotinic acid in the body are the two nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). I n view of the altered nicotinic acid metabolism in pellagra, studies were directed toward investigating the profile of these constituents. The total nucleotide concentration in the erythrocytes of pellagrins was similar to that in normals (Raghuramulu et al., 1965b). This is in line with the observations of Axelrod et al. (1941). The rate of nucleotide synthesis was, however, significantly lower in the pellagrins. Supplementation with L-leucine for 5 days did not alter the nucleotide concentration either in pellagrins or in normal subjects, but its rate of synthesis was suppressed in both groups to nearly
PELLAGRA AND AMINO ACID IMBALANCE
513
60% of the initial values. A rapid reversal to basal conditions occurred following withdrawal of leucine. Leucine added in vitro a t high concentratilons also inhibited the nucleotide synthesis (Belavady et al., 1973). As mentioned earlier, leucine supplementation led to an increased excretion of quinolinic acid and, i t may be logically assumed, to an increased concentration in blood. Quinolinic acid was found to inhibit nucleotide syntihesis in vitro, the inhibition being progressive with increasing concentrations of the compound (Raghuramulu e t al., 1965b). A similar mechanism may also be operative in v i m . The depressed rate of synthesis may, therefore, be due to a direct action of leucine or of quinolinic acid. The paradox of a normal concentration of the nucleotide in the face of a depressed synthesizing capacity of the erythrocytes was resolved in subsequent studies. Fractionation of the nucleotides was carried out by paper chromatography using the solvent system 95% ethanol: 1 M ammonium acetate, pH 5 , in the ratio of 7:3, as described by Preiss and Handler (1958a). Much of the nucleotide in the normal subjects resolved into NAD and NADP. In pellagrins a third spot corresponding to nicotinamide mononucleotide (NMN) was identified. Levels of NAD and NADP were lower than in normals (Srikantia et al., 1968a). Treatment with nicotinic acid brought about a marked reduction in the NMN content of the erythrocytes of pellagrins. NMN is normally not found in human and dog erythrocytes. However, the monkey, guinea pig, and rat have significant concentrations of NMN in their erythrocytes (Table 11). In dogs which developed blacktongue after feeding of jowar or maize diets, the total nucleotide concentration of the erythrocytes was not altered-a finding similar to that observed in pellagrins and in normal volunteers administered L-leucine. Nevertheless, NMN was detected in considerable concentrations. Monkeys fed jowar diets, however, showed a fall in the total concentration of nucleotides in the erythrocytes but no alteration in the NMN concentration (Belavady et al., 1968). Similar observations were also made in monkeys fed leucine (Belavady and Rao, 1973). It appears that there may be species variations in the changes in the nucleotide pattern following consumption of jowar. In species that normally have NMN in erythrocytes, the concentration is not changed, whereas those which normally do not have detectable quantities of the nucleotide show an increase. I n the absence of an adequate explanation for the normally observed species variations, it is not wise to speculate further on these divergent observations. The pathway proposed by Preiss and Handler (1958b) for the biosynthesis of nicotinamide nucleotides from nicotinic acid is depicted in Fig. 2. 'The incorporation of I4C-labeled nicotinic acid into nicotinic acid ribonucleotide as well as into nicotinic acid adenine dinucleotide
514
C. GOPALAN AND K. S. JAYA RAO
SPECIICS
~)IFFEHENCES I N
TABLE I1 NICOTINAMIDE NUCIXOTIDPCONCENTH ITIONS OF
ERYTHROCYTES
Species
Tot a1 nucleo tides (mg/100 1x11 of erythrocytes)
Chick (3)$ Dog (13) Iluckling (7) Guinea pig (3) Monkey (21) Rat (6) Man (12)
9 . r, 7.0 13.3 10.2 12.4 12.1 5.2
NAU (yoof total) 74
74 87.5 64 58 80.5 65. .i
NAIjP (70 of total)
NMN ( % of total)
26 22 12.5 24.5 28 12 34.3
Trace
