Niacin is unusual among the vitamins in that it was discovered as a chemical compound, nicotinic acid produced by the oxidation of nicotine, in 1867 -long before there was any suspicion that it might have a role in nutrition. Its metabolic function as part of what was then called coenzyme II [nicotinamide adenine dinucleotide phosphate (NADP)] was discovered in 1935, again before its nutritional significance was known.

It is not strictly correct to regard niacin as a vitamin. Its metabolic role is as the precursor of the nicotinamide moiety of the nicotinamide nucleotide coenzymes, nicotinamide adenine dinucleotide (NAD) and NADP, and this can also be synthesized in vivo from the essential amino acid tryptophan. At least in developed countries, average intakes of protein provide more than enough tryptophan to meet requirements for NAD synthesis without any need for preformed niacin. It is only when tryptophan metabolism is disturbed, or intake of the amino acid is inadequate, that niacin becomes a dietary essential.

The nicotinamide nucleotide coenzymes function as electron carriers in a wide variety of redox reactions. In addition, NAD is the precursor of adenine dinucleotide phosphate (ADP)-ribose for ADP-ribosylation and poly(ADP-ribosylation) of proteins and cADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). They act as second messengers and stimulate increases in intracellular calcium concentrations.

Pellagra was first described as mal de la rosa in Asturias in central Spain by Casal in 1735. He observed that the condition was apparently related to diet and was distinct from scurvy and other then known causes of superficially similar dermatitis. The name pellagra was coined by the Italian physician Frapolli in 1771 to describe the most striking feature of the disease: the roughened, sunburn-like appearance of the skin. Pellagra became common in Europe when maize was introduced from the New World as a convenient high-yielding dietary staple. By the nineteenth century, it was widespread throughout southern Europe and north Africa. The disease was unknown in southern Africa until the outbreak of rinderpest in 1897, which led to widespread death of cattle and a major change in the dietary habits of the Bantu.

From being a meat- and milk-eating community, they became, and have remained, largely maize eaters, and pellagra continued to be a major problem of public health nutrition in South Africa throughout much of the twentieth century. The other region where pellagra was a major problem at the beginning of the twentieth century was the southern part of the United States. The social and economic upheaval of the American Civil War led to a poor maize-based diet for large sections of the population, and it was not until the entry of the United States into the second World War that increasing employment and a rise in the general standard of living solved the dietary problem. Although Casal had considered pellagra to be due to a dietary deficiency, investigations at the beginning of the twentieth century started from the assumption that, like other diseases, it was an infection. It was the pioneering studies of Gold-berger and coworkers in the United States that showed that the condition was neither contagious nor infectious, but could be prevented or cured by dietary means.

After it had been established that pellagra was a nutritional deficiency disease, the next problem was to discover the missing nutrient. Additional dietary protein was shown to be beneficial, thus it was concluded that pellagra was because of a protein deficiency. This view, and later that it was more specifically from a deficiency of tryptophan, was held for some time. In 1938, Spies and coworkers showed that nicotinic acid would cure pellagra; thereafter it was gradually accepted that it was a niacin deficiency disease.

The role of additional dietary protein in curing pellagra was elucidated in 1947, when it was shown that the administration of tryptophan to human beings led to an increase in the urinary excretion of N -methyl nicotinamide, the major urinary metabolite of niacin. It is usual to regard pellagra as a niacin deficiency disease, and tryptophan as a substitute for niacin when the dietary intake of the vitamin is inadequate. However, this is not strictly correct, and pellagra should be regarded as being a deficiency of both tryptophan and niacin.


The term niacin is the generic descriptor for the two compounds that have the biological action of the vitamin: nicotinic acid and nicotinamide (see nicotinamide nicotinic acid nicotinamide nicotinic acid


phosphorylated in NADP

nicotinamide adenine dinucleotide (NAD)

Figure 8.1. Niacin vitamers, nicotinamide and nicotinic acid, and the nicotinamide nucleotide coenzymes. Relative molecular masses (Mr): nicotinic acid, 123.1; nicotinamide, 122.1; NAD, 663.4; and NADP 743.4.

Figure 8.1). Although the amino acid tryptophan is quantitatively the major precursor of the nicotinamide ring of the coenzymes (Section 8.3), it is not considered to be a niacin vitamer.

Nicotinic acid was discovered and named as a product of the chemical oxidation of nicotine in 1867. When it was later discovered to be the pellagra-preventing vitamin, it was not assigned a number among the B vitamins because its chemistry was already known. Niacin is generally placed between vitamins B2 and B6, although it is incorrect to call it vitamin B3, which was at one time assigned to pantothenic acid (Section 12.1).

There is confusion in the literature because of the North American usage of the name niacin to mean specifically nicotinic acid, whereas the amide is known as niacinamide. The name niacin was coined in the late 1940s when the role of deficiency in the etiology of pellagra was realized, and it was decided that dietary staples should be fortified with the vitamin. It was felt that nicotinic acid was not a suitable name for a substance that was to be added to foods, both because of its phonetic (and chemical) relationship to nicotine and because it is an acid.

Nicotinic acid and nicotinamide have equal biological activity. As discussed in Section 8.3, approximately 60 mg of tryptophan is equivalent to 1 mg of dietary preformed niacin. Requirements and intakes are calculated as mg niacin equivalents - the sum of preformed niacin + 1/60 x tryptophan.


8.2.1 Digestion and Absorption

Niacin is present in tissues, and therefore in foods, largely as the nicotinamide nucleotides. The postmortem hydrolysis of NAD(P) is extremely rapid in animal tissues, and it is likely that much of the niacin of meat (a major dietary source of the vitamin) is free nicotinamide.

Nicotinamide nucleotides present in the intestinal lumen are hydrolyzed to nicotinamide. A number of intestinal bacteria have high nicotinamide deami-dase activity, and a significant proportion of dietary nicotinamide may be deamidated in the intestinal lumen. Both nicotinic acid and nicotinamide are absorbed from the small intestine by a sodium-dependent saturable process. Unavailable Niacin in Cereals Chemical analysis reveals niacin in cereals (largely in the bran), but this is biologically unavailable, because it is bound as niacytin - nicotinoyl esters to a variety of macromolecules ranging between Mr 1,500 to 17,000. In wheat bran, 60% is esterified to polysaccharides, and the remainder to polypeptides and glycopeptides (Mason et al., 1973). In calculation of niacin intakes, it is conventional to ignore the niacin content of cereals completely.

A small fraction of the niacin in niacytin may be biologically available as a result of hydrolysis by gastric acid. About 10% of the total is released as free nicotinic acid after extraction of maize or sorghum meal with 0.1 mol per L of hydrochloric acid, and Carter and Carpenter (1982) have shown that about 10% of the total niacin content of maize is biologically available to humans beings.

Treatment of cereals with alkali (e.g., soaking overnight in a calcium hydroxide solution, as is the traditional method for the preparation of tortillas in Mexico) and baking with alkaline bakingpowder, releases much of the nicotinic acid. This may explain why pellagra has always been rare in Mexico, despite the fact that maize is the dietary staple. Roasting of whole grain maize has a similar effect, because there is enough ammonia released from glutamine to form free nicotinamide by ammonolysis.

8.2.2 Synthesis of the Nicotinamide Nucleotide Coenzymes

As shown in Figure 8.2, the nicotinamide nucleotide coenzymes NAD and NADP can be synthesized from either of the niacin vitamers or from quinolinic

Quinolinate Phosphoribosyl Transferase

Figure 8.2. Synthesis of NAD from nicotinamide, nicotinic acid, and quinolinic acid. Quinolinate phosphoribosyltransferase, EC; nicotinic acid phosphoribosyl-transferase, EC; nicotinamide phosphoribosyltransferase, EC; nicotinamide deamidase, EC; NAD glycohydrolase, EC; NAD pyrophosphatase, EC; ADP-ribosyltransferases, EC and EC; and poly(ADP-ribose) polymerase, EC PRPP, phosphoribosyl pyrophosphate.

Figure 8.2. Synthesis of NAD from nicotinamide, nicotinic acid, and quinolinic acid. Quinolinate phosphoribosyltransferase, EC; nicotinic acid phosphoribosyl-transferase, EC; nicotinamide phosphoribosyltransferase, EC; nicotinamide deamidase, EC; NAD glycohydrolase, EC; NAD pyrophosphatase, EC; ADP-ribosyltransferases, EC and EC; and poly(ADP-ribose) polymerase, EC PRPP, phosphoribosyl pyrophosphate.

acid, a metabolite of the amino acid tryptophan. Incorporation of nicotinic acid is catalyzed by nicotinate phosphoribosyltransferase to yield nicotinic acid mononucleotide, which is converted to nicotinic acid adenine dinucleotide (desamido-NAD) by the action of nicotinic acid mononucleotide pyrophosphorylase, and then amidated to NAD. Incorporation of nicoti-namide may either be direct, catalyzed by nicotinamide phosphoribosyltrans-ferase to yield nicotinamide mononucleotide, and then NAD by the action of nicotinamide mononucleotide pyrophosphorylase, or indirect, by deamida-tion to nicotinic acid, catalyzed by nicotinamide deamidase. The dicarboxylic acid intermediate in the quinolinate phosphoribosyltransferase reaction undergoes spontaneous decarboxylation to nicotinic acid mononucleotide.

In the liver, there is little utilization of preformed niacin for nucleotide synthesis. Although isolated hepatocytes will take up both vitamers from the incubation medium, they seem not to be used for NAD synthesis and cannot prevent the fall in intracellular NAD(P), which occurs during incubation. The enzymes for nicotinic acid and nicotinamide utilization are more or less saturated with their substrates at normal concentrations in the liver, and hence are unlikely to be able to use additional niacin for nucleotide synthesis. By contrast, incubation of isolated hepatocytes with tryptophan results in a considerable increase in the rate of synthesis of NAD(P) and accumulation of nicotinamide and nicotinic acid in the incubation medium. Similarly, feeding experimental animals on diets providing high intakes of nicotinic acid or nicotinamide has relatively little effect on the concentration of NAD(P) in the liver, whereas high intakes of tryptophan lead to a considerable increase. It thus seems likely that the major role of the liver is to synthesize NAD(P) from tryptophan, followed by hydrolysis to release niacin for use by extrahepatic tissues (Bender et al., 1982; McCreanor and Bender, 1986; Bender and Olufunwa, 1988).

In most extrahepatic tissues, nicotinic acid is a better precursor of nucleotides than is nicotinamide. However, muscle, brain, and to a lesser extent the testis are able to take up nicotinamide from the bloodstream effectively, and apparently utilize it without prior deamidation (Gerber and Deroo, 1970).

8.2.3 Catabolism of NAD(P)

The nicotinamide nucleotide coenzymes are catabolized by four enzymes, which act on the oxidized, but not the reduced, coenzymes:

1. NAD pyrophosphatase, which releases nicotinamide mononucleotide. This can either be hydrolyzed by NAD glycohydrolase to release nicotinamide, or can be a substrate for nicotinamide mononucleotide pyrophosphorylase, to form NAD.

2. NAD glycohydrolase, which releases nicotinamide and ADP-ribose. As discussed in Section 8.4.4, this enzyme also catalyzes the synthesis of cADP-ribose and nicotinic acid ADP which have roles in intracellular signaling.

3. ADP-ribosyltransferase, which catalyzes ADP-ribosylation of proteins, releasing nicotinamide (Section 8.4.2).

4. Poly(ADP-ribose) polymerase, whichcatalyzespoly-ADP-ribosylation of proteins, again releasing nicotinamide (Section 8.4.3).

The total NADase activity of tissues from these four enzymes is very high, and the total tissue content of nicotinamide nucleotides can be hydrolyzed within a few minutes. Two factors prevent this in vivo. Apart from NAD py-rophosphatase, the enzymes that catalyze the release of nicotinamide from NAD(P) are biosynthetic rather than catabolic, and their activity is highly regulated under normal conditions. Furthermore, the values of Km of the enzymes are of the same order of magnitude as those of many of the NAD(P)-dependent enzymes in the cell, so that there is considerable competition for the nucleotides. Only that relatively small proportion of the nicotinamide nucleotide pool in the cell that is free at any one time will be immediately available for hydrolysis.

8.2.4 Urinary Excretion of Niacin Metabolites

Under normal conditions, there is little or no urinary excretion of either nicotinamide or nicotinic acid, because both vitamers are actively reabsorbed from the glomerular filtrate. It is only when the concentration is so high that the transport mechanism is saturated that there is any significant excretion.

As shown in Figure 8.3, the principal metabolites of nicotinamide are N1-methyl nicotinamide and methyl pyridone carboxamides. N1 -Methyl nicotinamide is actively secreted into the urine by the proximal renal tubules. Nicotinamide N-methyltransferase is an S-adenosylmethionine-dependent enzyme that is present in most tissues. Very high intakes of nicotinamide may deplete tissue pools of one-carbon fragments - indeed, this was the basis for the use of nicotinamide in the treatment of schizophrenia (Section 8.8).

N1-Methyl nicotinamide can also be oxidized to methyl pyridone-2-carboxamide and methyl pyridone-4-carboxamide. The extent to which this oxidation occurs, and the relative proportions of the two pyridones formed,

Source Nicotinamide Formation

Figure 8.3. Metabolites of nicotinamide and nicotinic acid. Nicotinamide deamidase (nicotinamidase), EC; nicotinamide N-methyltransferase, EC; aldehyde dehydrogenase, EC Relative molecular masses (Mr): nicotinamide, 123.1; nicotinic acid, 122.1; nicotinamide N-oxide, 139.1; N1-methyl nicotinamide, 139.1; trigonelline, 137.1; nicotinuric acid, 179.2; and methyl pyridone carboxamides, 154.1.

Figure 8.3. Metabolites of nicotinamide and nicotinic acid. Nicotinamide deamidase (nicotinamidase), EC; nicotinamide N-methyltransferase, EC; aldehyde dehydrogenase, EC Relative molecular masses (Mr): nicotinamide, 123.1; nicotinic acid, 122.1; nicotinamide N-oxide, 139.1; N1-methyl nicotinamide, 139.1; trigonelline, 137.1; nicotinuric acid, 179.2; and methyl pyridone carboxamides, 154.1.

not only varies from one species to another, but shows considerable variation between different strains of the same species. Aldehyde oxidase catalyzes the formation of both pyridones, and some additional 2-pyridone arises from the activity of xanthine oxidase. Aldehyde oxidase is activated by androgens, and male mice excrete 2 to 3 times more pyridone than do females (Felsted and Chaykin, 1967; Stanulovic and Chaykin, 1971a, 1971b).

Nicotinamide can also undergo oxidation to nicotinamide N-oxide. This is normally a minor metabolite in human beings, unless large amounts (about 200 mg) of nicotinamide are ingested. In the mouse, nicotinamide N-oxide is the major excretory product of niacin metabolism. At high levels of nicotinamide intake, some 6-hydroxynicotinamide may also be excreted.

Nicotinic acid can be conjugated with glycine to form nicotinuric acid (nicotinoyl-glycine), or may be methylated to trigonelline (N1 -methyl nicotinic acid). It is not clear to what extent urinary excretion of trigonelline reflects endogenous methylation of nicotinic acid, because there are significant amounts of trigonelline in foods that may be absorbed, but cannot be utilized as a source of niacin, and are excreted unchanged. Small amounts of 6-hydroxynicotinic acid may also be formed.


As shown in Figure 8.2, NAD(P) can be synthesized from the tryptophan metabolite quinolinic acid. The oxidative pathway of tryptophan metabolism is shown in Figure 8.4. Under normal conditions, almost all of the dietary intake of tryptophan, apart from the small amount that is used for net new protein synthesis, is metabolized by this pathway, and hence is potentially available for NAD synthesis. About 1% of tryptophan metabolism is by way of 5-hydroxylation and decarboxylation to 5-hydroxytryptamine (serotonin), which is excreted mainly as 5-hydroxyindoleacetic acid.

A number of studies have investigated the equivalence of dietary trypto-phan and preformed niacin as precursors of the nicotinamide nucleotides, generally by determining the excretion of N1 -methyl nicotinamide and methyl pyridone carboxamide in response to test doses of the precursors, in subjects maintained on deficient diets.

The most extensive such study was that of Horwitt and coworkers (1956). They found that there was a considerable variation between subjects in the response to tryptophan and niacin, and suggested that in order to allow for individual variation, it should be assumed that 60 mg of tryptophan was equivalent to 1 mg of preformed niacin. This ratio has been generally accepted, and is the basis for expressing niacin requirements and intake in terms of niacin equivalents - the sum of preformed niacin and 1/60 of the tryptophan.

Changes in hormonal status may result in considerable changes in this ratio, between 7 to 30 mg of dietary tryptophan equivalent to 1 mg ofpreformed niacin in late pregnancy. The intake of tryptophan also affects the ratio. At low intakes, 1 mg of tryptophan may be equivalent to only 1/125 mg of preformed niacin (Nakagawa et al., 1969).

Tryptophan dioxygenase (Section 8.3.2) is only found in the liver; other tissues have an indoleamine dioxygenase, with lower specificity, that catalyzes the same reaction. However, the pathway for onward metabolism of kynure-nine is found only in liver and mononuclear phagocytes, and induction of in-doleamine dioxygenase by cytokines, such as interferon-y, leads to increased circulating concentrations and urinary excretion of kynurenine, with little or

Biosynthesis Niacin From Tryptophan

Figure 8.4. Pathways of tryptophan metabolism. Tryptophan dioxygenase, EC; formylkynurenine formamidase, EC; kynurenine hydroxylase, EC; kynureninase, EC; 3-hydroxyanthranilate oxidase, EC; picol-inate carboxylase, EC; kynurenine oxoglutarate aminotransferase, EC; kynurenine glyoxylate aminotransferase,; tryptophan hydroxylase, EC; and 5-hydroxytryptophan decarboxylase, EC Relative molecular masses (Mr): tryptophan, 204.2; serotonin, 176.2; kynurenine, 208.2; 3-hydroxykynurenine, 223.2; kynurenic acid, 189.2; xanthurenic acid, 205.2; and quinolinic acid 167.1. CoA, coenzyme A.

Figure 8.4. Pathways of tryptophan metabolism. Tryptophan dioxygenase, EC; formylkynurenine formamidase, EC; kynurenine hydroxylase, EC; kynureninase, EC; 3-hydroxyanthranilate oxidase, EC; picol-inate carboxylase, EC; kynurenine oxoglutarate aminotransferase, EC; kynurenine glyoxylate aminotransferase,; tryptophan hydroxylase, EC; and 5-hydroxytryptophan decarboxylase, EC Relative molecular masses (Mr): tryptophan, 204.2; serotonin, 176.2; kynurenine, 208.2; 3-hydroxykynurenine, 223.2; kynurenic acid, 189.2; xanthurenic acid, 205.2; and quinolinic acid 167.1. CoA, coenzyme A.

no formation of quinolinic acid and hence NAD(P). Induction of indoleamine dioxygenase may therefore be a factor in tryptophan depletion leading to the development of pellagra (Section 8.5).

8.3.1 Picolinate Carboxylase and Nonenzymic Cyclization to Quinolinic Acid

As shown in Figure 8.4, the synthesis of NAD from tryptophan involves the nonenzymic cyclization of aminocarboxymuconic semialdehyde to quinolinic acid. The alternative metabolic fate of aminocarboxymuconic semialdehyde is decarboxylation, catalyzed by picolinate carboxylase, leading into the ox-idative branch of the pathway, and catabolism via acetyl coenzyme A. There is thus competition between an enzyme-catalyzed reaction that has hyperbolic, saturable kinetics, and a nonenzymic reaction thathas linear, first-order kinetics.

The result of this is that at low rates of flux through the kynurenine pathway, which result in concentrations of aminocarboxymuconic semialdehyde below that at which picolinate carboxylase is saturated, most of the flux will be byway of the enzyme-catalyzed pathway, leading to oxidation. There will be little accumulation of aminocarboxymuconic semialdehyde to undergo nonenzymic cyclization. As the rate of formation of aminocarboxymuconic semialdehyde increases, and picolinate carboxylase nears saturation, there will be an increasing amount available to undergo the nonenzymic reaction and onward metabolism to NAD. Thus, there is not a simple stoichiometric relationship between tryptophan and niacin, and the equivalence of the two coenzyme precursors will vary as the amount of tryptophan to be metabolized and the rate of metabolism vary.

As might be expected, the synthesis of NAD from tryptophan is inversely related to the activity of picolinate carboxylase. Inhibition with pyrazinamide results in increased availability of aminocarboxymuconic semialdehyde, and hence increased NAD formation. Equally, activation of picolinate carboxylase results in reduced availability of aminocarboxymuconic semialdehyde for cyclization, and hence a reduced formation of NAD.

Cats, which have some 30- to 50-fold higher activity of picolinate carboxylase than other species, are entirely reliant on a dietary source of preformed niacin, and are not capable of any significant synthesis of NAD from tryptophan.

It is thus apparent that the utilization of tryptophan as a precursor for NAD synthesis depends on both the amount of tryptophan to be metabolized and also the rate of metabolic flux through the pathway. The activities of three enzymes (tryptophan dioxygenase, kynurenine hydroxylase, and kynureni-nase) may all affect the rate of formation of aminocarboxymuconic semialdehyde, as may the rate of tryptophan uptake into the liver.

8.3.2 Tryptophan Dioxygenase

The first enzyme of the pathway, tryptophan dioxygenase (also known as tryptophan oxygenase or tryptophan pyrrolase), is rate-limiting under normal conditions. In isolated hepatocytes, the control coefficient for flux through the pathway of tryptophan dioxygenase is 0.75 and that for tryptophan uptake into the cells is 0.25 (Salter et al., 1986).

Tryptophan dioxygenase has a short half-life (of the order of 2 hours) and is subject to regulation by three mechanisms: saturation with its heme cofactor, hormonal induction and feedback inhibition, and repression by NAD(P). Saturation of Tryptophan Dioxygenase with Its Heme Cofactor

Unlike many other heme enzymes, the hematin of tryptophan dioxygenase behaves more like a dissociating cofactor than a tightly bound prosthetic group, and dissociation of the holo-enzyme can occur in the presence of protoporphyrin or mesoporphyrin. The holo-enzyme is more resistant to proteolysis than is the apoenzyme. In the presence of relatively large amounts of heme, both the activity of the enzyme and the total amount of immunoreac-tive tryptophan dioxygenase protein in the liver are increased. It has been suggested that induction of tryptophan dioxygenase apoenzyme may provide a metabolic sink for excess heme synthesis (Badawy and Evans, 1975; Badawy, 1977).

Tryptophan and a number of tryptophan analogs also enhance the stability of tryptophan dioxygenase by enhancing conjugation of the apoenzyme with hematin and stabilizing the holo-enzyme. The tryptophan analogs that promote heme conjugation are not substrates, nor do they compete with tryp-tophan at the catalytic site of the enzyme. They appear to bind to a domain on the enzyme protein that is distinct from the catalytic site. The conjugation promoting site of the apoenzyme has a relatively broad specificity, and a Km for tryptophan of 26 ^mol per L, whereas the catalytic site on the holo-enzyme binds only L-tryptophan and has a 10-fold higher Km. Induction of Tryptophan Dioxygenase by Glucocorticoid Hormones The de novo synthesis of tryptophan dioxygenase is induced by glucocorticoid hormones (cortisol in human beings and corticosterone in the rat). This is true induction of new mRNA and protein synthesis; indeed, tryptophan dioxygenase was the first mammalian enzyme to be shown to be inducible.

In isolated hepatocytes, alter maximum induction of tryptophan dioxygenase by glucocorticoids, the uptake of tryptophan into the cells has a control coefficient of 0.75, whereas the control coefficient of tryptophan dioxygenase falls to 0.25. Therefore, the induction of tryptophan dioxygenase has only a limited effect on tryptophan catabolism and NAD synthesis (Salter and Pogson, 1985; Salter et al., 1986). In isolated perfused liver, although cortisol leads to a several-fold increase in tryptophan dioxygenase activity, there is only a relatively small increase in the rate of clearance of tryptophan from the perfusion medium (Kim and Miller, 1969). Induction of Tryptophan Dioxygenase by Glucagon Glucagon (mediated by cAMP) increases the synthesis of tryptophan dioxygenase after the administration of glucocorticoids, although it has little effect in unstimulated animals. The effect of glucagon appears to be the result of an increase in the rate of translation of mRNA rather than an increase in transcription and is antagonized by insulin. Repression and Inhibition of Tryptophan Dioxygenase by Nicotinamide Nucleotides High concentrations of the nicotinamide nucleotide coenzymes, and especially NADPH, both inhibit preformed tryptophan oxy-genase and also repress its synthesis. It is not clear how important this is in terms of physiological regulation, because concentrations of the nucleotides required to achieve significant inhibition are relatively high, but some of the effects of alcohol on tryptophan metabolism may be because of the increase in NADH and NADPH that follows the ingestion of relatively large amounts of alcohol (Badawy, 2002).

8.3.3 Kynurenine Hydroxylase and Kynureninase

The entry of tryptophan into the oxidative pathway is limited by tryptophan dioxygenase activity and the uptake of tryptophan; under basal conditions, these two processes have control coefficients of 0.75 and 0.25, respectively. Neither kynurenine hydroxylase nor kynureninase has a significant control coefficient in metabolic flux studies inhepatocytes isolated from normal animals (Salter and Pogson, 1985; Salter et al., 1986). However, the activity of both enzymes is only slightly higher than that of tryptophan dioxygenase under basal conditions, and increased tryptophan dioxygenase activity is accompanied by increased accumulation and excretion of kynurenine, hydoxykynurenine, and their transamination products, kynurenic and xanthurenic acids (see Figures 8.4 and 9.4).

Even without induction of tryptophan dioxygenase, impairment of the activity of either enzyme may impair the onward metabolism of kynurenine and thus reduce the accumulation of aminocarboxymuconic semialdehyde and synthesis of NAD.

During the first half of the twentieth century, when 87,000 people died from pellagra in the United States, there was a two-fold excess of females over males. Reports of individual outbreaks of pellagra show a similar sex ratio. This may well be the result of inhibition of kynureninase, and impairment of the activity of kynurenine hydroxylase, by estrogen metabolites, and hence reduced synthesis of NAD from tryptophan (Bender and Totoe, 1984b). Kynurenine Hydroxylase Kynurenine hydroxylase is an FAD-dependent mixed-function oxidase of the outer mitochondrial membrane, which uses NADPH as the reductant. The activity of kynurenine hydroxylase in the liver of riboflavin-deficient rats is only 30% to 50% of that in control animals, and deficient rats excrete abnormally large amounts of kynurenic and anthranilic acids after the administration of a loading dose of tryptophan, and, correspondingly lower amounts of quinolinate and niacin metabolites. Riboflavin deficiency may thus be a contributory factor in the etiology of pellagra when intakes of tryptophan and niacin are marginal (Section 8.5.1).

In a number of studies, sexually mature women show a higher ratio of urinary kynurenine:hydroxykynurenine than do children, postmenopausal women, or men, suggesting impairment of kynurenine hydroxylase activity by estrogens or their metabolites. In experimental animals, the administration of estrogens results in a reduction in kynurenine hydroxylase activity to about 30% of the control activity. The mechanism of this effect is unclear, because the addition of estrogens or their metabolites has no effect on the enzyme in vitro. It is possible that the effect is indirect, and due to the inhibition of kynureninase by estrogen conjugates, and hence an accumulation of hydroxykynurenine, and increased formation of kynurenic and xanthurenic acids. Kynurenine hydroxylase is inhibited by micromolar concentrations of xanthurenic acid (Bender and McCreanor, 1985). Kynureninase Kynureninase is apyridoxalphosphate (vitamin B6)-dependent enzyme that catalyzes the hydrolysis of 3-hydroxykynurenine to 3-hydroxyanthranilic acid, releasing the side chain as alanine. Impairment of kynureninase activity in vitamin B6 deficiency leads to accumulation of kynurenine and hydroxykynurenine, and their transamination products, kynurenic and xanthurenic acids. This is the basis of the tryptophan load test forvitaminB6 nutritional status (Section9.5.4). Vitamin B6 deficiency, orinhi-bition of kynureninase by estrogen metabolites, would therefore be expected to reduce the rate of metabolic flux through the oxidative pathway and reduce the formation of quinolinic acid and NAD from tryptophan.


Nicotinamide is the reactive moiety of the nicotinamide nucleotide coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate), which are coenzymes (or more correctly cosubstrates) in a wide variety of oxidation and reduction reactions (Section 8.4.1). The notation NAD(P) is used to mean either NAD or NADP, without specifying the oxidation state.

NAD is the source of ADP-ribose for the modification of proteins by mono-ADP-ribosylation, catalyzed by ADP-ribosyltransferases (Section 8.4.2), and poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (Section 8.4.3). It is also the precursor of two second messengers that act to increase the intracellular concentration of calcium, cADP-ribose, and nicotinic acid adenine dinucleotide phosphate (Section 8.4.4).

Nicotinic acid has been tentatively identified as the organic component of the (as yet uncharacterized) chromium-containing glucose tolerance factor that enhances the interaction of insulin with cell surface receptors.

8.4.1 The Redox Function of NAD(P)

The nicotinamide coenzymes are involved as proton and electron carriers in a wide variety of oxidation and reduction reactions. Before their chemical structures were known, NAD and NADP were known as coenzymes I and II. Later, when the chemical nature of the pyridine ring of nicotinamide was discovered, they were called diphosphopyridine nucleotide (DPN = NAD) and triphospho-pyridine nucleotide (TPN = NADP). The nicotinamide nucleotide coenzymes are sometimes referred to as the pyridine nucleotide coenzymes.

As shown in Figure 8.5, the oxidized coenzymes have a formal positive charge, and are represented as NAD+ and NADP+, whereas the reduced forms, carrying two electrons and one proton (and associated with an additional proton), are represented as NADH and NADPH. The two-electron reduction of NAD(P)+ proceeds by way of a hydride (H-) ion transfer to carbon-4 of the nicotinamide ring.

oxidized coenzyme NAD+ or NADP+

Figure 8.5. Redox function of the nicotinamide nucleotide coenzymes.

In general, NAD+ is involved as an electron acceptor in energy-yielding metabolism, and the resultant NADH is oxidized by the mitochondrial electron transport chain. The major coenzyme for reductive synthetic reactions is NADPH. An exception here is the pentose phosphate pathway (see Figure 6.4), which reduces NADP+ to NADPH and is the source of about half the reductant for lipogenesis.

In the reduced coenzymes, the hydrogen atoms at carbon-4 of the nicoti-namide ring lie above and below the plane of the ring. Isotope studies have shown that they are not equivalent, and enzymes specifically remove or add the pro-R hydrogen (above the plane of the ring) or the pro-S hydrogen (below the plane of the ring). The result of this is that, although NAD(P) acts as a cosubstrate, binding to and being released from the enzyme catalytic site during the reaction, rather than remaining enzyme bound, there can be considerable channeling between pairs of enzymes that use the opposite faces of the coenzyme and effectively sequester the coenzyme between them. Use of NAD(P) in Enzyme Assays The reduced coenzymes have an absorption maximum at 340 nm, whereas the oxidized coenzymes do not. This is widely exploited to provide sensitive and specific methods for determining a variety of analytes using purified NAD(P)-linked enzymes and following the change in absorption at 340 nm as the coenzyme is either reduced or oxidized by the substrate.

8.4.2 ADP-Ribosyltransferases

ADP-ribosylation is a reversible modification of proteins, as shown in Figure 8.6, and there are specific hydrolases that remove the ADP-ribose from target proteins.

ADP-ribosyltransferases are enzymes of the cytosol, plasma membrane, and nuclear envelope that catalyze the transfer of ADP-ribose onto arginine,

Poly Adp Ribose Polymerase
Figure 8.6. Reactions of ADP-ribosyltransferase (EC and poly(ADP-ribose) polymerase (EC

lysine, or asparagine residues in acceptor proteins to form N-glycosides. The plasma membrane ADP-ribosyltransferases are ecto-enzymes, anchored in the membrane by a glycosyl phosphoinositol tail, and have been implicated in cell adhesion and also in chemotaxis in lymphocytes. In addition to endogenous ADP-ribosyltransferases, a number of bacterial toxins, including diphtheria and cholera toxins, Escherichia coli enterotoxin LT and Pseudomonas aeruginosa exotoxin A also have ADP-ribosyltransferase activity.

In the absence of an acceptor protein, ADP-ribosyltransferase catalyzes the hydrolysis of NAD+ to release nicotinamide and free ADP-ribose. The carboxy terminal region of the enzyme has NAD glycohydrolase activity, but does not catalyze the transfer of ADP-ribose onto target proteins.

The ribosomal elongation Factor II is the acceptor protein for the ADP-ribosyltransferase activity of diphtheria toxin and P. aeruginosa exotoxin A, as well as a mammalian cytosolic ADP-ribosyltransferase. ADP-ribosylation results in loss of activity. The uncontrolled action of the bacterial toxins causes the cessation of protein synthesis and hence cell death. The more regulated action of the endogenous ADP-ribosyltransferase is part of the normal regulation of protein synthesis.

A variety of guanine nucleotide binding proteins (G-proteins) involved with the regulation of adenylate cyclase activity and transducin in the retina (Section 2.3.1) are substrates for ADP-ribosylation. Cholera toxin and E. coli enterotoxin LT ADP-ribosylate, and hence activate, the stimulatory G-protein of adenylate cyclase, whereas pertussis toxin ADP-ribosylates, and inactivates the inhibitory G-protein of adenylate cyclase. The result of ADP-ribosylation by either mechanism is increased adenylate cyclase activity, and an increase in intracellular cAMP and the opening of membrane calcium channels. Again, there are endogenous ADP-ribosyltransferases that modify the same G-proteins, but in a controlled manner (Moss et al., 1997, 1999).

8.4.3 Poly(ADP-ribose) Polymerases

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze transfer ofmultiple ADP-ribose units onto target proteins, as shown in Figure 8.6. They are DNA-binding proteins with a zinc-finger motif and require nicked DNA (with single- or double-strand breaks) for activity. They are present in the cell in high concentrations; about one molecule of enzyme for each kilobase of DNA (Hayaishi and Ueda, 1977; D'Amours et al., 1999).

Poly(ADP-ribose) polymerases catalyze three reactions:

1. Initial ADP-ribosylation of the y-carboxyl group of glutamate residues (or the carboxyl group of a C-terminal lysine) in the target protein, forming an O-glycoside.

2. Elongation of the poly-(ADP-ribose) chain by reaction with the 2 -hydroxyl group of the nonreducing end of the growing chain, with up to 200 elongation events catalyzed per initiation event.

3. Introduction of branch points every 30 to 40 ADP-ribose units.

Poly(ADP-ribose) formed by the polymerase turns over rapidly in DNA-damaged cells, with a half-life of the order of a minute. Cells contain a gly-cohydrolase with both endo- and exoglycosidase activity, which acts initially to remove the complete branched poly(ADP)ribose from the protein, then, more slowly, hydrolyze it to oligomers and free ADP-ribose.

The best-established function of poly(ADP-ribose) polymerase is in repair of damaged DNA; it is activated by DNA strand breaks, and acts to clear histones and other nucleoproteins away from the DNA to permit access of the DNA repair enzymes. Both in vitro and in experimental animals, niacin deficiency leads to increased genomic instability, as the ability to repair damaged DNA is impaired, and may increase tumor risk. There is little information about genomic instability and cancer risk in human niacin deficiency (Hageman and Stierum, 2001).

Activation of poly(ADP-ribose) polymerase also provides a mechanism of cell death in response to severe DNA damage. In undamaged cells, the concentration of NAD is 400 to 500 ^mol per L, with a half-life of the order of an hour. In response to DNA damage, it may fall by 80% within 5 to 15 minutes, resulting in significant impairment of oxidative metabolic activity, hence depletion of ATP and cell death.

The main target forpoly(ADP-ribosylation) ispoly(ADP-ribose) polymerase itself; the enzyme has up to 28 sites for automodification, and several thousand molecules of ADP-ribose may be added to each molecule of enzyme. Both while bound to the enzyme, and more importantly after release from the enzyme by glycohydrolase action, poly(ADP-ribose) has a considerably higher affinity for the basic amino acids in the a-helical tail of histones than does DNA, and serves to remove histones from DNA binding and hence unravel nucleosomes.

A variety of other nuclear proteins are also targets for poly(ADP-ribo-sylation), including histones, topo-isomerases, DNAligases, and DNA-depen-dent RNA polymerase, suggesting that in addition to its role in DNA repair, poly(ADP-ribose) polymerase may be important in DNA replication and transcription. In DNA replication, it controls the progression of the replication fork, and in preadipocytes, there is a considerable increase in activity in response to the induction of differentiation (Simbulan-Rosenthal et al., 1996).

Poly(ADP-ribose) polymerase may also have a role in regulating the activity of nuclear-acting hormone receptors; it binds directly to retinoid X receptors (Section and inhibits the transcriptional activity of retinoid X receptor-thyroid hormone dimers. It also acts as a transcription factor in pancreatic P-islet cells (Miyamoto et al., 1999).

The diabetogenic compounds streptozotocin and alloxan cause DNA strand breaks by radical generation, leading to necrosis of pancreatic p-islet cells as a result of NAD, and hence ATP, depletion. Release of cell contents from necrotic cells leads to the development of anti-p-cell antibodies and the autoimmune development of diabetes mellitus. In poly(ADP-ribose) polymerase knockout mice, these compounds are not diabetogenic, and inhibitors of poly(ADP-ribose) polymerase, such as nicotinamide, protect against streptozotocin- and alloxan-induced diabetes (Pieper et al., 1999). This has led to trials of nicotinamide as a protective agent against insulin-dependent diabetes mellitus in people at high risk (Section 8.8).

8.4.4 cADP-Ribose and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)

Cells contain high activities of enzymes that were originally identified as NAD glycohydrolases, catalyzing the hydrolysis of NAD(P)+ to nicotinamide and ADP-ribose (phosphate). As shown in Figure 8.7, these glycohydrolases also catalyze two additional reactions that lead to products that have a role in calcium release from intracellular stores, and act as second messengers (Dousa et al., 1996; Lee, 1996, 1999, 2000, 2001):

1. Cyclization of ADP-ribose arising from NAD to cADP-ribose.

2. Exchange of the nicotinamide moiety of NADP for nicotinic acid, forming NAADP. Early studies also showed that the enzyme can catalyze exchange of nicotinamide with a variety of other bases, including his-tamine. Formation of histamine adenine dinucleotide phosphate was thought to provide an alternative to amine oxidase for rapid inactivation of histamine.

The intracellular NAD glycohydrolase is now known as ADP-ribose cyclase; there is also a cell surface ectozyme, identical with the lymphocyte CD38 antigen. CD38 also occurs intracellularly, in endosomes. Both enzymes catalyze the formation of both cADP-ribose and NAADP, as well as the glycohydrolase reaction. The cyclase reaction predominates at neutral pH, and the nicotinamide/nicotinic acid exchange reaction at acid pH, suggesting that in cytosol the main product of the soluble enzyme is cADP-ribose. In endosomes,

Niacin Nadp
Figure 8.7. Reactions catalyzed by ADP ribose cyclase (NAD glycohydrolase, EC When the substrate is NADP, the base exchange reaction leads to the formation of nicotinic acid adenine dinucleotide phosphate.

the main product of CD38 is NAADP (Lee, 1999). In sea urchin eggs, cyclase activity is increased by a cGMP-dependent kinase, whereas NAADP formation is activated by a cAMP-dependent kinase, although in mammalian cells cGMP has no effect on the enzymes (Wilson and Galione, 1998).

Both cADP-ribose and NAADP act to increase cytosolic calcium concentrations, releasing calcium from intracellular stores via a receptor distinct from that which responds to inositol trisphosphate (Section 14.4.1). The responses to cADP-ribose and NAADP are additive, and they seem to act on different intracellular calcium stores (Jacobson et al., 1995; Patel et al., 2001).

The role of cADP-ribose and NAADP in regulating cytosolic calcium may provide an alternative explanation to the serotonin hypothesis for the psychiatric and neurological signs of the niacin deficiency disease pellagra (Section 8.5; Petersen and Cancela, 1999).

cADP-ribose and NAADP act as second messengers in response to nitric oxide, acetyl choline, and cholecystokinin, and are therefore involved in responses to neurotransmitters and hormones (Cancela, 2001). Pancreatic f-islet cells express CD38, and cADP-ribose has been implicated in the calcium release that signals insulin secretion. In response to increased intracellular ATP, as a result of increased uptake and metabolism of glucose, there is inhibition of the NAD glycohydrolase activity of the enzyme, and increased formation of cADP-ribose. This acts synergistically withpalmitate, formed as a result of increased glucose uptake and metabolism, to release calcium from intracellular stores (Okamoto, 1999a, 1999b).

All-frans-retinoic acid (Section stimulates the synthesis of cADP-ribose in kidney cells in culture, apparently as a result of the induction of CD38 (Beers et al., 1995; Takahashi et al., 1995); in ovariectomized rats, estradiol induces cytosolic ADP-ribosyl cyclase in the uterus, but not in estrogen unresponsive tissues (Chini et al., 1997). If this induction of ADP-ribose cyclase by estrogens leads to significant depletion of nicotinamide nucleotides, it may provide an additional explanation for the 2:1 excess of females to males in the incidence of pellagra (Section 8.5).


Pellagra is characterized by a photosensitive dermatitis, like severe sunburn, typically with a butterfly-like pattern of distribution over the face, affecting all parts of the skin that are exposed to sunlight. Similar skin lesions may also occur in areas not exposed to sunlight, but subject to pressure, such as the knees, elbows, wrists, and ankles. Advanced pellagra is also accompanied by a dementia or depressive psychosis, and there may be diarrhea. Untreated pellagra is fatal.

Pellagra was a major problem of public health in the early part of the twentieth century and continued to be a problem until the 1980s in some parts of the world. It is now rare, although there were reports of outbreaks among refugees in Africa (Malfait et al., 1993), and occasional cases are reported in alcoholics in developed countries and among people being treated with isoniazid (Section 8.5.6) and some other drugs, and people with chronic gastrointestinal disease.

Despite our understanding of the biochemistry of niacin, we still cannot account for the characteristic photosensitive dermatitis in terms of the known metabolic lesions. There is no apparent relationship between reduced availability of tryptophan and niacin, and sensitivity of the skin to ultraviolet (UV) light. The only biochemical abnormalities that have been reported in the skin of pellagrins involve increased catabolism of the amino acid histi-dine leading to a reduction in the concentration of urocanic acid, a histidine metabolite that is the major UV-absorbing compound in normal dermis (see Figure 10.6).

The other characteristic feature of pellagra is the development of a depressive psychosis, superficially similar to schizophrenia and the organic psychoses, but clinically distinguishable by the sudden lucid phases that alternate with the most florid psychiatric signs. The mental symptoms may be the result of tryptophan depletion, and hence a lower availability of tryptophan for synthesis of the neurotransmitter serotonin (5-hydroxytryptophan). But the role of cADP-ribose and NAADP in controlling calcium release in response to neurotransmitters (Section 8.4.4) and impaired energy-yielding metabolism in the central nervous system as a result of depletion of NAD(P) may also be important.

The diarrhea associated with pellagra is caused by rectal inflammation; within 5 to 7 days of starting treatment with niacin, rectal histology is normalized and the diarrhea ceases (Segal et al., 1986).

8.5.1 Other Nutrient Deficiencies in the Etiology of Pellagra

Although the nutritional etiology of pellagra is well established, and additional tryptophan or niacin will prevent or cure the disease, there are a number of reports that suggest that additional factors may be involved. Carpenter and Lewin (1985) reexamined the diets associated with the development ofpellagra in the United States during the early part of the twentieth century and showed that the total intake of tryptophan and niacin was apparently adequate, as judged by current knowledge of requirements. They suggested that deficiency of riboflavin (and hence impaired activity of kynurenine hydroxylase; Section or vitamin B6 (and hence impaired activity of kynureninase; Section may have been a significant factor in the etiology of pellagra when intakes of tryptophan and niacin were only marginally adequate.

Iron deficiency may also be a factor, because impairment of heme synthesis will both reduce the activity of the enzyme and increase its susceptibility to proteolysis (Section 8.3.2; Oduho et al., 1994).

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  • Brian
    What the coenzyme roles of the niacin and their specific reaction in which they are involve?
    2 years ago
  • rhoda
    Which Vitamin acts as a precursor for formation of NAD?
    1 year ago
  • Ulpu
    How to lower xanthurenic acid?
    1 month ago

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