TRYPTOPHAN NUTRITION AND METABOLISM: AN OVERVIEW

J.C. Peters The Procter & Gamble Company Miami Valley Laboratories Cincinnati, Ohio 45239 USA Tryptophan (TRP) was the first amino acid to be recognized as being essential for normal growth of young animals when Wilcock and Hopkins (1906) and later Osborne and Mendel (1914) observed its ability to stimulate weight gain in mice and rats when added to low TRP rations. Subsequent studies in a variety of species confirmed that TRP was essential for normal growth and, furthermore, was required for maintenance of nitrogen equilibrium in mature animals. Some years after those early animal studies, Rose and collaborators (1957) demonstrated that TRP was an essential amino acid for human nutrition. Tryptophan's role in maintaining normal physiologic function goes beyond its role as a substrate for tissue protein synthesis. Tryptophan has been suggested to playa unique role in regulating protein synthesis in the liver, and has been shown to affect protein synthesis in other tissues in a fashion that appears unrelated to its function as a precursor amino acid. Tryptophan gives rise to a wide array of metabolites involved in a variety of aspects of normal nutrition and metabolism. For example, pico1inic acid, a product of TRP's oxidative metabolism, is involved in normal intestinal absorption of zinc. Another TRP metabolite, quino1inic acid, is involved in the regulation of gluconeogenesis. Tryptophan can also contribute to the body's pool of the nicotinamide nuc1eotides through its metabolic conversion to niacin. Finally, TRP is the precursor of several neuroactive compounds including serotonin (5-hydroxytryptamine, 5-HT) , which functions as a neurochemical substrate for a variety of normal behavioral and neuroendocrine functions. In light of the neurotransmitter precursor function of TRP it is not surprising that many of the effects of treatments or conditions which severely alter TRP nutrition and metabolism are expressed as behavioral effects reflecting altered central nervous system function. The purpose of this brief overview is to highlight some of the major functions of TRP in the body, to outline key pathways of TRP utilization and to discuss some of the mechanisms involved in the integration of whole-body TRP metabolism and the responses of those systems to variations in diet. TRYPTOPHAN OCCURRENCE AND REQUIREMENT Tryptophan is the least abundant amino acid in most proteins (Block and Weiss, 1956) accounting for roughly 1% to 1.5% of the total amino acids in Kynurenine and Serotonin Pathways Edited by R. Schwarcz el 01•• Plenum Press. New York. 1991

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typical plant and animal proteins, respectively. Despite its scarcity, it is rarely the most limiting amino acid for maintenance or growth when the dietary amino acid source is from naturally occurring proteins. For example the corn protein zein is nearly devoid of TRP, yet lysine is limiting for growth in this protein since the content of lysine in zein in the lowest of any of the essential amino acids in relation to its requirement. It is important to keep in mind however, that while TRP may not be limiting for growth in some poor quality proteins, its supply may be insufficient for normal functioning of other pathways which depend on an adequate supply of this amino acid. As might be expected due to TRP's scarcity in dietary proteins, the requirement of mammals for TRP is correspondingly the lowest among the indispensable amino acids. In adult man, the minimal daily requirement for TRP has been estimated to be 250 mg/day in males and 160 mg/day in females (Harper, 1977). In human infants, the requirement for growth is roughly 1240 mg/kg. The recommended dietary allowance for protein in adult man ranges from 44 g/day for women to 56 g/day for men, an amount that would supply between 500 and 700 mg/day of TRP if the protein was of high quality. PATHWAYS OF TRYPTOPHAN UTILIZATION Protein synthesis Tryptophan is one of 20-22 amino acids required for the synthesis of tissue proteins. In an average adult male at nitrogen equilibrium, approximately 225-250 grams of protein are synthesized each day (approx. 3 g/kg/day, Young et al., 1983). If TRP is assumed to represent 1.5% of the total amino acids in tissue protein, then approximately 3.5 grams of TRP would be utilized daily for protein synthesis. Thus, despite the fact that in an adult at nitrogen balance, no net accretion of body protein takes place, an appreciable flux of amino acids flows through this pathway each day. In the case of TRP, this amounts to more than 15 times the minimum intake requirement and more than 3 times the average daily intake of TRP in well nourished individuals, making this pathway quantitatively the most significant in the utilization of TRP. It has long been recognized that protein synthesis in the whole animal is sensitive to nutritional factors, including the supply of energy and the amount and pattern of amino acids provided in relation to amino acid requirements. Over the past 30 years, a variety of studies have yielded information suggesting a unique role for TRP in the regulation of protein synthesis in a number of tissues including liver (Sidransky et al., 1984), muscle (Lin et al., 1988) and brain (Blazek and Shaw, 1978). Much of the work focusing on the role of TRP has come from studies of protein synthesis in the liver and suggests that TRP may act at several different points in the overall process. In early studies of the effect of amino acids on hepatic protein synthesis, Munro and associates (1975) observed that tube-feeding fasted rats a complete mixture of amino acids caused a shift in the ribosomal pattern of liver from lighter to heavier aggregates. This response did not occur when animals were force-fed a complete amino acid mixture devoid of TRP. Furthermore, the response to the amino acid mixture including TRP was not influenced by treatment of the animals with Actinomycin D, suggesting that the effect of TRP was most likely at a post-transcriptional step in protein synthesis. In a systematic study by Pronczuk et al. (1968), the response of liver ribosomes was studied when fasted animals were fed 10 different amino acid mixtures, each one lacking in a single indispensable amino acid. These workers observed that impairment of polyribosome aggregation occurred only when TRP was 346

omitted from the amino acid mixture. In view of the fact that the free TRP content of serum and tissues is the lowest of any indispensable amino acid (Munro, 1970), these findings raised the possibility that TRP might be the limiting amino acid for protein synthesis under conditions of fasting. However, these studies did not establish whether or not TRP was unique in its effect on protein synthesis. In subsequent investigations, Pronczuk et a1. (1970) found that when animals were fed threonine or isoleucine imbalanced diets, which depleted tissue pools of these amino acids, hepatic po1yribosomes were disaggregated and were stimulated by addition of the limiting amino acid to the meal. Ip and Harper (1974) extended these findings in studies of rats fed a threonine-imbalanced diet. Feeding animals a threonine imbalanced diet for 7 days resulted in hepatic ribosomes that were largely disaggregated. Oral administration of threonine caused ribosomes to reaggregate and stimulated the incorporation of 14C-1eucine into tissue proteins. Administration of TRP to threonine-depleted animals did not improve protein synthesis, suggesting that liver protein synthesis was sensitive to the supply of the amino acid most limiting in the tissue. Collectively, these studies established that TRP's ability to affect hepatic polyribosomal aggregation was not a unique effect of this amino acid on the protein synthetic machinery. However, the observation that TRP is normally the least abundant amino acid in the liver free amino acid pool when animals are fed nutritionally adequate diets, and the finding that the TRPtRNA content of liver falls more rapidly during food deprivation than do the t-RNAs of other indispensable amino acids (Rogers, 1976), suggests that TRP may be an important effector of hepatic protein synthesis under many physiological circumstances. In studies in which rats or mice were given solutions containing single amino acids, Sidransky and coworkers (1971) found that administering TRP alone stimulated ribosome aggregation and protein synthesis in liver while giving isoleucine, methionine or threonine alone did not. A somewhat lesser response was observed with certain TRP metabolites including 5-HT, 5-hydroxytryptophan (5-HTP), indole and 3-hydroxyanthranilic acid. This effect was still intact in adrenalectomized animals and thus could not be attributed to an effect of adrenal corticosteroid secretion. Protein synthesis was stimulated both in fed and fasted animals when TRP was given, and thus was apparently not due simply to increasing tissue TRP content. Sidransky and associates have investigated the mechanism of this response to TRP and have found that TRP administration affects a number of aspects of hepatic RNA metabolism, including DNA-dependent RNA polymerase activity, polyribosomal RNA and nuclear RNA synthesis, cytoplasmic po1y(A) and poly(A)-mRNA concentrations, nucleocytoplasmic translocation of po1y(A)-mRNA and levels of nucleoside triphosphatase activity in the nuclear envelope (Sidransky et a1., 1984). These workers have hypothesized that TRP can stimulate hepatic protein synthesis by at least two mechanisms: 1) increasing the synthesis of mRNA, and 2) increasing nucleocytoplasmic translocation of mRNA, which would increase the supply of message to locations in the cell where translation occurs. Recent evidence from this group indicates that TRP's effect involves its specific binding to a nuclear membrane glycoprotein (Sidransky et a1., 1984). The serotonin pathway The conversion of TRP to 5-HT occurs in several tissues throughout the body including the enterochromaffin cells of the gut, blood platelets and the central nervous system. In the central nervous system, 5-HT functions as a neurotransmitter and is believed to be involved in a variety of normal brain functions. For example, serotoninergic neurons are thought to participate in 347

regulating pain perception, aggressive behavior, sleep, and appetite (Sved, 1983). Furthermore, the serotoninergic system plays an important role in certain neuroendocrine systems (Fernstrom, 1981). Based on measurements in man of urinary excretion of the major endproduct of 5-HT metabolism, 5-hydroxyindo1eacetic acid (5-HlAA), it can be estimated that roughly 3.6 mg of 5-HT are turned over each day (Udenfriend et a1., 1955). This represents the conversion of an equivalent molar amount of TRP to 5-HT, which corresponds to the utilization of less than 1% of dietary TRP intake. The proportion of total urinary 5-HlAA arising from 5-HT turnover in the central nervous system compared to that stemming from other sources such as the gut is probably variable but has been estimated to be 10% in the rat and as much as 30% in man (Bosnan, 1978). Serotonin synthesis in brain occurs via a two-step reaction beginning with hydroxylation of L-TRP by the enzyme TRP hydroxylase, to form 5-hydroxytryptophan (5-HTP). This reaction is followed by the decarboxylation of 5-HTP to 5-HT carried out by aromatic L-amino acid decarboxylase. The principal product of 5-HT degradation is 5-HlAA, which is formed by the sequential action of monoamine oxidase and aldehyde dehydrogenase. The reaction catalyzed by TRP hydroxylase is rate limiting in brain and regulates the flux of TRP through the 5-HT pathway. Studies in animals have shown that the Km of TRP hydroxylase for its substrate TRP is about 50 ~, which is close to the normal concentration of TRP in brain (Kaufman, 1974). Thus, 5-HT synthesis under normal conditions is controlled by the availability of TRP to serotoninergic neurons. The supply of TRP to 5-HT releasing cells is in turn dependent on many factors, some of which are influenced by diet composition and the previous nutritional status of the animal. Because 5-HT synthesis is sensitive to precursor supply, the possibility exists that a number of behavioral functions dependent on serotoninergic neuronal activity may be sensitive to variations in TRP availability to the brain. Thus, despite the quantitative insignificance of this pathway in terms of who1ebody TRP disposal, derangements in this pathway have the potential to influence a wide variety of normal biological functions dependent on 5-HT mediated neurotransmission. At least three factors are important in determining the supply of TRP to brain, and hence 5-HT synthesis. These include: 1) the plasma TRP concentration, 2) the plasma concentrations of other large neutral amino acids (LNAA), which compete with TRP for uptake into brain, and 3) the extent of binding of TRP to serum albumin, which can influence the pool of unbound TRP that interacts with the amino acid carrier mechanism situated at the b1oodbrain barrier. Each of these factors, in turn, can be influenced by the nutritional and hormonal status of the animal, and by interorgan relationships in the metabolism of amino acids. For example, the concentration of TRP in plasma is a function not only of dietary TRP intake, but of the extent of removal of TRP from blood by body tissues. Since there is little net utilization of TRP by non-hepatic tissues owing to the relatively limited capacity of these tissues to oxidize TRP (Miller, 1962), the liver is the most important organ influencing plasma TRP concentration. The extent of removal of TRP by the liver following a meal is influenced by several factors at least one of which is the extent to which TRP stimulates the activity of TRP oxygenase, the principal enzyme regulating TRP oxidation. The direct relationship between dietary TRP intake and TRP catabolism will tend to limit its entry into the general circulation following a meal. The plasma concentrations of the principal LNAA (LNAA - leucine, isoleucine, valine, tyrosine and phenylalanine) which compete with TRP for uptake 348

into brain, are also influenced by the extent of their uptake and net utilization by tissues. Tyrosine and phenylalanine are similar to TRP in that the liver is the primary site for their metabolism (Miller, 1962). The branched chain amino acids (BCAA) on the other hand escape significant liver metabolism and are taken up and metabolized predominately by skeletal muscle (Harper et a1., 1984). Because of this, following a protein-containing meal the BCAA rise more in peripheral blood than the other LNAA. The fact that the BCAA's rise in peripheral blood in proportion to their content in the diet, while the increase in other indispensable amino acids is blunted by liver metabolism, means that these amino acids dominate the effect of the LNAA's as a group on brain TRP uptake. Finally, the binding of TRP to serum albumin, a phenomenon first described by McMenamy and Onc1ey (1958), can under some circumstances influence the carrier mediated transport of TRP across blood-brain barrier. In normal fed animals, the proportion of total plasma TRP bound to albumin is about 8590%. This equilibrium can be shifted under conditions which raise plasma non-esterified fatty acid (NEFA) concentrations, such as during fasting or stress (McMenamy, 1965). This is because NEFA's compete with TRP for binding sites on the albumin molecule; therefore, when the concentration of NEFA's rise, TRP is displaced from the albumin molecule raising the concentration of free TRP. A number of investigations have been carried out to determine the relative importance of albumin binding and of the fraction of plasma TRP which exists in the unbound or free form, on the uptake of TRP by brain under various conditions. Studies designed to mimic physiological situations in which the equilibrium between free and bound TRP was perturbed by altering plasma NEFA concentrations have shown that brain TRP concentration is not affected by changes in plasma free TRP concentration (Fernstrom et a1., 1975), but is more closely predicted by the plasma ratio of TRP/LNAA. Other studies in which various drugs were used to displace TRP from albumin and increase the plasma free pool, have led to the opposite conclusion, namely that the size of the plasma free pool of TRP is the most important determinant of TRP supply to the brain (B10xam et a1., 1980). Although there is no universal agreement on this point, it is probable that under normal circumstances the binding of TRP to albumin has some influence (even if small) on the carriermediated transport of TRP into brain. Nutritional effects on brain 5-HT synthesis It has been known for many years that diets deficient in TRP lead to depletion of brain 5-HT and hence to disturbances in 5-HT-mediated brain function (Gal and Drewes, 1962). However, it wasn't appreciated until the work of Fernstrom and Wurtman that variations in plasma and brain TRP and brain 5-HT synthesis could occur under normal physiologic circumstances. In the early 1970's, Fernstrom and Wurtman (1971a,b, 1972) began a systematic investigation of the relationship between TRP supply and 5-HT synthesis under a variety of circumstances. In their initial studies, they found that injecting rats with a small dose of TRP (12.5 mg/kg), only 5% of the daily intake of an adult rat, led to significant increases in both brain TRP and 5-HT content. They later observed that giving fasted rats a single injection of insulin rapidly elevated serum and brain TRP concentrations and produced a corresponding rise in brain 5-HT content. Giving fasted rats a single meal of a protein-free, high carbohydrate diet, produced the same effect as did insulin treatment, indicating that the response occurred under normal physiological conditions. Subsequent studies revealed that diet-induced increases in brain 5-HT content actually reflected an increased rate of 5-HT synthesis.

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The ability of various treatments to alter brain TRP and brain 5-HT was not simply a function of their effects on serum TRP however, but turned out to be dependent on the concentration of TRP in plasma relative to the concentrations of other large neutral amino acids that compete with TRP for transport into brain via a common carrier (Pardridge, 1977). For example, it was found that in overnight fasted rats fed a meal containing 18-24% protein, plasma TRP concentrations were considerably higher than in animals fed a protein-free meal, yet brain TRP and 5-HT concentrations were unchanged compared to fasting (Fernstrom and Wurtman, 1972). Furthermore, feeding fasted rats a 40% protein meal actually decreased brain TRP and 5-HT content, despite the high TRP content of the meal (Fernstrom and Faller, 1978). Other studies showed that feeding animals diets in which the LNAA competitors were omitted resulted in a large increase in brain TRP and 5-HT compared to the response of animals fed a complete amino acid mixture having the same level of TRP (Fernstrom and Wurtman, 1972). Collectively, these observations demonstrated that changes in brain TRP and 5-HT concentrations were directly related to changes in the plasma ratio of TRP to LNAA (TRP/LNAA), and that alterations in this ratio induced by diet were predictive of changes in brain 5-HT synthesis. Based on the pioneering work of Fernstrom and Wurtman, other workers began to investigate the possibility that diet-induced changes in brain TRP content and 5-HT synthesis might be involved in the control of normal animal feeding behavior. In long-term studies in weanling rats that were allowed to self-select between high and low protein diets, Ashley and Anderson (1975) observed a strong inverse correlation between chronic cumulative protein intake and the plasma TRP/LNAA ratio. These authors proposed that the plasma TRP/LNAA ratio, and hence 5-HT synthesis were involved in regulating protein intake and selection in rats. The results of these and other studies by Anderson and associates (Anderson, 1979) led to the hypothesis that changes in brain TRP and 5-HT brought about by single meals constituted a behavioral feedback loop by which animals regulated the selection of protein and carbohydrate. According to their hypothesis, ingestion of a protein-free or lowprotein diet would increase the plasma ratio of TRP/LNAA and brain 5-HT which would cause a shift in diet selection toward a diet having a higher protein content, a move that presumably would restore the premeal level of brain 5-HT. Their hypothesis also predicted that high protein diets should reduce the plasma TRP/NAA ratio and 5-HT, and should shift diet selection toward a lower protein ration. Peters and Harper (1985, 1987a) carried out a series of investigations of the effects of dietary protein content on the plasma TRP/LNAA ratio, brain TRP and brain 5-HT concentrations. In rats that were previously adapted to 20% casein diets, consumption of single meals of diets containing from 0 to 55% of casein led to increases in plasma TRP concentrations that were proportional to dietary protein level (i.e., TRP intake). The plasma TRP/LNAA ratio, however, remained unaffected by differences in dietary protein level across a wide range from 10% to 55% of casein. Likewise, brain TRP, 5-HT and 5-HIAA concentrations were unchanged over the range of increasing dietary protein levels. Feeding rats the protein-free diet on the other hand led to an elevation in the plasma TRP/LNAA ratio and brain 5-HT, despite causing a reduction in the absolute concentration of TRP in blood. This finding was consistent with the earlier work of Fernstrom and Wurtman (197lb). The results of these acute studies indicated that neither the plasma TRP/LNAA ratio nor brain 5-HT are likely to play an important role in directing protein intake or selection in rats when the dietary choices offered have protein contents within the range compatible with optimum growth. However, the data raise the possibility that elevations in brain TRP and 5-HT caused by consumption of a protein-free diet may act as a signal to alter food intake or selection.

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The response of TRP and 5-HT to a protein-free, high-carbohydrate meal has been suggested to be primarily an effect of the carbohydrate on insulin secretion (Fernstrom and Wurtman, 1971b). According to this idea, the carbohydrate load induces the secretion of insulin which raises the plasma level of TRP/LNAA by stimulating uptake of the competing LNAA into muscle while having a relatively lesser effect on tissue uptake of TRP. If this effect were due entirely to the secretion of insulin per se, then one would predict that the TRP/LNAA ratio would also be elevated by a diet containing 5% or 10% protein since such a diet is still high in carbohydrate and would most likely lead to an equivalent stimulation of insulin secretion. In fact, diets containing substantial amounts of both protein and carbohydrate stimulate insulin release to a greater extent than do diets having carbohydrate alone (Spiller et a1., 1987). Thus, as has been pointed out previously (Harper and Peters, 1989), the response of rats to meals lacking in protein would seem to be a response to protein deficiency rather than a response to the high level of carbohydrate. The potential of such a response to act as a signal to modify food intake or selection would then seem to make sense as it could function as a mechanism that would allow animals to avoid diets that are incompatible with survival. It is noteworthy that the response of plasma TRP, the TRP/LNAA ratio and brain 5-HT are different when rats are fed different levels of protein for many days compared to the acute responses described above. When young rats were allowed to adapt to diets containing from 5% to 75% of casein for 11 days, plasma TRP did not rise in proportion to dietary protein level, but remained within a fairly narrow range when the diet contained between 15% and 75% of casein (Peters and Harper, 1985). Animals fed the 5% and 10% protein diets had lower plasma TRP concentrations reflecting the inadequacy of the diet to support rapid growth. The plasma TRP/LNAA ratio however, did show a relationship with dietary protein content and was significantly inversely correlated with dietary casein level. Furthermore, brain TRP, 5-HT and 5-HIAA showed a similar relationship, and although the absolute changes in their concentrations were small in comparison to the effects brought about by single meals, they declined in proportion to increasing dietary protein level. An explanation for the apparently opposite effects of dietary protein content in the short and long term on plasma TRP, the TRP/LNAA ratio, brain TRP and 5-HT has been discussed in detail elsewhere (Harper and Peters, 1989). The different responses of animals fed increments of protein in the long-term compared to single meals of the same diets, results from adaptive responses in the activities of several amino acid degrading enzymes which increase over time in rats fed high protein diets. The activities of the enzymes responsible for degrading the BCAA do not increase with increasing dietary protein content to the same extent as do enzyme systems which dispose of other dietary indispensable amino acids (Harper et a1., 1970, 1984). Thus, after several days of high protein diets, the plasma concentrations of most amino acids, including TRP, return to near normal owing to an increased capacity for their oxidation, but concentrations of the BCAA remain elevated. Because the BCAA quantitatively represent the largest fraction of plasma LNAA, the plasma TRP/LNAA ratio, brain TRP and 5-HT would be expected to decline as the protein content of the diet was increased. The oxidative pathway via kynurenine The oxidation of TRP via the kynurenine pathway is quantitatively the most significant route of TRP disposal in the body, accounting for 95% or more of daily TRP metabolism. Tryptophan catabolism via this route leads to a number of end products including the nicotinamide nuc1eotides and acety1CoA which can be further oxidized to CO 2 to yield energy. Furthermore, several intermediates in this pathway have been shown to play other important

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roles in nutrition and metabolism. Picolinic acid, the product of the nonenzymatic cyclization of aminomuconic semialdehyde, is involved in normal intestinal uptake of zinc (Evans and Johnson, 1980). Another pathway intermediate, quinolinic acid, can form a chelate with iron, which has been shown by Lardy and coworkers (Veneziale et al., 1967) to be involved in the physiological regulation of gluconeogenesis. Whole-body TRP oxidation occurs almost exclusively in the liver (Miller, 1962) owing to the localization of TRP oxygenase in that tissue. There is however, some capacity in other visceral and peripheral tissues to degrade TRP. Hayaishi and coworkers (1980) have identified an indole oxygenase that is distributed widely throughout the body which can also oxidize TRP. This enzyme differs from the liver enzyme in that it uses superoxide anion instead of molecular oxygen as the oxidizing agent, and its substrate specificity is much broader than that of TRP oxygenase. Indole oxygenase will oxidize D-TRP as well as L-TRP and also demonstrates activity toward 5-hydroxytryptophan, tryptamine, and 5-HT. Before TRP can be utilized by liver tissue, it must first gain entry into hepatic cells, a process influenced by extra-cellular factors. For example, the uptake of TRP by the liver, while not sensitive to competitive inhibition by other LNAA, appears to be influenced by the binding of TRP to serum albumin. Smith and Pogson (1980) have shown that TRP oxidation by isolated hepatocytes is significantly reduced when albumin is included in the incubation medium. L-tryptophan-2,3-oxygenase (tryptophan oxygenase) is the first and rate controlling enzyme in TRP degradation and its activity regulates the overall flux of TRP through the oxidative pathway. Tryptophan oxygenase is somewhat unique among enzymes catalyzing degradation of essential amino acids in that both the amount and activity of the enzyme are controlled by its substrate, TRP. Knox and Mehler (1951) were the first to observe that the activity of rat liver TRP oxygenase was stimulated by TRP. Later, Greengard and Feigelson (1961) discovered that heme acts as a cofactor for the enzyme and is required for conversion of the apoenzyme to the active holoenzyme. In a series of investigations that followed, it was shown that TRP affects the activity of TRP oxygenase both by increasing heme saturation of the enzyme (thus increasing the level of active holoenzyme) as well as by stabilizing the enzyme against degradation (Schimke et al., 1965). Tryptophan oxygenase is also responsive to various hormones, the most studied of which are the corticosteroids. Administration of hydrocortisone to rats induces liver TRP oxygenase activity (Schimke et al., 1964), an effect that has been shown to result from synthesis of new apoenzyme protein. Furthermore, induction of enzyme activity by glucocorticoids was shown to be additive with the effect of TRP (Schimke et al., 1964). Tryptophan oxygenase is also subject to feedback inhibition by many intermediates and products of the oxidative pathway, including NADH and NADPH (Badawy, 1977). The product of TRP oxygenase, formylkynurenine, under conditions of normal flux of TRP through the oxidative pathway, is converted to kynurenine which is metabolized further to acroleyl aminofumarate. An important branch point occurs at this step in the pathway which commits the carbon skeleton either to complete oxidation to CO 2 or to synthesis of niacin and NAD. The rate limiting step in the branch of the pathway leading to complete oxidation is catalyzed by picolinic carboxylase, while the alternate pathway leading to NAD involves the non-enzymatic cyclization of acroleyl amino fumarate to yield quinolinic acid. The partitioning of acroleyl amino fumarate between the two possible metabolic routes appears to depend on the substrate supply in relation to the ~ and capacity of the carboxylase. Thus, significant conversion of TRP to niacin only occurs when the capacity of picolinic carboxylase 352

becomes limiting (Bender, 1982). One of the most well known species differences in the TRP to niacin conversion is in the cat, in which picolinic carboxylase has both a high activity and high capacity to metabolize acroleyl aminofumarate. In this species, virtually no conversion of TRP to niacin takes place (Ikeda et al., 1965). The efficiency of conversion of TRP to niacin has been a subject of several investigations. Horwitt et al. (1956) originally reported a conversion efficiency of 60:1 in young men, meaning that ingestion of 60 mg of TRP was required for each 1 mg of niacin formed. Subsequent work has shown values for the efficiency of conversion to be as high as 122:1, depending on the amount of TRP provided by the diet (Nakagawa et al., 1969). In a recent study of young adult men, Patterson et al. (1980) showed that when TRP intake was increased from 245 to 845 mg/day, urinary N1-methyl-nicotinamide excretion increased from 5.4 to 17.1 ~mole/24 hours while N1-methyl-2-pyridone-5-carboxamide output increased from 11.7 to 38.6 ~mole/24 hours. Based on the urinary excretion of these niacin metabolites, the authors calculated an efficiency of conversion of TRP to niacin which averaged 72:1, although there was a trend toward increased conversion efficiency at the higher TRP intakes. In general, it appears that conversion of TRP to niacin depends on the surplus of dietary TRP provided in excess of the body's needs for maintenance of nitrogen equilibrium and for the synthesis of other important molecules such as 5-HT (Bender, 1982). INTEGRATION AND REGULATION OF WHOLE-BODY TRYPTOPHAN UTILIZATION The utilization of TRP by the whole-body, like the utilization of other amino acids, is subject to relatively few points of regulation (Harper, 1974). Unlike certain other essential dietary nutrients, such as calcium or iron whose whole body utilization is regulated primarily at the level of absorption, there is little regulation of amino acid metabolism exerted at the level of amino acid absorption from the intestine. The digestibility of most naturally occurring proteins is high and amino acid uptake by the gut is nearly quantitative. Excretion of amino acids, including TRP, by the kidney is also not a significant route of amino acid disposal owing to the efficient reabsorption of amino acids in the renal tubule. Amino acid excretion by the kidney can occur, but only when blood amino acid levels are extremely high such as might be encountered in some inborn errors of metabolism or other pathological states. In the young animal, utilization of TRP and other amino acids for protein synthesis is substantial, but in mature animals, there is no net increase in body protein or free amino acid contents. Also, unlike the situation for carbohydrate and fat, there is no storage pool in the body for amino acids that are ingested in excess of immediate needs. Therefore, even in the young animal, protein synthesis, while sensitive to total amino acid supply, is not a major site of regulation of amino acid disposal. The low Km's of the amino acid synthetases insure that amino acid needs for protein synthesis are met even under conditions of low amino acid intake. Thus, in both growing and mature animals, once the body's demand for amino acids to supply protein synthesis or for the synthesis of other biologically important molecules such as 5-HT have been met, the surplus amino acids are oxidized to provide energy or are converted to fat. It is evident then, that the primary mechanism by which utilization of amino acids is regulated is at the level of amino acid catabolism. The flux of TRP and other essential amino acids through their respective degradative pathways is determined largely by the characteristics of the various enzymes involved in their metabolism (Krebs, 1982), and by factors that determine the accessibility of the various amino acids to the enzymatic machinery. The availability of substrates for metabolism can be influenced by interorgan relationships in the transport of substrates and by factors that modify uptake

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of substrates into the tissues in which the appropriate enzyme systems are located. In the case of TRP, the liver is the major organ responsible for its degradation, which means that hepatic TRP disposal must be responsive to fluctuations in dietary TRP supply. The localization of TRP degrading enzymes in liver also means that hepatic TRP disposal is an important regulator of TRP supply to the rest of the body. The rate controlling enzyme in TRP catabolism, TRP oxygenase, is not saturated with its substrate under normal conditions (Krebs, 1972). Because the Km of the enzyme is considerably above the normal tissue TRP concentration, TRP degradation will increase with increasing availability of substrate. The supply of TRP in turn is determined by intake of TRP from the diet, and the extent to which TRP is bound to serum albumin. Liver extraction and degradation of TRP will determine the rise in peripheral blood TRP concentration after a meal which determines the availability of TRP to extrahepatic tissues. As pointed out previously, the extent to which peripheral blood TRP concentration increases after a protein-containing meal depends not only on the amount of TRP ingested, but also on the adaptive metabolic state of the animal (i.e., the capacity to degrade surplus amino acids). The Km of the LNAA transport system in muscle is considerably higher than the average plasma concentrations of the neutral amino acids. Thus, uptake into this tissue is relatively insensitive to substrate competition effects and should be determined primarily by the plasma TRP concentration relative to the Km of the muscle transporter for TRP. As discussed previously, the situation for brain is different because the Km of the neutral amino acid transport carrier for TRP and the other LNAA is low, and approximates the normal plasma concentration of TRP (Pardridge, 1977). Therefore, uptake of TRP into brain, which regulates 5-HT synthesis, is influenced by the concentrations in blood of the other LNAA. Because there are significant differences in the location and extent of metabolism of the LNAA as a function of dietary amino acid supply, the conversion of TRP to serotonin is influenced by the interaction between different organ systems (esp. muscle and liver) in the metabolism of TRP and the competing LNAA. Furthermore, the binding of TRP to serum albumin offers an additional mechanism by which TRP availability to tissues can be regulated. The possible survival advantage that albumin binding of TRP may confer upon the animal is not readily apparent. At low TRP intakes, binding of TRP to albumin may protect TRP from hepatic catabolism, insuring an adequate supply to peripheral tissues. In the periphery, the low Km for TRP uptake into brain compared to the ~ for transport into muscle would help to insure adequate TRP for normal 5-HT synthesis and hence normal brain function. The albumin-bound pool of TRP may therefore serve as a buffer which protects the brain from TRP and 5-HT depletion when dietary intake of TRP is low. Under normal conditions, when animals are maintained on relatively low protein diets, the activities of most amino acid degrading enzymes are low, although activities are sufficient to dispose of amino acids in excess of requirements. However, when the protein content of the diet is very high, or when individual indispensable amino acids are given individually in large amounts above the requirement, the capacity of the animal to catabolize the surplus amino acids may be exceeded, and those amino acids present in excess accumulate in blood and tissues (Harper et al., 1970). When animals are offered only a single diet of fixed composition, accumulation of amino acids, including TRP, in body fluids and tissues is associated with depressed food intake. Alternatively, if animals are allowed to self-select their diet, elevated tissue amino acid levels lead to altered diet preference, such that animals choose a diet that will restore amino acid concentrations toward normal. These responses describe an additional mechanism by which animals can

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regulate whole-body amino acid metabolism; regulation through alterations in food intake and diet selection (Harper, 1974). If animals are fed high protein diets for several days or longer, activities of several amino acid catabolizing enzymes increase, including those for degrading TRP. Increased catabolic capacity favors a reduction in the blood concentrations of amino acids toward normal, and food intake is restored to near control levels (Anderson et a1., 1968). The ability of amino acid catabolic enzymes to adjust to amino acid intake over time allows the animal to maintain plasma and tissue amino acid concentrations within an acceptable range despite wide fluctuations in dietary protein content. However, under some circumstances the capacity of these systems may still be exceeded and plasma and tissue amino acid concentrations would remain elevated. In this situation, food intake would remain depressed and normal function and possibly survival would become threatened. It is clear that modification of food intake is a mechanism involved in the regulation of metabolism of all of the indispensable amino acids, and is not unique for TRP (Harper et a1., 1970). The mechanism by which changes in feeding behavior are brought about by alterations in amino acid intake and metabolism is however, not known. The question of specificity of the relationship between TRP metabolism and utilization and food intake is complicated by the fact that TRP (unlike most other dietary amino acids) can be converted to 5-HT, a neurotransmitter involved in the central nervous system control of feeding behavior (Blundell, 1977). Serious doubts have been raised as to whether diet-induced changes in brain TRP and 5-HT concentrations play any significant role in the control of normal food intake and selection (Peters and Harper, 1987b). Further work in this area is needed to identify those conditions under which feeding behavior might be specifically controlled by diet-induced alterations in brain TRP supply and 5-HT synthesis, and whether such a mechanism might playa more general role in regulating whole-body amino acid metabolism. REFERENCES Anderson, G.H., 1979, Control of protein and energy intake: role of plasma amino acids and brain neurotransmitters, Can J. Physiol. Pharmacol., 57:1043-1057. Anderson, H.L., Beneverga, N.J., and Harper, A.E., 1968, Associations among food and protein intake, serine dehydratase, and plasma amino acids, Am. J. Physiol., 214:1008-1013. Ashley, D.V.M., and Anderson, G.H., 1975, Correlation between the plasma tryptophan to neutral amino acid ratio and protein intake in the se1fselecting weanling rat, J. Nutr., 105:1412-1421. Badawy, A.A.-B., 1977, The functions and regulation of tryptophan pyrro1ase, Life Sci., 21:755-768. Bender, D.A., 1982, Biochemistry of tryptophan in health and disease, Molec. Aspects Med., 6:101-197. Blazek, R., and Shaw, D.M., 1978, Tryptophan availability and brain protein synthesis, Proc. Brit. Assoc. Psychopharmacol., 17:1065-1068. Block, R.J., and Weiss, K.W., 1956, "Amino Acid Handbook", The Ryerson Press, Toronto. B1oxam, D.L., Trick1ebank, M.D., Patel, A.J., and Curzon, G., 1980, Effects of albumin, amino acids, and clofibrate on the uptake of tryptophan by the rat brain, J. Neurochem., 34:43-49. Blundell, J.E., 1977, Is there a role for serotonin (5-hydroxytryptamine) in feeding?, Int. J. Obes., 1:15-42. Bosnan, T., 1978, Serotonin metabolism, in: "Serotonin in Health and Disease", Vol. 1, Essman, W.B., ed., SP Medical and Scientific Books, New York, pp. 181-300. 355

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