Tryptophan plays a unique role in defense against infection because of its relative scarcity compared to other amino acids. During infection, the body induces tryptophan-catabolizing enzymes which increase tryptophan's scarcity in an attempt to starve the infecting organisms [Brown, et al., 1991]. In unresolved chronic infections, tryptophan metabolism remains disturbed. The biological disturbances caused by widespread tryptophan deficiency may be substantially responsible for some of the cognitive deficits, neuroendocrine dysregulation, and immune incompetence associated with AIDS, autoimmune disease, and other chronic disease states.
Tryptophan is metabolized in several tissues by different enzyme systems. The primary site of tryptophan catabolism is the liver where tryptophan oxidase metabolizes tryptophan with molecular oxygen as the oxidizing agent. The oxygen is used to split the 5-member nitrogen-containing ring on the tryptophan molecule (see illustration page 4) generating kynurenine (KYN) derivatives.
A little over a decade ago, tryptophan oxidase was widely believed to be the only tryptophan-catabolizing enzyme. Then Japanese researchers discovered indoleamine-2,3-dioxygenase (IDO), also called indole oxidase. In peripheral tissues and in the brain, IDO is the only tryptophan-catabolizing enzyme, using superoxide anion as the oxidizing agent. IDO is a more general enzyme. It has a limited capacity to oxidize a broad class of compounds called indoles (see illustration), which are chemically related to tryptophan. IDO has less specificity for tryptophan than the hepatic tryptophan oxidase enzyme.
Small amounts of tryptophan (approximately 1%) are metabolized by brain tissue into serotonin (a neurotransmitter) and melatonin (a neurohormone). During infection, tryptophan deprivation reduces the activity of these pathways and may influence vital regulatory functions. In addition, tryptophan catabolism in the brain and peripheral tissues produces toxic chemicals which stimulate excitatory neurotransmitter pathways.
Modern neuroscientists have come to realize that glutamate (glutamic acid) and aspartate (aspartic acid) play a global role as excitatory neurotransmitters in the brain. The receptors for glutamate and aspartate regulate ion channels which control the flow of sodium, potassium, calcium, magnesium and chloride ions across nerve membranes. They are named NMDA receptors after their natural agonist N-methyl-D-aspartate, the stimulating chemical for which they have affinity.
Some chemicals have extremely high affinity for the NMDA receptor. They are called excitotoxins because of their ability to bind to the NMDA receptor and overstimulate the excitatory nervous system. Overstimulation of the central nervous system is believed to be involved in the development of numerous neurodegenerative conditions like Huntington's disease, cerebral ischemia and hypoxia, and temporal-lobe epilepsy. Such excitatory overstimulation may also be involved in Alzheimer's disease, Parkinson's disease, AIDS dementia, hypoglycemia, schizophrenia, and anxiety disorders [Schwarcz and Du, 1991]. Although glutamate and aspartate are excitatory neurotransmitters and may play a limited role in neurodegeneration, excitotoxins are dramatically more potent. The tryptophan metabolite quinolinic acid, for example, is one hundred times more potent than glutamate in rats.
Another tryptophan metabolite, kynurenic acid (KYNA), is a nonselective blocker of NMDA receptors. Interestingly, it also decreases nervous system susceptibility to excitotoxins, especially quinolinic acid. KYNA and quinolinic acid are both naturally present at concentrations several orders of magnitude lower than glutamate and aspartate. Quinolinic acid levels are similar across species, but KYNA varies substantially. Man has 50 times higher KYNA levels than rats. Although some researchers have suggested that deficiencies of KYNA may be associated with the development of neurodegenerative diseases, the specific roles that quinolinic and kynurenic acids play in chronic infection or degenerative disease remains to be elucidated.
In most proteins, tryptophan is the least abundant essential amino acid, comprising approximately 1% of plant proteins and 1.5% of animal proteins. Although the minimum daily requirement for tryptophan is 160 mg for women and 250 mg for men, 500-700 mg are recommended to ensure high-quality protein intake. Actual tryptophan utilization is substantially higher. Men use approximately 3.5 grams of tryptophan to make one days's worth of protein [Peters, 1991]. The balance is obtained by hepatic recycling of tryptophan from used (catabolized) proteins.
Dietary tryptophan is well absorbed intestinally. About 10% of the tryptophan circulating in the bloodstream is free, and 90% is bound to the protein albumin. The tryptophan binding site on albumin also has afinity for free fatty acids (FFAs), so tryptophan is displaced when FFAs rise, as when fasting.
Tryptophan transport into the brain is regulated by an active transport enzyme system which also transports other large neutral amino acids (LNAAs) like phenylalanine, tyrosine, leucine, isoleucine, and valine. Paradoxically -- because of the competitive influence of LNAAs -- brain uptake of tryptophan is inhibited after consumption of tryptophan-rich (and LNAA-rich) high-protein foods. Carbohydrates and insulin, on the other hand, lower LNAAs and enhance tryptophan transport into the brain.
Although tryptophan is not usually the limiting amino acid in protein synthesis, tryptophan may become insufficient for the normal functioning of other tryptophan-dependent pathways. Numerous lines of research point to tryptophan's central role in regulation of feeding and other behaviors. Tryptophan is not only typically the least abundant amino acid in the liver's free amino acid pool, but liver tryptophan-tRNA levels fall faster during food deprivation than other indispensable amino acids [Rogers, 1976]. Under fasting conditions, and possibly in wasting syndromes, tryptophan may become the rate-limiting amino acid for protein synthesis [Peters, 1991].
The LNAA transport enzyme responsible for tryptophan transport across the blood-brain barrier also has high affinity for kynurenine and 3-hydroxykynurenine, the two early amino-acid metabolites in the tryptophan catabolic pathway. LNAA-mediated transport of kynurenines concentrates peripheral tryptophan metabolites in the central nervous system.
The changing bio-availability of tryptophan has significant influence on neuroendocrine function. The rate-limiting step in the synthesis of the brain neurotransmitter serotonin is the conversion of tryptophan to 5-hydroxytryptophan by tryptophan hydroxylase (with tetrahydrobiopterin -- a form of folic acid -- as co-factor). Under normal conditions, tryptophan hydroxylase is not saturated (filled with substrates), so extra tryptophan increases serotonin synthesis. This sensitivity of serotonin production to tryptophan availability appears to be biologically optimized. Under average conditions, a large percentage of the tryptophan hydroxylase is not saturated and therefore available for tryptophan binding.
It has long been recognized that tryptophan-deficient diets lead to depletion of brain serotonin and disturbance in serotonergic neural function, but now it is becoming recognized that normal physiologic variations in plasma tryptophan produce biologically significant variations in serotonin synthesis.
Tryptophan metabolites play other physiologically important roles. Picolinic acid, a product of tryptophan's oxidative metabolism, is involved in the normal intestinal absorption of zinc. Quinolinic acid itself has been reported to be involved in the regulation of gluconeogenesis.
Abnormalities of tryptophan metabolism have been observed in both monkeys with simian retrovirus type-D (SRV-D) and humans with HIV infections. Simian retrovirus infections are associated with the same immunodepression, T4-cell-count reductions, neoplasms, and opportunistic infections that occur in human AIDS. Heyes et al.  have found that cerebrospinal fluid quinolinic acid concentrations are increased in SAIDS. This observation has been confirmed in human AIDS patients.
The concentration of quinolinic acid present in HIV and SRV-D infections is sufficient for it to function as an excitotoxin to NMDA neuroreceptors. During opportunistic infections, quinolinic acid levels can rise even further. Overstimulation of NMDA receptors is known to interfere with long-term learning and may account for some of the cognitive deficit seen in AIDS dementia.
The regulation of tryptophan availability during infection is largely mediated by interferon-gamma (IFN-gamma). IFN-gamma induces IDO in peripheral blood monocytes and their derived macrophages and has an anti-proliferative effect on tumor cells [Ozaki, et al., 1991].
Tryptophan and kynurenine metabolites have been reported to be essential for the IFN-gamma-mediated activation of macrophage tumor-cell killing [Leyko and Varesio, 1989]. Increased IDO may be contributed by mature (differentiated) tissue macrophages rather than from pluripotent (undifferentiated) monocytes (see Forefront #32 for more information).
The actions of type-I IFNs (IFN-alpha and IFN-beta) are antagonistic to IFN-gamma. Modulation of IFN-gamma influence by type-I IFNs may represent feedback control of immune response and an immunoregulatory function of cytokines.
Long-term expression of IFN-gamma and IDO may represent a failure of immune feedback control. Dr. Ozaki and colleagues state, "It is conceivable that the overexpression of IDO and elimination of the least abundant amino acid tryptophan may be more harmful than beneficial to the integrity of macrophages." IDO increases one hundred fold during viral infection, endotoxin (lipopolysaccharide) shock, or interferon administration. During this stress reaction, tryptophan catabolism is markedly enhanced. Kynurenine and xanthurenic acid increase several-fold. This negative influence may become critical in unresolved long-term infections.
In cell cultures, tryptophan depletion has been found to be the mechanism by which IFN-gamma acts against replication of such parasites as Toxoplasma gondii, Chlamydia trachomatia and Clamydia psittaci. Supplemental tryptophan reverses this inhibition [Brown, et al., 1991].
The antiproliferative effect of IFN-gamma against several human tumor cells also depends on the tryptophan-deprivation mechanism. In mice, transplanted tumor cells exhibit a marked increase in IDO induction during rejection from the host. The host cells infiltrating the transplanted tissue show no such increase. The active agent affecting this process is IFN-gamma [Takikawa, 1991].
While tryptophan deprivation is engineered by IDO induction, there may be other mechanisms involved. Serum tryptophan falls significantly in only 2-4 hours, long before IDO induction is observed in vitro. Preliminary tryptophan deprivation through absorption into mononuclear cells may be the initial mechanism [Finocchiaro, et al., 1988].
Cancer patients given type-I and II interferons experience a 50-80% decrease in serum tryptophan and a 5-500% increase in urinary kynurenine metabolites. Tumor cells transplanted into allogeneic (genetically different) mice produce large amounts of IDO when being rejected by the host animal. No such IDO production occurs in the infiltrating host immune cells or when tumor cells are transplanted into syngeneic mice. Human lung-cancer patients have been reported to have significantly elevated IDO levels in their lung tissue compared to normal tissue [Yasui, et al., 1986]. These observations support the hypothesis that IFN-gamma-induced tryptophan restriction is an in vivo antitumor mechanism [Takikawa, et al. 1991].
Patients with autoimmune disease exhibit increased IFNs and tryptophan metabolites. Although there is some disagreement in the literature about IFN levels with these diseases, many of the factors known to be associated with IFN production are evident (increased neopterin, elevated serum beta-2-microglobulin, and increased tryptophan metabolites). Cells isolated from synovia (joint membranes) of rheumatoid arthritis patients have been reported to exhibit increased IDO activity, even though IFNs are not usually detected in general circulation. Such observations suggest that IFN expression and IDO induction can be extremely localized. Despite localization, this tryptophan depletion likely contributes to the degeneration, wasting and other symptoms of these diseases.
IFNs are also increased in HIV disease; the higher the levels the worse the prognosis [Brown, et al. 1991]. Tryptophan and serotonin are depleted and kynurenine and quinolinic acid are elevated. This excess may account for the cachexia, dementia, diarrhea and some of the immunosuppression of AIDS.
In many respects, AIDS resembles classic pellagra caused by niacin/tryptophan deficiency. But is tryptophan supplementation equally appropriate in AIDS? Is there any long-term survival value in continued tryptophan restriction during unresolved chronic infection or autoimmune disease? How toxic are tryptophan's metabolites? How significant are the neuroendocrine deficits caused by long-term tryptophan deprivation?
Given the known adverse influences of some IDO-induced tryptophan metabolites, supplementing tryptophan may be an inappropriate strategy. Even if tryptophan and its metabolites inhibit IDO, increased substrate availability might result in increased quinolinic acid production. The possible neuroendocrinological benefit of increasing endogenously produced serotonin and melatonin might be offset by simultaneous increased production of quinolinic acid and exacerbation of the excitatory insult.
As alternatives to tryptophan, 5-hydroxytryptophan (5-HTP), serotonin, N-acetylserotonin and melatonin may be used to fortify the serotonergic nervous system. These chemicals are not able to function as substrates for IDO and may be able to stabilize neuroendocrine function without excitatory side effects. Oral use of 5-hydroxytryptophan in humans has been studied. In obese adult women, 300 mg of 5-HTP 30 minutes before eating produced an anorexic influence and early satiety during the meal [Cangiano, et al., 1991]. Nausea was reported in 70% of the women, but this had disappeared 6 weeks into the study.
Despite the unwanted anorexic influence of 5-HTP in patients with wasting syndromes, 5-HTP therapy may be beneficial in patients with elevated IDO activity. Administration of 5-HTP 30 minutes before eating is designed to optimize its adverse effect on appetite. Administration of 5-HTP 30 minutes after eating may avoid this consequence. Additionally, obese and lean people may react oppositely to serotonergic stimulation.
Melatonin has also been studied in people as a remedy for jet lag. Melatonin is a neurohormone produced from serotonin by the pineal gland, a neuroendocrine gland closely involved in regulation of the biological circadian (daily) rhythm. Melatonin release is periodic, triggered by environmental factors relating to the cessation of light exposure. In normal, healthy people, melatonin release begins shortly after it gets dark, increases for several hours, peaks in the early hours of the morning and then drops back to minimal levels by sunrise.
Melatonin deficits are known to cause sleep disturbances and may be responsible for some of the cognitive deficit seen in jet lag. Oral melatonin, in 5-10 mg doses, is able to rapidly and efficiently correct jet-lag-induced cognitive impairment. The same doses in HIV patients have produced anecdotal reports of improved quality of sleep and feelings of well-being. No significant side effects were noted with melatonin use at this dose.
Inhibition of IDO may also be a good therapeutic strategy. Tryptophan catabolism in the liver is known to be feedback-inhibited by intermediates and products of the tryptophan degradation pathway, like NAD and NADH. IDO may function similarly. NADH carries hydrogen and perform reducing (anti-oxidizing) functions in cellular metabolism. Perhaps it is the reducing power that NAD and NADH provide which inhibits IDO.
Oxidation reactions are known to trigger immune activation. Oxygen free radicals interact with the FFAs (like arachidonic acid) to produce prostaglandins, tissue hormones which induce inflammation, fever, and immune activation. Macrophages release oxygen free radicals (superoxide and hydroxyl radicals) when they come in contact with their antigen targets. Antibodies themselves may be more reactive in an oxidizing environment, and the elevated temperature of fever promotes free-radical reactions.
Oxidizing conditions, then, appear to be the on switch for immune activity. Do reducing conditions turn it off?
The best clinical method of supplementing reducing power is intravenous ascorbate (vitamin C) [Cathcart, 1991]. Each ascorbate molecule is capable of donating 2 hydrogen atoms, and doses of up to 200 grams per 24 hours have been given without serious side effects. Such doses have shown the ability to dramatically arrest the clinical course of several life-threatening autoimmune diseases [Cathcart, 1992].
The maximum tolerated oral dose of ascorbate is limited by intestinal absorption, causing diarrhea when bowel tolerance is exceeded. Interestingly, the vitamin C bowel-tolerance dose escalates with the severity of the disease: 4-15 grams in healthy people, 30-100 grams in people sick with colds, and 100+ grams in people sick with serious viral illnesses like influenza and mononucleosis [Cathcart, 1985]. Perhaps the bowel tolerance dose of vitamin C directly tracks the degree of oxidation in the body.
Because IFN-gamma triggers IDO, antiIFN-gamma agents might be effective in decreasing IDO expression. IFN-gamma antibodies have been used in scientific cell-culture experiments, but they have not yet been investigated in clinical trials with humans. IFN-alpha and IFN-beta (Type-I IFNs) are known to antagonize IFN-gamma (Type-II IFN) activity in lymphocytes. Several forms of IFN-alpha are available in the world drug market.
Endogenous production of IFN-alpha can also be encouraged. Multi-gram doses of vitamin C are known to stimulate IFN-alpha production.
Abnormalities in tryptophan metabolism are widespread in AIDS and autoimmune disease. These abnormalities contribute to neurodegenerative processes, cognitive impairment and immune incompetence. There are numerous possible interventions which might ameliorate these effects, many of which are of a low order of toxicity. Further research into this area is badly needed.
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