Kynurenine pathway and human systems

Journal Pre-proof Kynurenine pathway and human systems

Abdulla A.-B. Badawy PII:

S0531-5565(19)30596-0

DOI:

https://doi.org/10.1016/j.exger.2019.110770

Reference:

EXG 110770

To appear in:

Experimental Gerontology

Received date:

4 September 2019

Revised date:

31 October 2019

Accepted date:

1 November 2019

Please cite this article as: A.A.-B. Badawy, Kynurenine pathway and human systems, Experimental Gerontology(2018), https://doi.org/10.1016/j.exger.2019.110770

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© 2018 Published by Elsevier.

Journal Pre-proof Kynurenine pathway and human systems Abdulla A-B Badawy1* 1

Formerly School of Health Sciences, Cardiff Metropolitan University, Western Avenue, Cardiff CF5 2YB, Wales, UK __________________________________________________________________________________________

ABSTRACT

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The essential amino acid L-tryptophan (Trp) appears to play an important role in aging by acting as a general regulator of protein homeostasis. The major route of Trp degradation, the kynurenine pathway (KP), produces a range of biologically active metabolites that can impact or be impacted by a variety of body systems, including the endocrine, haemopoietic, immune, intermediary metabolism and neuronal systems, with the end product of the KP, NAD+, being essential for vital cellular processes. An account of the pathway, its regulation and functions is presented in relation to body systems with a summary of previous studies of the impact of aging on the pathway enzymes and metabolites. A low-grade inflammatory environment characterized by elevation of cytokines and other immune modulators and consequent disturbances in KP activity develops with aging. The multifactorial nature of the aging process necessitates assessment of factors determining the progression of this mild dysfunction to age-related diseases and developing strategies aimed at arresting and reversing this progression.

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Keywords: Aging Indoleamine 2,3-dioxygenase Kynurenine pathway enzymes Plasma free tryptophan Tryptophan binding Tryptophan 2,3-dioxygenase

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*Corresponding author’s e-mail address: [email protected]

Journal Pre-proof 1. Introduction

2.1. General description

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2. The kynurenine pathway

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Please insert Figure 1 near here

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The kynurenine (Kyn) pathway (KP) (Figures 1 and 2) is quantitatively the major route of tryptophan (Trp) metabolism, accounting for ~ 95% of dietary Trp degradation (Badawy, 2017a). In liver, the KP accounts for ~ 90% of Trp degradation, whereas in extrahepatic tissues it makes only a minor contribution (< 5%) under normal physiological conditions. However, upon immune activation, the extrahepatic KP makes a more significant contribution. Whereas the hepatic KP contains all the enzymes required for NAD+ synthesis, this is not the case in extrahepatic tissues. Accordingly, the number of Kyn metabolites formed in these tissues will depend on the enzymes present. Early studies of the KP in humans centred round assessment of urinary excretion of intermediates of the pathway, particularly after acute Trp loading, to establish the capacity of the pathway for Trp disposal and to identify abnormalities in Trp metabolism in various disease states. More recently, the KP has become the focus of research on the important biological activities of Kyn and its metabolites, especially in relation to the immune system and neuronal function, and their impacts on health. While various contributions to this issue on the KP and aging focus on specific age-related aspects of the pathway in health and disease, this particular contribution will be of a more general and introductory nature. In the following text, a description of the KP, its regulatory mechanisms and functional aspects linking it to various body systems will precede an assessment of age-related changes in enzymes and metabolites of the pathway and their physiological determinants. Extensive reviews of the functional and regulatory aspects of the KP (Badawy, 2017a) and its provision of multiple targets for pharmacotherapeutic intervention (Badawy, 2019a) have been published.

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As illustrated in Figure 1, dietary Trp is first oxidised to N-formylkynurenine (NFK) by the action of Trp 2,3-dioxygenase (TDO: EC 1.13.11.11; formerly Trp pyrrolase) in liver and by indoleamine 2,3-dioxygenase (IDO: EC 1.13.11.17) elsewhere. NFK is then hydrolysed by NFK formamidase to Kyn. Kyn is metabolised mainly by oxidation and to a lesser extent by transamination. Kyn monooxygenase (KMO: also known as Kyn hydroxylase) oxidises Kyn to 3-hydroxykynurenine (3-HK). The latter is then hydrolysed to 3-hydroxyanthranilic acid (3-HAA) by kynureninase. Kynureninase also hydrolyses Kyn to anthranilic acid (AA). 3-HAA is further oxidised by 3-HAA 3,4-dioxygenase (3-HAAO) to 2-amino-3-carboxymuconic acid-6-semialdehyde (ACMS: also known as acroleyl aminofumarate). Kyn and 3-HK are also transaminated by Kyn aminotransferase (KAT) respectively to kynurenic acid (KA) and xanthurenic acid (XA) in the KAT A and B reactions. The KAT reactions are limited by the relatively high Km values of their substrates Kyn and 3-HK, compared with the corresponding Km values of these 2 substrates for KMO and kynureninase respectively (Badawy, 2017a). The KAT reactions therefore become more substantial when their substrate concentrations are increased, e.g. after acute Trp or Kyn loading, TDO or IDO induction or KMO inhibition (Badawy, 2017a). Trp loading in humans, however, inhibits the KAT B reaction (Badawy and Dougherty, 2016). ACMS occupies a central position in the KP at 2 crossroads, the predominant one involving non-enzymic cyclisation of ACMS to quinolinic acid (QA), which undergoes a variety of transformations leading to production of the important redox cofactor oxidised

Journal Pre-proof nicotinamide-adenine dinucleotide (NAD+). This part of the KP is referred to as the de novo (NAD+- biosynthetic) pathway. The less predominant crossroad involves decarboxylation of ACMS to AMS (2-amino-3-muconic acid-6-semialdehyde) by ACMS decarboxylase (ACMSD: also known as picolinate carboxylase). AMS can undergo non-enzymic cyclisation to picolinic acid (PA) or further metabolism leading eventually to formation of acetyl CoA. PA formation is generally limited, but can be enhanced if saturation of AMS dehydrogenase with its AMS substrate is maximal. Please insert Figure 2 near here NAD+ can also be synthesized from nicotinic acid or nicotinamide via the so-called salvage pathway through the transformations depicted in Figure 2.

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2.2. Tryptophan availability to the KP

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Trp availability to the KP is determined by its circulating levels and, in particular, free (non-albumin-bound) Trp, which is immediately available for uptake by tissues and organs. Free Trp represents 5-10% of the total circulating amino acid, with the remainder (90-95%) being albumin-bound. The physiological binder of Trp is therefore albumin, whereas the physiological displacers of albumin-bound Trp are nonesterified fatty acids (NEFA). Studies by the group of C. I. Pogson in the UK in isolated rat hepatocytes under a variety of experimental conditions suggest that the flux of Trp down the KP is determined primarily by plasma free Trp (for references, see Badawy, 2017a). However, Trp loading studies in humans suggest that plasma total Trp is equally important for this flux (Badawy and Dougherty, 2016; Badawy, 2017a). This is not surprising, given the rapid equilibration between the free and albumin-bound fractions. Furthermore, free Trp is a labile parameter easily influenced by many factors. These include nutritional, hormonal, physiological, pharmacological and pathological modulators of albumin binding (Badawy, 2010). Trp binding is usually expressed as the percentage free Trp (100 X [Free Trp]/[Total Trp]). Table 1 gives examples of these modulators and the changes in Trp binding they induce. Please insert Table 1 near here

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Briefly as shown in Table 1, Trp binding is decreased by direct displacement by NEFA or drugs such as salicylate or other agents acting via NEFA, i.e. lipolytic agents, e.g. adrenaline, noradrenaline, other sympathomimetic amines and phosphodiesterase inhibitors such as the methylxanthines, and c-AMP. Trp binding is also decreased if circulating albumin is depleted, e.g. in pregnancy and liver, kidney and certain other diseases including cancer. By contrast, Trp binding is increased by antilipolytic agents such as insulin and nicotinic acid. Induction of TDO or IDO and inhibition of TDO do not alter Trp binding, because induction decreases whereas inhibition increases both plasma free and total [Trp] by relatively similar proportions in each case. When displacement of albumin-bound Trp is strong and sustained, the rapid equilibration between free and bound Trp coupled with the associated increase in tissue uptake combine to cause depletion of total Trp. This is best illustrated in rats treated acutely with ethanol (Badawy and Evans, 1975) or sodium salicylate (Gessa and Tagliamonte, 1974; Badawy, 1982) or in late pregnancy (Badawy, 1988). A situation could therefore arise in which observing a decrease in total [Trp] without having measured free Trp can only lead to the incorrect conclusion of occurrence of Trp depletion (Munn et al., 1998, Badawy et al., 2016). It is therefore important to

Journal Pre-proof measure both free and total [Trp] and determinants of Trp binding to albumin, not only for accurate interpretation of changes in Trp disposition, but also to gain an insight into the Trp status and its biological determinants in normal and disease states (Badawy, 2010). 2.3. Flux of plasma tryptophan through the KP

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As stated above, studies in isolated rat hepatocytes have led to the conclusion that plasma Trp and, in particular, free Trp is the major determinant of flux of Trp through the KP (see Badawy, 2017a for references). However, in view of the rapid equilibration between free and bound Trp, total Trp in human plasma appears to play an equally important role in this flux. This latter conclusion is based on correlations between plasma free or total Trp and parameters of the Trp flux, such as [Kyn], TDO, TDO relative to free Trp, total Trp oxidation and total Trp oxidation relative to free Trp (Badawy, 2017a) and also on data on plasma [Trp] and [Kyn] previously reported in acute Trp loading studies collated to assess the diagnostic value of the [Kyn]/[Trp] ratio as an expression of IDO activity (Badawy and Guillemin, 2019). As IDO activity is inhibited by [Trp] of ≥ 50 µM (Ozaki et al., 1986; Efimov et al., 2012), the flux of plasma Trp through the human KP after acute Trp loading, or in other situations in which plasma levels similarly exceed 100 µM, is almost certain to take place through TDO (Badawy and Guillemin, 2019). 2.4. Regulation of the KP

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The regulation of the KP has been reviewed in detail by Badawy (2017a) and the following accounts provide brief summaries of the roles of the KP enzymes and intermediates. In addition to regulation by enzymes and intermediates, which is exerted in part via body system components, these components can exert further regulatory effects, which will be included in this section.

2.4.1.1. TDO and IDO

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2.4.1. Regulation of the KP by pathway enzymes

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As the most rate-limiting enzymes of the KP, TDO and IDO determine the extent to which Trp is metabolised along the pathway and hence levels of its biologically-active metabolites. TDO has been the subject of intense study that led to the concepts of enzyme induction and metabolic regulation, because of its short half-life and its rapid response to induction and activation by various agents. Such response is facilitated by the presence of the enzyme in livers of rats, mice, humans and some, but not all, other animal species in two forms, the active haem-containing holoenzyme and inactive haem-free apoenzyme (Badawy and Evans, 1976b). The apoenzyme becomes activated by addition in vitro of a source of haem, such as haematin or methaemoglobin, or after administration in vivo of haematin or the haem precursor 5-aminolaevulinate (5-ALA) and also of Trp. Thus, TDO activity is enhanced by 3 mechanisms: glucocorticoid induction of synthesis of new apoenzyme, substrate activation and stabilisation by Trp and cofactor activation by haem. It is thought that the Trp activation of TDO, which involves conjugation of the apoenzyme with haem, involves enhancement of haem biosynthesis, most probably by stimulating 5-ALA dehydratase activity (Badawy and Evans, 1975; Badawy et al., 1981). Stabilisation of TDO by Trp prolongs its T1/2. Thus, the half-life of the rat liver apoenzyme under basal conditions (2.3h) is not altered by cortisol, haematin or 5-ALA administration, but is prolonged to 6.7h with a large dose of Trp (500 mg/kg) and to 4.9h with a smaller dose (50 mg/kg) that does not activate TDO in rats (Badawy and Evans, 1975). The half-life of the basal holoenzyme (7.7h) is similarly unaltered by cortisol and the haem sources, but is prolonged only by the large Trp dose to

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11.4h. Human studies, however, suggest that doses of Trp of 25-50 mg/kg may activate, as well as stabilise, TDO and this may have important implications for the flux of Trp down the KP and production of Kyn metabolites in subjects receiving Trp on a long-term basis. The ability of repeated Trp administration to male subjects to enhance Kyn formation was demonstrated by Green et al. (1980) in a study showing that plasma and urinary [Kyn] following a single 50 mg/kg dose of Trp was considerably (2.3-fold and 4-fold respectively) higher in subjects previously receiving this Trp dose twice daily, compared with subjects who did not. TDO is regulated, negatively, by end-product allosteric inhibition by NAD(P)H (Cho-Chung and Pitot, 1967). Agents that increase the hepatic concentrations of these two reduced dinucleotides, such as glucose, nicotinamide (Badawy and Evans, 1976a) and chronic ethanol administration (Punjani et al., 1979) inhibit TDO activity. By contrast, IDO is not induced by glucocorticoids or activated by haem and is only activated moderately by a large Trp dose (Yoshida et al., 1980; Cook et al., 1980). This limited activation could, as stated above, be due to enzyme inhibition by [Trp] of ≥ 50 µM. IDO is induced by the major cytokine interferon- (IFN-) (Pfefferkorn et al., 1986) and certain other cytokines (see below) and inhibited by NO (Hucke et al., 2004; Thomas et al., 2007). 2.4.1.2. Other enzymes

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NFK formamidase: Although present in abundance in liver, this formamidase, if inhibited, can block the flux of Trp down the KP and thus prevent the formation of neuroactive and other Kyn metabolites. A range of compounds including organophosphate insecticides and metal cations are potent inhibitors of the enzyme and development of safer inhibitors for use in humans has been suggested (Han et al., 2012).

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Kynurenine monooxygenase: KMO is an important regulatory enzyme which, by controlling Kyn levels, can influence those of the transamination product KA and hydrolytic product AA on the one hand, and those of 3-HK and subsequent metabolites on the other. Modulation of KMO could therefore have important clinical consequences. KMO is upregulated in many inflammatory conditions (Badawy and Guillemin, 2019) and its inhibition can be beneficial in these and other conditions (Badawy, 2019a). As far as I could ascertain, the only clinical condition in which KMO is down-regulated is Down’s syndrome (Powers et al., 2019).

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Kynureninase: Modulation of kynureninase can influence levels of Kyn, AA, 3-HK and subsequent KP metabolites, with important clinical consequences. The enzyme is up-regulated in chronic inflammatory skin disease and many human autoimmune and auto inflammatory diseases (see Badawy and Guillemin, 2019) Kynurenine aminotransferase: Upregulation of KAT can increase [KA] provided that adequate amounts of Kyn are available. This may be desirable for combating QA-related excitotoxicity. However, excessive production of KA could lead to a state of glutamatergic hypoactivity, as occurs in schizophrenia. This is most likely due to upregulation of TDO, as well as of KAT and down-regulation of KMO, thus increasing the availability of the Kyn substrate (for references, see Badawy and Guillemin, 2019). 3-Hydroxyanthranilic acid 3,4-dioxygenase: 3-HAAO is the most active of the KP enzymes (Nishizuka and Hayaishi, 1963; Bender, 1980), which may explain the rapid conversion of 3-HAA to QA. While 3-HAAO inhibition is desirable to limit QA production, it can result in 3-HAA accumulation with potential proinflammatory effects. A better strategy is therefore to inhibit the KP at the earlier step of KMO. However, 3-HAAO inhibition is justified if it is up-regulated, e.g.

Journal Pre-proof in the hepatic proteome of the hyperlipidemic substance for normal cell function and integrity mouse HcB19 model (Van Greevenbroek et al., 2004) and brain of the triple transgenic Alzheimer’s Disease mouse model (Wu et al., 2013). ACMSD or picolinate carboxylase: ACMSD can exert significant effects on intermediates of the KP. Its inhibition diverts ACMS towards QA formation and thus leads to increased NAD+ synthesis (Pellicciari et al., 2018). While this may be desirable in certain situations, it is important to guard against excessive accumulation of QA, as shown with the use of inhibitory phthalate esters (Fukuwatari et al., 2004) and with a genetic mutation of ACMSD in a family with Parkinson’s Disease (Thirtamara-Rajamani et al., 2017). 2.4.2. Regulation of the KP by its intermediates and end products

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End-product inhibition of TDO activity involves allosteric inhibition by NAD(P)H (Cho-Chung and Pitot, 1967). Of a range of NAD+-related metabolites and immediate precursors, NADPH exerts the strongest inhibition (60% at 2 x 10-4M) followed by NADH (30% at a similar concentration). As stated above, this is the mechanism by which chronic administration of glucose, nicotinamide (Badawy and Evans, 1976a) and ethanol (Punjani et al., 1979) inhibit TDO activity in rat liver. Additionally, as inhibition is associated with increased Trp availability to the brain, it may be concluded that inhibition by NAD(P)H also occurs in vivo.

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Intermediate metabolites have also been reported to inhibit TDO activity. With TDO purified from Xanthomonas pruni, inhibition was demonstrated with Kyn and 3-HAA, in addition to NAD(P)H (Wagner and Brown, 1970). However, with the rat liver enzyme, strong inhibition occurs only with 3-HK and 3-HAA (Wagner, 1964). Acute administration to rats of 3-HK and 3-HAA, however, causes changes in liver Trp and/or Kyn metabolites inconsistent with TDO inhibition (Badawy and Bano, 2016). In short: 3-HK does not alter TDO or KMO activities, but inhibits that of kynureninase. 3-HK inhibition of the human recombinant kynureninase has been reported (Walsh and Botting, 2002). 3-HAA stimulates TDO but inhibits KMO and kynureninase activities. Kynureninase inhibition by 3-HAA has previously been demonstrated in pig liver (Tanizawa and Soda, 1979). KA administration also influences KP enzyme activities (Badawy and Bano, 2016). It stimulates TDO, KMO and kynureninase activities and acts on TDO by increasing [3-HAA]. These novel eff ects of Kyn metabolites require further appraisal and suggest that the KP may be regulated from within by its own intermediates as well as its enzymes and this may have further implications in clinical situations associated with activation of the pathway. Please insert Table 2 near here 2.4.3. Regulation by body systems Table 2 lists the dual relationship between the KP and body systems. Body systems can influence the KP (indicated by italics), be modulated by the KP (indicated by plain letters) or both (bold letters). The following account gives a summary of various aspects including the ones described above. TDO may also be subject to control by endocrine hormones other than glucocorticoids, some of which act by modulating glucocorticoid induction, while others act by different mechanisms (see Badawy, 2017a). Thus, glucocorticoid induction in primary rat hepatocyte cultures is potentiated by glucagon but inhibited by insulin and adrenaline (Nakamura et al., 1987). In cultured hepatocytes, however, glucagon may also act by a different mechanism, involving enhancing TDO

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activity at the translational step synergistically with glucocorticoid induction (Nakamura et al., 1980). The insulin effects on TDO are controversial. Thus, although it inhibits glucocorticoid induction of TDO in primary rat hepatocyte cultures, its administration to rats does not inhibit TDO activity (Labrie et al., 1969; Broqua et al., 1990) and it along with glucagon and dibutyryl c-AMP may enhance TDO by increasing haem synthesis (Yamamoto et al., 1982). In experimental diabetes induced by streptozotocin, rat liver TDO activity (Badawy and Evans, 1977) and flux of Trp through TDO, kynureninase and ACMSD in isolated hepatocytes (Smith and Pogson, 1981) are all enhanced. TDO enhancement in liver homogenates may also be secondary to removal of the KP feedback inhibition by NAD(P)H following depletion of these dinucleotides by streptozotocin (Badawy and Evans, 1977). The effects of adrenaline and noradrenaline on TDO activity are also controversial: while they inhibit TDO activity in vitro (Satoh and Moroi, 1969), their administration exerts the opposite effects by substrate activation initiated by enhancement of lipolysis (Badawy and Evan, 1976c).

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Body systems other than the endocrine and haemopoietic systems can also influence TDO activity. Among these is the intermediary metabolism of carbohydrates, lipids and proteins. In rats, high carbohydrate and high fat diets inhibit TDO activity (Chiancone, 1964; Badawy et al., 1984), wherea high protein diets enhance it (Chiancone, 1964; Satyanarayana and Rao, 1980). In mice, a decrease in plasma [Trp] and [Kyn] with protein-rich food suggests a TDO enhancement, whereas no significant changes in these 2 parameters are observed in humans receiving a high protein diet (Poesen et al., 2015).

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2.5. Functions of the KP

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The immune system is another modulator of the KP. A number of reviews can be consulted for detailed accounts (Mándi and Vécsei, 2012; Campbell et al., 2014; Fujigaki et al., 2017). Briefly, as well as by IFN-, IDO is inducible by other proinflammatory cytokines, notably interleukin IL-1, IL-2 and TNF-, but is inhibited by IL-4 and IL-13. IFN- also upregulates KMO, kynureninase and 3-HAAO, but downregulates TDO, KAT and QPRT. IL-4 can downregulate KMO (see also Asp et al., 2011; Mándi and Vécsei, 2012; Adams et al., 2014).

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As functional and regulatory aspects of the KP have been reviewed in detail (Badawy, 2017a), the following accounts provide brief outlines of these aspects. 2.5.1. Control of plasma tryptophan availability Liver TDO plays the major role in control of plasma Trp availability under normal conditions. This is suggested by the findings that deletion of the mouse TDO gene causes a 9.3-12.7-fold increases in plasma [Trp] (Kanai et al., 2009; Terakata et al., 2013) and a 10.6-fold elevation of brain [Trp] (Too et al., 2016). In this latter study, no increase in brain [Trp] was observed after deletion of the IDO1 and IDO2 genes. Under conditions of immune activation, however, IDO induction enhances Trp catabolism leading to decreased plasma [Trp] and increased [Kyn] (for various examples, see Badawy and Guillemin, 2019). Trp availability to the brain is an important function of the KP in relation to cerebral 5-hydroxytryptamine (5-HT or serotonin) synthesis. The importance of brain [Trp] in 5-HT synthesis is underpinned by the existence of the rate-limiting enzyme Trp hydroxylase (TPH1) partially (≤50%) saturated with its Trp substrate (Carlsson and Lindqvist, 1978). It follows therefore that minor changes in Trp availability to the brain can influence 5-HT synthesis

Journal Pre-proof significantly. At the primary level, TDO activity is the major determinant of Trp availability to the brain. At the secondary, but more immediate, level, Trp availability to the brain is determined by extent of binding to albumin, i.e. the plasma free [Trp], and concentrations of a number of amino acids (CAA) that compete with Trp for entry into the brain, namely the branched-chain amino acids Val, Leu and Ile, and the aromatic amino acids Phe and Tyr. Trp availability to the brain is usually expressed as the [Trp]/[CAA] ratio and serves as an indicator of likely changes in brain [Trp]. An inverse relationship exists between liver TDO activity and brain [5-HT] involving the appropriate changes in plasma and brain [Trp] (for a detailed account, see Badawy, 2013 and references cited therein). 2.5.2. Detoxification of tryptophan

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2.5.3. Control of hepatic haem biosynthesis

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Excess Trp is toxic to species lacking the free apoenzyme form of TDO and its glucocorticoid induction mechanism. These species include the cat, frog, golden hamster, guinea pig, ox, sheep and rabbit (Badawy and Evans, 1976b). By contrast, species possessing both the TDO holoenzyme and haem-free apoenzyme and the glucocorticoid induction mechanism, e.g. rat, mouse, pig, turkey, chicken and human, can handle Trp through glucocorticoid induction of new enzyme protein. Thus, loss of this induction mechanism underpins Trp toxicity. For example, adrenalectomy renders the rat vulnerable to Trp toxicity and cortisol administration to adrenalectomized rats restores safety (Knox, 1966). This protective effect of TDO does not apply to IDO for 2 reasons: (1) Trp-sensitive species are vulnerable to Trp toxicity despite having a higher IDO activity than rats; (2) as stated above, [Trp] ≥ 50 µM inhibits IDO activity.

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The TDO apoenzyme in rat liver has been shown to utilise the small regulatory haem pool in the hepatic cytosol whose concentration is ~ 10-7 M (Badawy, 1979). This pool controls haem biosynthesis by a negative feedback mechanism involving repression of synthesis of the rate-limiting enzyme 5-aminolaevulinate synthase (5-ALAS). By utilising this haem, TDO removes the negative feedback control, thereby allowing haem synthesis to proceed unhindered. It follows that prevention of this haem utilisation by TDO may form the basis of a therapy of acute hepatic porphyric attacks, wherein 5-ALAS induction occurs leading to overproduction of 5-ALA and occurrence of 5-ALA-related symptoms. Currently, these attacks are treated by targeting 5-ALAS with glucose (an inhibitor of TDO), haematin or haem arginate (direct 5-ALAS repressor) and, most recently, proposed 5-ALAS1 gene silencing. Metabolic targeting of 5-ALAS with Trp and inhibitors of haem utilisation by TDO has recently been proposed (Badawy, 2019b) in a hypothesis that provides detailed accounts of the biochemistry of haem and the preferential utilisation of the regulatory pool by TDO. 2.5.4. Modulation of immune and neuronal function The KP produces a variety of metabolites which modulate neuronal and immune functions. The pioneering work of T W Stone and his group on glutamate function has identified the modulation of the NMDA (N-methyl-D-aspartate) type of receptors of this excitatory amino acid by the Kyn metabolites KA and QA (Stone, 1993), with the former acting as antagonist and the latter as agonist. It is thought that the balance between KA and QA determines the state of neuronal excitability. A decrease in QA and its immediate precursors can be achieved by KMO inhibition and could result in protection against CNS diseases, as suggested in studies of models of cerebral malaria, neuropathic pain, and Alzheimer’s and Huntington’s diseases (for references, see

Journal Pre-proof Badawy, 2019a). KMO inhibition can also lead to accumulation of Kyn and a potential activation of the KAT A reaction Kyn  KA, especially if TDO is simultaneously upregulated. While this situation may be desirable for dual neuronal protection, it can also lead to a state of glutamatergic hypoactivity, as is the case in schizophrenia (Badawy, 2017, 2019a).

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As well as playing opposite roles in modulating neuronal function, KA and QA may also play opposite roles in the immune system, with KA being in the main antiinflammatory and QA proinflammatory (for review, see Badawy, 2018). Initially, QA along with 3-HK and 3-HAA have been shown to possess immunosuppressive properties (see extensive review by Yeung et al., 2015). Both 3-HK and 3-HAA were shown to suppress allogeneic T-cell proliferation in an additive manner possibly by an apoptotic mechanism (Terness et al., 2002) that was suggested to explain the ability of 3-HAA and QA to undermine T helper type 1 (Th1) cells (Fallarino et al., 2002). These latter authors showed that, at the smallest concentration used (10 µM), only 3-HAA and QA induced apoptosis in thymocytes. As this concentration is much higher than circulating levels of Kyn metabolites, an important question is whether higher concentrations can be reached in cell microenvironments. This question was addressed satisfactorily for QA by the group of J R Moffett in the USA (Moffett et al., 1994, 2003), who showed by immunohistochemistry that very high levels of QA can be detected in cells of the immune system. Detection and quantification of QA by this technique are due to the nature of the QA structure, in particular the absence of an NH2 group, which enabled the production of specific antibodies to QA. With other Kyn metabolites, except KA and PA, the presence of both an amino and a carboxyl group allowed orientation in several directions during coupling to a protein (Moffett et al., 1994), thus leading to non-specific epitopes.

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An interesting aspect of Kyn metabolite immune activity is the role of the aryl hydrocarbon receptor (AhR). The AhR can control immune responses in both protective and destructive ways, with endogenous ligands facilitating a dampening of the immune response to prevent excessive inflammation and autoimmunity and exogenous ligands acting as signals to enhance inflammatory responses to infection and resistance of cancer to its own destruction (for references, see Badawy, 2019a). Kyn is an endogenous ligand of the AhR that can influence various immune responses. However, the structure of Kyn suggests that its binding affinity to the AhR is low, with an EC50 of ~13 µM (Seok et al., 2018). In the DRE-luciferase assay in H4IIE rat hepatoma cells, a significant stimulation was observed with a [Kyn] of 50 µM, with no effect at 5 µM (Opitz et al., 2011). In this system, KA was without effect at both concentration levels. By contrast, in the CEE luciferase assay in primary human hepatocytes, KA was shown to be a potent agonist, causing significant stimulation at 1 and 10 µM concentrations (DiNatale et al., 2010). Kyn, 3-HK, AA and QA were ineffective at 10 µM. XA was also shown to be effective. Based on the ability of KA to remarkably alter the transcription of an AhR target gene (CYP1A1 mRNA in HepG2 cells) by ~ 66-fold at a 100 nM concentration, the above authors suggested that KA is the primary KP activator of the AhR. PA also possesses antiinflammatory properties (see Badawy et al., 2016), but along with XA they are the 2 least studied Kyn metabolites in inflammation. They do, however, exert important effects on carbohydrate metabolism as described below. 2.5.5. Modulation of carbohydrate metabolism Carbohydrate metabolism and its impact on diabetes can be influenced significantly by PA, XA

Journal Pre-proof and QA. Thus, QA inhibits the key enzyme of gluconeogenesis phospho-enol-pyruvate carboxykinase. The group of C. I. Pogson in the UK has shown that Trp administration inhibits gluconeogenesis in species capable of producing large amounts of QA, such as rats, but not in those with poor QA production, such as gerbil, guinea pig or sheep (for references, see Badawy, 2019a). It is therefore of interest that animal models of diabetes are mainly those of rodents and pigs, but not species of limited QA production.

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XA is diabetogenic, presumably by binding and thus inactivating insulin (Bender, 1983). High insulin resistance and high odds of having diabetes are associated with high plasma [XA] (Reginaldo et al., 2015) and both plasma and urinary [XA] are raised in diabetic patients and experimental diabetes in rats (Ikeda and Kotake, 1986; Oxenkrug, 2015). High Zn excretion accompanies that of XA (Ikeda and Kotake, 1986). Zn is vital for insulin function and its absorption and bioavailability are controlled by PA. Although no information is available on PA in diabetes, current evidence suggests that it may be increased, because ACMSD activity and mRNA expression are increased in experimental diabetes, although PA production in liver is not impaired and ACMSD activity is greater in kidney than in liver (for references, see Badawy, 2017a). Plasma [PA] is however elevated in hepatitis viral infection, and to a greater extent if diabetes is present (Zuwała-Jagiello et al., 2012).

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2.5.6. Control of niacin biosynthesis and pellagra prevention

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The pellagra-preventing factor, “vitamin B3” in its two forms, nicotinic acid (niacin) and nicotinamide, can be synthesized from Trp through the QA arm of the KP (Figure 1). As QA is formed in the predominant arm, sufficient niacin can always be synthesized even in the presence of dietary niacin deficiency. However, dietary deficiency is not always limited to niacin, but could also include Trp. With niacin deficiency coupled with Trp-deficient staples, such as maize of sorghum, incidence of pellagra is inevitable. This was the case in Southern Europe during the 18th century and the USA following the American civil war. Although maize and sorghum are deficient in Trp, they contain adequate levels of niacin, but in a polysaccharide bound form (niacytin) that cannot be hydrolysed by mammalian digestive enzymes. Those who introduced maize to Southern Europe failed to follow the liming process to release niacin, which has been practiced by the peasants of Central America for millennia (for review and references, see Badawy, 2014). 2.5.7. Control of NAD+ biosynthesis Because of the vital roles of NAD+ at multiple cellular and system levels, it is no exaggeration to suggest that NAD+ synthesis is the most important function of the KP. Quantitatively, NAD+ synthesis from Trp is more efficient that that from niacin. Thus, dietary Trp is more effective than dietary nicotinamide or nicotinic acid in elevating liver nicotinamide-adenine dinucleotides and urinary levels of N1-methylnicotinamide (Williams et al., 1950; Bender, 1982; McCreanor and Bender, 1986). Details of activities of enzymes of NAD dinucleotide synthesis have been presented in the latter 2 studies by the group of D. A. Bender. 3. Effects of aging on enzymes, precursors and metabolites of the kynurenine pathway and the role of body systems 3.1. Plasma tryptophan

Journal Pre-proof 3.1.1. Plasma tryptophan concentration and binding In a previous review (Badawy, 2017b), plasma total [Trp] was shown in a number of studies to be decreased by aging by 7-32% in males (n=10 studies) by 0-38% in females (n=7 studies), thus averaging -13% in males and -17% in females. Free [Trp] was measured in only 3 studies. In females, negligible changes were observed (+5%, -4% and 0%), whereas in males, it was decreased in 2 studies by 12% and 16% and increased in the third by 6%. Thus, overall, a moderate decrease in total [Trp] occurs with aging. The ~ proportionate decreases in free and total [Trp] reported so far suggest that TDO or IDO activity may be enhanced in humans by aging (see below).

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As regards Trp binding, the study by Demling et al. (1996) showed that the percentage free Trp is higher in aged males and females (by 42% and 59% respectively). However, because older subjects were patients tested after undergoing surgery, with some receiving the lipolytic agent heparin and other medications, attributing the above increases to aging is unjustified. In the study by Thomas et al. (1986) in healthy volunteers, there were no significant differences in Trp binding between young and older subjects (the % free Trp was 19.47% and 18.06% respectively in males and 20.61% and 20.28% respectively in females). This suggests that Trp binding to albumin is not impaired by aging.

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3.1.2. Determinants of plasma tryptophan binding

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Determinants of Trp binding are albumin and NEFA. Gomi et al. (2007) reported that aging lowers serum [albumin] in both males and females. Gradual decreases from age 65-67 to age ≤ 90 years occur, with the overall decreases being 9.30% in males and 6.98% in females. In an earlier study by Salive et al. (1992), decreases of 7% in males and 5% in females were observed in normal populations aged 71-74 and ≥ 90 years. Data from a previous study in pregnant rats suggest that a decrease in Trp binding to albumin requires a drop in serum [albumin] of ≥ 19% (Badawy, 1988). The small decreases in albumin reported above in older people are therefore unlikely to influence Trp binding, which is consistent with the absence of altered binding in the above studies. A review and meta-analysis by Cabrerezo et al. (2015) concludes that age is not the cause of hypoalbuminemia, but that factors such as the nutritional status and various pathologies are more likely determinants. The other determinants of plasma Trp binding are NEFA. Their concentration increases with aging in rats (Hardy et al., 2002). In humans, studies of the effect of aging on [NEFA] have been controversial, with either an increase or no change having been reported (for references, see Rosenthal and woodside, 1988). Serial measurements by the above authors over the 8h period from midnight showed that older males (mean age: 69 years) had lower [NEFA] than younger males (mean age 26 years). Plasma cortisol and catecholamines were generally unaltered by aging, with noradrenaline being slightly higher and adrenaline slightly lower in older subjects. Plasma insulin was, however, significantly higher in older subjects, which may explain their lower [NEFA]. It may therefore be concluded that moderate decreases in plasma [Trp] occur in aging in humans, but that their extent is unlikely to decrease Trp availability to the brain, as expressed by the [Trp]/[CAA] ratio. Published data of this ratio are controversial (see Badawy, 2017b). The likely absence of changes in plasma Trp availability to tissues, however, does not exclude changes occurring within these tissues through altered enzyme activities. Additionally, in liver, it is even

Journal Pre-proof possible that the moderately lower plasma [Trp] may be caused by TDO enhancement, as suggested below. 3.2. Enzymes of the kynurenine pathway 3.2.1. TDO

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TDO activity and extent of its induction by cortisol are lower in aged rats (Green and Curzon, 1975; Patnaik and Patnaik, 1989; Comai et al., 2005; Brady et al., 2011). Data on serum corticosterone in aged rats are, however, controversial, with a decrease (Reaven et al., 1988; de Almeida et al., 1998), an increase (for various references, see Issa et al., 1990) or no change (Issa et al., 1990; Kmiec et al., 2006, Buechel et al., 2014) having all been reported. Little is known about the TDO status in humans in aging. The moderate decrease in plasma [Trp] in older subjects described above suggests that TDO activity could be elevated. If so, the most likely cause is the higher circulating cortisol in older subjects. The moderate increase in serum cortisol levels reported in the elderly (Bergendahl et al., 2000; see also the review by Yiallouris et al., 2019) may be sufficient to cause TDO induction. Elevated cortisol in the elderly is but one feature of the complex changes in adrenal function that could impact various aspects of health (Yiallouris et al., 2019).

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3.2.2. IDO

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Although TDO is activated by its heme cofactor, it appears that aging is unlikely to influence hepatic heme levels to undermine this activation. Thus, although 5-ALAS activity is decreased, and that of heme oxygenase is increased (Scotto et al., 1983; Abraham et al., 1985; Bloomer et al., 2004), by aging in rats, hepatic microsomal heme and cytochrome P-450 levels are unimpaired (Abraham et al., 1985). While P-450 does not utilise the regulatory-heme pool, it is likely that the loss of this pool by heme oxygenase induction may be overcome by removal of the negative feedback control of 5-ALAS synthesis. The role of heme oxygenase in the stress response in aging (see Bloomer et al., 2004 and references cited therein) suggests the need for further studies of the heme-metabolic pathways in aging.

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The moderate decrease in plasma [Trp] in the elderly could also result from IDO induction. Surprisingly very few studies of the effect of aging on IDO activity have been performed in experimental animals. Comai et al. (2005) reported variable IDO activities with age in rat intestine, the richest source. Activity in 12- and 18-month-old rats was lower than that in 2-3-month-old ones, with that of 18-month- being higher than that of 12-month-old rats. Brady et al. (2011) reported an increase in brain IDO activity and a decrease in that in kidney. Surprisingly, these authors reported a comparable or higher IDO activity in liver, given that this organ possesses little IDO. In this latter study, the reported decreases in liver [Trp] and [Kyn] cannot be reconciled with the reported decrease in TDO activity and in that assumed to be IDO activity. Miura et al. (2008) reported that aging decreases levels of Trp and Kyn, and increases those of 5-HT in certain brain areas, suggesting brain IDO inhibition. Thus the IDO status in experimental models of aging is in need of clarification. The situation in humans is much clearer in that the immunological and neurological dysfunctions occurring in the elderly are associated with IDO induction. Thus, ample evidence exists for IDO induction and consequent changes in neuroactive and immuno-reactive Kyn metabolites in a variety of disease states (Darlington et al., 2010; Platten et al., 2015; Fujigaki et

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al., 2017; Lovelace et al., 2017). Experimental animal models of these diseases also demonstrate IDO induction (see, e.g., Dobos et al., 2012; Forteza et al., 2018; see also the reviews by Remus and Dantzer, 2016 and Mellor et al., 2017). Evidence for IDO induction in aging normal subjects is, however, limited. Frick et al. (2004) reported a significant increase in the plasma [Kyn]/[Trp] ratio in normal subjects aged 72-93 years, compared with younger subjects. Pertovarra et al. (2006) reported that IDO activity in nonageraians is markedly increased and predicts mortailty. The IDO increase, however, could not be correlated with the mRNA expression (Marttila et al., 2011). This latter finding supports the notion that increased mRNA expression (of IDO) is not synonymous with increased enzyme catalytic activity. A similar example is that of 1-methyltryptophan preventing the increase in the [Kyn]/[Trp] ratio (an expression of IDO activity), but not the increased IDO gene expression in mice treated with Bacillus Calmette-Guérin (BCG) (O’Connor et al., 2009). These and other examples emphasize the need to appraise critically the various expressions of IDO (see the discussion by Badawy and Guillemin, 2019). It is not unreasonable to assume that the extent of IDO induction in well elderly subjects, as reported in the above 3 papers, is lower than in those with manifest disease, where an increase in the [Kyn]/[Trp] ration) could be as much as 560% (see Table 1 in Badawy and Guillemin, 2019).

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3.2.3. Other enzymes

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As far as I could ascertain, only two animal studies of these enzymes have been published. Comai et al. (2005) and Brady et al. (2011) reported that, compared to 2-3-month-old rats, those of 12-18-months of age exhibit a lower KMO in liver and kidney, a normal liver KAT and a higher brain, liver and kidney KAT, a lower liver kynureninase, a higher liver ACMSD and a lower brain and liver QPRT, but a higher kidney enzyme. Other than IDO, little work has been done on other KP enzymes in elderly humans. While access to peripheral organs is not an option, studies could be performed in peripheral blood cells. However, not all these cells contain the full set of KP enzymes. For example, Whereas IDO1 is expressed in several such cells, KMO, kynureninase and 3-HAAO are expressed only in macrophages, with little or no activity in THP-1 monocytes, lymphocytes or astrocytes (Heyes et al., 1997; Murakami and Saito, 2013). A more recent study (Minhas et al., 2019) reported that human macrophages possess the complete set of enzymes of the KP for conversion of Trp to NAD+ and that aging impairs this conversion. A pivotal role of the KP in regulating macrophage immune function in resting, immune-challenged, and aged macrophages was suggested. 3.3. Metabolites of the kynurenine pathway 3.3.1.Animal studies Effects of aging in experimental animal studies on KP metabolites have been reviewed by Ortega et al. (2015). The higher brain, liver and kidney KAT activities reported by Brady et al. (2011) explain the increase in [KA] in these 3 organs reported by these authors. Similarly, the lower brain and liver QPRT could explain the increase in [QA] in these 2 organs. The higher liver ACMSD reported by Comai et al. (2005) may explain the increased [PA] in liver reported by Brady et al. (2011). 3.3.2. Human studies In human aging, two studies have examined various KP metabolites. In the Hordaland Community Health Study (Theofylaktopoulou et al., 2013), compared to subjects aged

Journal Pre-proof 45-46-years (n=3723), those aged 70-72-years (n = 3229) had increased plasma concentrations of Kyn, AA, KA, 3-HK, 3-HAA and neopterin, an increased [Kyn]/[Trp] ratio and a lower [Trp] and [XA]. Similar age-related changes were observed in both men and women. The higher CSF [KA] in older subjects (> 50-years) compared with younger ones (< 50-years) has previously been reported by Kepplinger et al. (2005), who found no difference in serum levels. De Bie et al. (2016) compared CNS levels of KP metabolites in a relatively small sample (n=49) of healthy women across various age groups (20-40, 40-60, and 60-80-years-old). Increases in the latter group were observed in neopterin, quinaldic acid, QA, PA, 3-HAA and Kyn, with decreases in Trp, KA and 3-HK, and elevation of the [Kyn]/[Trp] ratio. Overall, the above findings in humans strongly suggest that KP activity is increased with aging, though more studies are required to establish a greater concordance of data.

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3.4. Cytokines and other modulators of inflammation

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An important determinant of KP activity initiated by IDO and other intermediate enzymes is level of cytokines and other immune modulators. Aging is associated with a low-grade inflammation and cytokine dysfunction (Morley and Baumgartner, 2004; Álvarez-Rodríguez et al., 2012; Xia et al., 2016). Age is associated with raised levels of the proinflammatory cytokines IL-12, IL-1, TNF-. IL-6 and C-reactive protein (CRP), with the latter three modulators being particularly associated with frailty (Álvarez-Rodríguez et al., 2012; Michaud et al., 2013). A detailed account of studies of changes in inflammatory markers in human aging has been published by Singh and Newman (2011). These changes and their impact on KP enzymes and metabolites provide the link between the aging process and development of age-related diseases. Aging is a multifactorial phenomenon and it is noteworthy that under- or malnutrition in the elderly can impact the immune system and modulate cytokine levels (Marcos et al., 2003).

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4. General conclusions and comments

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Tryptophan is an important essential amino acid and evidence suggests that it plays an important role in the aging process. Thus, through control of Trp availability, TDO functions as a metabolic switch of age-related protein homeostasis and lifespan (van der Goot et al., 2012). Trp degradation results in production of a range of metabolites with wide biological activities, including neuronal- and immuno-reactivity. The kynurenine and other pathways of Trp-degradation exhibit a dual relationship with a variety of body systems: it can impact, or be impacted by, these system. The outcome of this dual relationship is therefore twofold: (1) disturbances in body systems induced by disease can be reflected in KP enzymatic and metabolite changes, which could be utilized as disease markers; (2) dysfunction of KP enzymes due to genetic or environmental causes can induce changes in body systems, which if strong enough, could result in disease, in which case the KP can be targeted for pharmacological and other interventions. Changes in KP enzymes and metabolites occur in normal aging but to a level lower than that seen in age-related diseases. While these moderate changes may be the precursors of stronger ones, it is important that future efforts are made to identify factors mediating the transition to age-related disease states and target them to arrest the transition process. Acknowledgements The author held an honorary professorship at Cardiff Metropolitan University during the period September 2006 and September 2016.

Journal Pre-proof Conflict of interest The author has no conflict of interest to declare regarding this paper. References

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Journal Pre-proof Figure 1. The kynurenine pathway of tryptophan degradation Adapted from Figure 1 in Badawy, A.A.-B., 2017. Kynurenine pathway of tryptophan metabolism: regulatory and functional aspects. Int J Tryptophan Res 10, 1-20. https://doi.org/10.1177/1178646917691938

Figure 2. NAD+ synthesis in the salvage pathway Adapted from Figure 2 in Badawy, A.A.-B., 2017. Kynurenine pathway of tryptophan metabolism: regulatory and functional aspects. Int J Tryptophan Res 10, 1-20.

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https://doi.org/10.1177/1178646917691938

Journal Pre-proof Table 1 Modulators of plasma tryptophan binding __________________________________________________________________________________________ Binding

% Free Free Total Modulators and examples Trp Trp Trp __________________________________________________________________________________________ ,

,

-,

Increased





-

Direct displacement (NEFA, salicylate) Via NEFA (catecholamines, ethanol, phosphodiesterase inhibitors) Decreased albumin (pregnancy, liver and kidney diseases, cancer) Antilipolytic agents (insulin, nicotinic acid)

Unaltered

-





TDO/IDO induction (glucocorticoids/interferon-)



TDO inhibition (allopurinol, antidepressants, nicotinamide)



ro

-

of

Decreased

-p

__________________________________________________________________________________________

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Abbreviations used: IDO (indoleamine 2,3-dioxygenase), NEFA (nonesterified fatty acids), TDO (Trp 2,3-dioxygenase, Trp (tryptophan). Symbols:  increase,  decrease, - no change.

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Table 2 Interaction of the kynurenine pathway with body systems __________________________________________________________________________________ A. KP Enzymes Body system

Effects, modulation or both1

TDO

Induction by glucocorticoids Modulation of above induction by other hormones Activation by substrate and cofactor Control of haem biosynthesis Inverse relationship through altered Trp availability Inhibition Inhibition Induction Upregulation Upregulation and down-regulation Production of immune-reactive Kyn metabolites Control of immuno-reactive Kyn metabolites KA and QA Neuronal protection by KA Control of immuno-reactive Kyn metabolites Excitability through QA Control of NAD+ synthesis

Endocrine

B. KP Metabolites

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KAT Kynase ACMS

lP

KMO

na

IDO

Brain serotonin Lipids Carbohydrates Proteins Cancer biology Immune system Cancer biology Immune system Neuronal system Neuronal system Immune system Neuronal system Vital body functions

of

Heme synthesis

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Trp (free plasma) Protein synthesis Albumin Lipid metabolism NEFA Kyn Immune system (M) Proinflammatory 3-HK Immune system (M) Proinflammatory 3HAA Immune system (M) Proinflammatory KA Immune system Antiinflammatory AA Immune system? Antiinflammatory (?) XA Carbohydrate metabolism Insulin activity QA Immune system Proinflammatory Neuronal system Neurotoxicity by NMDA agonism Carbohydrate metabolism Gluconeogenesis NAD+ Various systems vital cellular functions PA Immune system Second signal for IFN- activity Carbohydrate metabolism Zn absorption and bioavailability for insulin actions __________________________________________________________________________________________ 1 Text in italics represents effects of body systems on components of the KP, whereas that in plain letters represents modulation by the KP of body systems. Bold letters represent both effects and modulation.

Journal Pre-proof Highlights

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The kynurenine pathway (KP) produces many biologically active metabolites Current emphasis is on changes in brain disorders, infectious diseases and cancer The pathway performs important physiological functions across many body systems Normal aging influences the KP activity through low-grade inflammatory changes Future research should focus on arresting the transition to age-related diseases

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Figure 1

Figure 2