TRYPTOPHAN LOADING: CONSEQUENT EFFECTS ON THE SYNTHESIS OF KYNURENINE AND 5-HYDROXYINDOLES IN RAT BRAIN

Abstract— Tryptophan loading of rats resulted in a continuous non-linear uptake of l -tryptophan from plasma into the brain. The optimum tryptophan load for increasing cerebral 5-hydroxytryptamine (5-HT) level was 25 mg/kg. Above this, there was a gradual decrease both in the levels and synthesis of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) as assessed from simultaneous intraperitoneal or intraventricular injections of l [¹⁴C]tryptophan. A 5–10 fold increase in cerebral tryptophan produced a limited stimulation of 5-HT synthesis. When the cerebral tryptophan level reached 1 ± 10 -4 , substrate inhibition in vivo of the tryptophan monooxygenase (tryptophan-5-hydroxylase) but not of the indoleamine-2,3-dioxygenase occurred. Cerebral synthesis of kynurenine increased linearly with increasing tryptophan load. At a plasma ratio of 50:1 tryptophan to kynurenine, tryptophan loading interfered with the entry of peripheral kynurenine. Tryptophan loading also increased the efflux of 5-hydroxyindoles from the brain. One hour after intraperitoneal injection of l -kynurenine sulfate (5 mg/kg) into rats, there was a shift in the plasma ratio of l -tryptophan to l -kynurenine to 4:1. In these rats, a 20% reduction of cerebral tryptophan was noted.     

Tryptophan metabolism, from nutrition to potential therapeutic applications

Tryptophan is an indispensable amino acid that should to be supplied by dietary protein. Apart from its incorporation into body proteins, tryptophan is the precursor for serotonin, an important neuromediator, and for kynurenine, an intermediary metabolite of a complex metabolic pathway ending with niacin, CO₂, and kynurenic and xanthurenic acids. Tryptophan metabolism within different tissues is associated with numerous physiological functions. The liver regulates tryptophan homeostasis through degrading tryptophan in excess. Tryptophan degradation into kynurenine by immune cells plays a crucial role in the regulation of immune response during infections, inflammations and pregnancy. Serotonin is synthesized from tryptophan in the gut and also in the brain, where tryptophan availability is known to influence the sensitivity to mood disorders. In the present review, we discuss the major functions of tryptophan and its role in the regulation of growth, mood, behavior and immune responses with regard to the low availability of this amino acid and the competition between tissues and metabolic pathways for tryptophan utilization.     

SYNTHESIS AND METABOLISM OF l-KYNURENINE IN RAT BRAIN

Abstract— A method for the quantitative analysis of femtomole amounts of kynurenine (along with tryptophan, 3-hydroxykynurenine and kynuramine) in rat brain using high pressure liquid chroma-tography and electron-capture GLC is described. Endogenous concentrations of these substances in rat brain regions were measured, and their formation after the injection of radioactive tryptophan or kynurenine was determined. Kynurenine was formed from tryptophan in brain and was also taken up from the periphery. Extracerebral kynurenine was calculated to account for 60% of the cerebral pool of kynurenine. The cerebral rates of synthesis of kynurenine and 3-hydroxykynurenine were 0.29 and 0.17nmol/g/h. The turnover rate of kynurenine in the brain was 1.02 nmol/g/h measured from [¹⁴C]tryptophan or 1.14 nmol/g/h from [³H]kynurenine injected intraperitoneally. Kynuramine levels in different areas of the brain were similar to those of tryptamine. Following intraperitoneal injection of [¹⁴C]tryptophan, the presence of anthranilic, 3-hydroxyanthranilic, xanthurenic, kynurenic and quinaldic acids was demonstrated in the brain.     

Effect of food restriction on serotonin metabolism in rat brain

The brain concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) increased in rats maintained on restricted volume of low-protein or normal-protein diet, whereas these two agents decreased in rats fed low-protein diet ad libitum. In these two food-restricted groups brain 5-HT and 5-HIAA concentrations were not correlated with brain tryptophan hydroxylase activity, but the concentrations correlated closely with cerebral tryptophan concentrations. The cerebral tryptophan concentration in the two food-restricted groups was not consistent with the total or free tryptophan concentration in plasma. In these restricted rats cerebral tryptophan concentration was elevated, and, unlike the plasma tryptophan, it showed no diurnal variation. These results suggested that tryptophan uptake into the brain from plasma was enhanced by limiting food volume intake. Tryptophan uptake was increased by glucagon injection without changing the plasma tryptophan level, but injection of hydrocortisone or insulin had little or no effect on tryptophan concentration in either the plasma or brain.d-Glucose injection elevated plasma tryptophan concentration but decreased brain tryptophan concentration.     

Model of Tryptophan Metabolism,Readily Scalable Using Tissue-specific Gene Expression Data

Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease.     

Kynurenine metabolism in vitamin-B-6-deficient rat liver after tryptophan injection.

Tryptophan contents of liver, serum and kidney were determined in normal and vitamin-B-6-deficient rats after tryptophan injection. Tryptophan contents of normal and B-6-deficient liver were different, but not those in serum and kidney. Both kynurenine and 3-hydroxykynurenine accumulated in B-6-deficient liver more than in the normal. The 3-hydroxykynurenine contents after tryptophan injection (30 mg/100 g body wt.) increased to 1380 nmol/g of liver at 1-1.5 h, a value sufficient to produce xanthurenate, in view of the Km value of kynurenine aminotransferase. The enzymes metabolizing kynurenine were assayed at various times after tryptophan injection. The activity of kynureninase holoenzyme in B-6-deficient liver was much decreased, but the activity of total enzyme was not changed. It appeared that a high dose of tryptophan in B-6-deficient rats could cause a greater deficiency of pyridoxal 5-phosphate. Tryptophan metabolism in B-6-deficient rat liver after tryptophan administration is discussed.     

The effect of repeated tryptophan administration on body weight, food intake, brain lipid peroxidation and serotonin immunoreactivity in mice

Tryptophan as a circulating precursor of serotonin (5-HT) may suppress food intake and body weight. Tryptophan administration can enhance the generation of reactive oxygen species (ROS) by inducing oxidative pathway in vivo and in vitro. We have examined the effect of repeated tryptophan administration on food consumption, body weight, brain lipid peroxidation and 5-HT immunoreactivity. Tryptophan was given at the dose of 100 mg/kg/24 hr in 0.2 ml saline solution i.p. for 7 days to mice. Control mice received 0.9% NaCL solution at the same manner and volume. Body weights were recorded at the beginning and end of the experiments. Thiobarbituric acid reactive substance (TBARS), the last product of lipid peroxidation, was measured spectrophotometrically. Brain 5-HT levels were determined by the immunohistochemical method. Our findings indicate that the tryptophan suppresses food intake significantly in mice. Body weight decreased and brain TBARS levels increased significantly by repeated tryptophan treatment. Immunohistochemical detection showed that 5-HT levels increased by tryptophan administration. There is a link between increased 5-HT level and oxidative stress by tryptophan administration on brain tissue. Tryptophan at repeated doses should be exercised carefully in clinical practice.     

Type I Interferon-Mediated Skewing of the Serotonin Synthesis Is Associated with Severe Disease in Systemic Lupus Erythematosus

Serotonin, a highly pro-inflammatory molecule released by activated platelets, is formed by tryptophan. Tryptophan is also needed in the production of kynurenine, a process mediated by the type I interferon (IFN)-regulated rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO). The aim of this study was to investigate levels of serotonin in patients with the autoimmune disease systemic lupus erythematosus (SLE), association to clinical phenotype and possible involvement of IDO in regulation of serotonin synthesis. Serotonin levels were measured in serum and plasma from patients with SLE (n=148) and healthy volunteers (n=79) by liquid chromatography and ELISA, as well as intracellularly in platelets by flow cytometry. We found that SLE patients had decreased serotonin levels in serum (p=0.01) and platelets (p<0.0001) as compared to healthy individuals. SLE patients with ongoing type I IFN activity, as determined by an in-house reporter assay, had decreased serum levels of serotonin (p=0.0008) as well as increased IDO activity (p<0.0001), as determined by the kynurenine/tryptophan ratio measured by liquid chromatography. Furthermore, SLE sera induced IDO expression in WISH cells in a type I IFN-dependent manner (p=0.008). Also platelet activation contributed to reduce overall availability of serotonin levels in platelets and serum (p<0.05). Decreased serum serotonin levels were associated with severe SLE with presence of anti-dsDNA antibodies and nephritis. In all, reduced serum serotonin levels in SLE patients were related to severe disease phenotype, including nephritis, suggesting involvement of important immunopathological processes. Further, our data suggest that type I IFNs, present in SLE sera, are able to up-regulate IDO expression, which may lead to decreased serum serotonin levels.     

Kynurenine Pathway Measurements in Huntington''s Disease Striatum: Evidence for Reduced Formation of Kynurenic Acid

Recent evidence suggests that there may be overactivation of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16-sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p less than 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.     

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.     

Factors controlling the selection of tissue-specific metabolic pathways in early embryonic cells

Rana pipiens embryos from the mid-blastula to the early gastrula stage were dissociated into cell cultures, and incubated with ¹⁴C-labeled tryptophan. The uptake of the tryptophan by the cells, its incorporation into protein and its metabolism by enzymes of the serotonin and kynurenine pathways were measured as a function of time, tryptophan concentration, and embryonic stage. It was found that the intracellular concentration of tryptophan was a constant fraction of the extracellular level except for a brief period around stage , during which the cells accumulated the amino acid to a higher concentration than in the external medium. The dominant metabolic pathway of tryptophan was a function of the intracellular concentration; at the lowest levels reported here most of the tryptophan was metabolized via the kynurenine pathway; at the highest levels most was metabolized via the serotonin pathway.     

Tryptophan catabolism via kynurenine production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>: identification of three genes coding for the enzymes of tryptophan to anthranilate pathway

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.     

Serotonin synthesis and its light-dark variation in the rat retina

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.     

Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux cycle

Mammalian cells rely on cellular uptake of the essential amino acid tryptophan. Tryptophan sequestration by up-regulation of the key enzyme for tryptophan degradation, indoleamine 2,3-dioxygenase (IDO), e.g., in cancer and inflammation, is thought to suppress the immune response via T cell starvation. Additionally, the excreted tryptophan catabolites (kynurenines) induce apoptosis of lymphocytes. Whereas tryptophan transport systems have been identified, the molecular nature of kynurenine export remains unknown. To measure cytosolic tryptophan steady-state levels and flux in real time, we developed genetically encoded fluorescence resonance energy transfer nanosensors (FLIPW). The transport properties detected by FLIPW in KB cells, a human oral cancer cell line, and COS-7 cells implicate LAT1, a transporter that is present in proliferative tissues like cancer, in tryptophan uptake. Importantly, we found that this transport system mediates tryptophan/kynurenine exchange. The tryptophan influx/kynurenine efflux cycle couples tryptophan starvation to elevation of kynurenine serum levels, providing a two-pronged induction of apoptosis in neighboring cells. The strict coupling protects cells that overproduce IDO from kynurenine accumulation. Consequently, this mechanism may contribute to immunosuppression involved in autoimmunity and tumor immune escape.     

Differential effects of short and long term lithium on tryptophan uptake and serotonergic function in cat brain

The effcts of short and long term lithium treatment on tryptophan uptake and on tissue levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) were studied in twelve brain regions of the cat. Tryptophan uptake and 5-HIAA were significantly correlated in control cats. Short term treatment caused parallel increases or decreases in tryptophan uptake and 5-HIAA. Long term treatment consistently increased tryptophan uptake without corresponding changes in 5-HIAA. Relatively low cumulative doses of lithium may reduce the degree to which tryptophan uptake is a limiting factor in the the regulation of serotonin synthesis.     

Tryptophan degradation in patients infected by human immunodeficiency virus

Tryptophan and kynurenine were measured retrospectively in sera of 11 male patients with advanced human immunodeficiency virus (HIV) infection (Walter Reed stages 4 and 6). Tryptophan levels are significantly reduced to less than 50% in patients with HIV infection and kynurenine levels significantly elevated when compared to sex and age matched controls. The decrease of tryptophan levels might contribute to neurologic symptoms often associated with HIV infection. Since interferon-gamma induces degradation of tryptophan via the kynurenine pathway, the present results may be consistent with enhanced endogenous production of interferon-gamma in advanced HIV infection.     

Developmental role of tryptophan hydroxylase in the nervous system

The serotonin 5-hydroxytryptamine (5-HT) neurotransmitter system contributes to various physiological and pathological conditions. 5-HT is the first neurotransmitter for which a developmental role was suspected. Tryptophan hydroxylase (TPH) catalyzes the rate-limiting reaction in the biosynthesis of 5-HT. Both TPH1 and TPH2 have tryptophan hydroxylating activity. TPH2 is abundant in the brain, whereas TPH1 is mainly expressed in the pineal gland and the periphery. However, TPH1 was found to be expressed predominantly during the late developmental stage in the brain. Recent advances have shed light on the kinetic properties of each TPH isoform. TPH1 showed greater affinity for tryptophan and stronger enzymic activity than TPH2 under conditions reflecting those in the developing brain stem. Transient alterations in 5-HT homeostasis during development modify the fine wiring of brain connections and cause permanent changes to adult behavior. An increasing body of evidence suggests the involvement of developmental brain disturbances in psychiatric disorders. These findings have revived a long-standing interest in the developmental role of 5-HT-related molecules. This article summarizes our understanding of the kinetics and possible neuronal functions of each TPH during development and in the adult.     

Determination of tryptophan and several of its metabolites in physiological samples by reversed-phase liquid chromatography with electrochemical detection

A new method for the concurrent assay of three tryptophan metabolites at the picomole level is described. The method has been developed for blood, urine, cerebrospinal fluid, and tissue samples such as whole brain, brain parts, and endocrine glands. Tryptophan itself, serotonin, and 5-hydroxyindoleacetic acid are isolated initially on extraction columns, eluted with a suitable solvent, and injected onto a liquid chromatograph with an amperometric detector. This general approach may be applicable to a variety of other tryptophan metabolites and should be useful in both research and clinical investigations.     

Tryptophan Uptake and Hydroxylation in Rat Forebrain Synaptosomes

Tryptophan uptake, hydroxylation, and decarboxylation in isolated synaptosomes were studied to assess how their properties may determine the rate of serotonin synthesis in the presynaptic nerve terminals of the brain. Simultaneous measurements of the rates of uptake, hydroxylation, and decarboxylation in the presence and absence of various inhibitors showed that tryptophan hydroxylase is rate-limiting for serotonin synthesis in this model system. There was significant direct decarboxylation of tryptophan to tryptamine. Measurement of tryptophan hydroxylase flux with varying internal concentrations of tryptophan allowed the determination of the Km of tryptophan hydroxylase in synaptosomes for tryptophan of 120 +/- 15 microM. Depolarisation of synaptosomes with veratridine caused both a reduction in the internal tryptophan concentration and an apparent activation of tryptophan hydroxylase. This activation did not occur in the absence of Ca2+ or in the presence of trifluoperazine. Synaptosomal serotonin synthesis and brain stem-soluble tryptophan hydroxylase were inhibited by low concentrations of noradrenaline or dopamine. Dibutyryl cyclic AMP, glucagon, insulin, and vasopressin were observed to have no effect on tryptophan uptake or hydroxylation in synaptosomes.     

Systemic metabolism of tryptophan and its catabolites,kynurenine and 3-HAA,in mice with inflammatory arthritis

Tryptophan is an essential amino acid. The liver is primary organ involved the oxidative catabolism of tryptophan. However, in the immune system, tryptophan and its catabolites, kynurenine and 3-hydroxyanthranilic acid (3-HAA), play an anti-inflammatory role. Rheumatoid arthritis (RA) is an autoimmune disease. Collagen induced arthritis (CIA) is an animal model of RA. Therefore, it was of interest to measure concentration of tryptophan, kynurenine and 3-HAA in mice with CIA. Concentration of tryptophan and 3-HAA was measured with HPLC methods. Concentration of kynurenine was measured with colorimetric test. mRNA expression for the kynurenine pathway genes was assessed using qRT-PCR. It has been found that in sera from diseased mice concentration of tryptophan was not changed. Concentration of kynurenine and 3-HAA was decreased. Moreover, in the livers from mice with CIA, concentration of tryptophan and kynurenine was decreased. These observations coincided with decreased mRNA expression for Ido2 and Afm and increased mRNA expression for Kynureninase in the liver. It has been also shown that in CIA the concentration of 3-HAA was increased in the kidneys.