Tesi sul tema "Kynurenine - Metabolism"

A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

BACKGROUND: Acute pancreatitis (AP) is a sterile initiator of systemic inflammation that can trigger multiple organ dysfunction syndrome (MODS). In the acute phase of AP, the kynurenine pathway of tryptophan metabolism plays an important role in the genesis of AP-MODS in experimental animal models, but it is unknown whether the pathway is activated in human AP. Human data are required to support the rationale for kynurenine 3- monooxygenase (KMO) inhibition as a treatment for AP-MODS and reinforce the translational potential. Additionally, as respiratory dysfunction is frequent in severe AP, the role of lung ultrasonography in severity stratification deserves investigation. Furthermore, the effect of AP-MODS on long-term survival is unknown. OBJECTIVES: My objectives were to: 1) Define the temporal and quantitative relationship of kynurenine metabolites with the onset and severity of APMODS, 2) Investigate the value of lung ultrasonography in the early diagnosis of respiratory dysfunction in human AP-MODS, and 3) Examine whether early AP-MODS impacts on long-term survival. METHODS: 1) A prospective, observational, clinical experimental medicine study titled “Inflammation, Metabolism, and Organ Failure in Acute Pancreatitis” (IMOFAP) was performed. For 90 days, consecutive patients with a potential diagnosis of AP were recruited and venous blood was sampled at 0, 3, 6, 12, 24, 48, 72 and 168 hours post-recruitment. Kynurenine metabolite concentrations were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and analysed in the context of clinical data, disease severity indices, and cytokine profiles. 2) In a nested cohort within IMOFAP, 41 participants underwent lung ultrasonography to evaluate whether this imaging modality can detect respiratory dysfunction in AP. 3) Survival data for a prospectively maintained database of patients with AP was analysed after accounting for in-hospital deaths. RESULTS: 1) During the IMOFAP study, 79 patients were recruited with an elevated serum amylase, of which 57 patients met the diagnostic criteria for AP; 9 had severe disease. Temporal profiling revealed early tryptophan depletion and contemporaneous elevation of plasma concentrations of 3- hydroxykynurenine, which paralleled systemic inflammation and AP severity. 2) Lung ultrasonography findings correlated with respiratory dysfunction. 3) 694 patients were followed up for a median of 8.8 years. AP-MODS conferred a deleterious effect on overall survival which persisted after the exclusion of inhospital deaths (10.0 years, 95% C.I. = 9.4-10.6 years) compared to AP without MODS (11.6 years, 95% C.I. = 11.2-11.9 years; P = 0.001). This effect was independent of age. CONCLUSIONS: In the acute phase of AP, metabolic flux through KMO is elevated and proportionate to AP severity. Lung ultrasonography may be a useful technique for evaluating AP-MODS. AP-MODS is an independent predictor of long-term mortality. Together, this work reinforces the rationale for investigating early phase KMO inhibition as a therapeutic strategy in humans.

Kynurenine 3-monooxygenase (K3MO) lies on the kynurenine pathway, the major pathway for the catabolism of L-tryptophan. It converts kynurenine to 3-hydroxy kynurenine. Inhibition of K3MO is important in several neurological diseases and there is evidence that inhibition of K3MO could also be targeted for the prevention of multiple organ failure, secondary to acute pancreatitis. A structure activity relationship based upon the 1,2,4-oxadiazoles motif was carried out which revealed amide 207 as an inhibitor of P. fluorescens K3MO. Further structure activity relationships were developed based upon 207. This revealed 3,4-dichloro substitution in 235 and 245 as optimum for inhibition. Co-crystalisation of these inhibitors with P. fluorescens K3MO revealed their interactions with the enzyme. It also highlighted new, potential interactions between the inhibitors and K3MO. This led to the synthesis of 271 and 272, which were also potent inhibitors of K3MO. These amides were successfully co-crystalised with P. fluorescens K3MO. Further development of the amides followed, with amide 282 providing the most potent inhibitor of P. fluorescens K3MO to date (Kᵢ = 29.1 nM).

Prenatal exposure to maternal infection has been thought as a major risk factor for neurodevelopmental brain damage and thus contributes to the pathophysiology of neurodegenerative diseases including schizophrenia and autism. The mechanisms of aberrant neurodevelopmental processes on the offspring, in which primary cerebral insults occur during early brain development, are not fully understood. In the present investigation, maternal infection was modelled in timed-pregnant rats at embryonic day (E) 14, 16 and 18 by administering intraperitoneal injections of polyriboinosinic-polyribocytidilic acid,poly(I:C), a viral mimetic double stranded RNA complex which activates Toll-Like-Receptor-3 (TLR-3). The aim was to examine the impact of maternal inflammatory response on the regulation of expression of neurodevelopmental proteins that play important roles in many neurodevelopment aspects, including maintenance of synaptic plasticity, intracellular signalling and neurogenesis which may be relevant in cognitive and behavioural functions. An examination of embryo brains 5 h after maternal poly(I:C) showed significant differences in expression of the NMDA receptor NR2 subunits. The expression of NR2A subunits was reduced, whereas infection induced during pregnancy enhanced NR2B subunit expression. Expression levels of both subunits at postnatal day 21 (P21) were not affected by prenatal poly(I:C) exposure. In utero viral challenge led to significant changes among neurogenesis factor only at P21. In the fetal brain, acute poly(I:C) exposure had no effect on the expression of SHH, PCNA and also SOX2 proteins. However, when poly(I:C) was administered during mid and late gestation in the rodent model, long term effects of prenatal viral challenge on survival and maintenance of cell in the brain as indicated by the expression of SOX2 and SHH was clearly demonstrable. Expression of SOX2 level was increased,while SHH was significantly decreased, suggesting possible increase in the number of cells and changes in the rate of differentiation, respectively. The results demonstrate that poly(I:C) challenge in pregnant dams results in selective molecular changes in the brain, with transient alteration in the levels of NMDA receptor subunit NR2A and NR2B in the foetal brain, and also affecting molecules associated with cell genesis processes at later stages of developmental age of offspring. On the other hand, recent pharmacological interest in kynurenines with respect to CNS diseases has mainly focussed on two neuroactive molecules: quinolinic acid (QUIN) and kynurenic acid (KYNA). Manipulation of the kynurenine pathway and its neuroactive metabolites has been associated with N-methyl-D-aspartate (NMDA) receptor neurotoxicity and dysfunction which linked to the development of various neurological disorders. An early developmental event has been proposed to precipitate alterations in the NMDA receptor function. In this respect, early development during the gestational period of rats is most suitable for investigating the modulating effect of kynurenine pathway inhibition by compound Ro61-8048 (3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulphomide) an inhibitor of kynurenine-3-monooxygenase (KMO) in shifting the balance towards the production of neuroprotective, kynurenic acid. Western blots were generated to indicate the expression of a range of proteins relevant to different aspects of CNS development including neuritogenesis, axon guidance, maintenance of synaptic plasticity, intracellular signalling and cell proliferation and migration. Within 5 h of Ro61-8048, there was a significant decrease in NR2A expression and increased NR2B in the embryo brains, with subsequent changes in SHH and NFB at 24 h post treatment. The litters were left undisturbed until weaning on P21 and other groups were allowed to develop to P60, at which time they euthanized and the brains removed for analysis. At P21, western blot analysis revealed significantly increased protein expression of the NR2A and NR2B subunits and postsynaptic density protein (PSD95). Among several neurodevelopmental proteins, the expression of NFB and proliferating cell nuclear antigen (PCNA) was increased, while reduced level of SHH was detected. We demonstrate here persisting changes in NR2A expression, with reduced level in the hippocampus while a raised level was noted in the cortex suggesting prenatal modulation of kynurenine pathway causes long lasting modifications of NMDA receptor composition and function. It is important to note that kynurenine pathway inhibition can generate a consistent set of long term changes in the SHH in which the levels of this protein remained repressed in some regional areas of the brain including hippocampus, cerebellum and cortex. We show that there are some common pathways that are affected by kynurenine pathway inhibition, and this early modulation tends to disrupt critical molecular processes that are known to be actively occurring at each specific developmental time. Overall, given these selective and differing developmental profile, an early life modulation of the kynurenine pathway might be expected to cause a sufficient disturbance of biological processes that are actively occurring at the time of exposure and also able to leave a series of molecular changes that persist into adulthood. This disruption is likely to influence the resulting physiology of the adolescent and adult brain and subsequently can lead to impairments in social behaviour. It is hoped that this study provides a broad analysis of the long term molecular effects of developmental kynurenine metabolism, and that it allows for a viable opportunity of potential therapeutic targets for disease intervention.

A via das Quinureninas (Via Quin) representa a principal via catabólica do metabolismo do triptofano (Trp) e é essencial para diversos processos fisiológicos. No fígado, o Trp é catalisado por triptofano 2,3-dioxigenase (TDO) quinurenina (Quin). A mesma reação também pode ser catalisada pela enzima indolamina 2,3-dioxigenase (IDO), produzida por células imunológicas. Em alguns processos patológicos, há um aumento do consumo de Trp pela Via Quin, que gera compostos que estão relacionados ao processo de imunotolerância. No presente estudo, foram selecionados dois modelos que mimetizam situações associadas às alterações da resposta imunológica: a restrição de sono e a obesidade. A partir do conhecimento das alterações na resposta imune nessas condições, geramos a hipótese de que parte do mecanismo se dê a partir da indução do catabolismo de Trp pela via Quin. Desse modo, foram investigadas as concentrações séricas e hepáticas de Trp nesses modelos experimentais, modelos esses que foram utilizados em outros projetos do nosso grupo de pesquisa. Não houve diferença significativa na concentração de Quin sérica e hepática entre os camundongos C57BL/6J restritos de sono (3 hs/15 dias), privação de sono paradoxal (72 hs) e período rebote (24 hs). A razão Quin/Trp também não diferiu entre os grupos RS e controle. Igualmente não houve diferenças estatísticas na concentração de Quin plasmática nos modelos privação de sono paradoxal e período rebote realizados em ratos Wistar. O mesmo foi observado em camundongos Swiss e camundongos C57BL/6J submetidos a protocolos experimentais de obesidade: ração hiperlipídica (21 dias) e de síndrome metabólica (20 semanas de ração hiperlipídica). Tais resultados sugerem que as alterações na resposta imunológica nesses quadros não estão associadas ao catabolismo de Trp.
The Kynurenine pathway (Kyn pathway) is the major catabolic pathway of tryptophan metabolism (Trp) and it is essential for many physiological processes. In the liver, Trp is catalyzed by tryptophan 2,3-dioxygenase (TDO), producing kynurenine (Kyn). The same reaction can also be catalyzed by the enzyme indoleamine 2,3-dioxygenase (IDO), produced by immune cells. In some pathological conditions, there is a high Trp consumption by Kyn pathway, that generate compounds related to immune tolerance. In this study, we chose two models strongly associated with changes in the immune response: sleep restriction and obesity. From the knowledge that there are immune response alterations in those conditions, we generated the hypotesis that in part, those alterations are correlated with induction the Trp catabolism by Kyn pathway. Thus, serum and liver concentrations of Trp and Kyn were investigated in these experimental models that have been used in other projects of our research group. There was no significant difference in concentration of Kyn in serum and liver among mice C57BL/6J induced to restricted sleep (3 hours / 15 days), paradoxical sleep deprivation (72 hours) and rebound period (24 hours). The Kyn/Trp ratio did not differ between control group and RS group. Also there were no statistical differences in plasma concentration of Kyn in paradoxical sleep deprivation and rebound period models performed in rats Wistar. The same profile was also observed in Swiss e C57BL/6J mice subjected to experimental obesity protocols: fat diet (21 days) and metabolic syndrome (20 weeks of fat diet). These results suggest that changes in the immune response in the conditions tested above are not associated with Trp catabolism.

ABSTRACT: During inflammation, the kynurenine pathway (KP) metabolises the essential amino acid tryptophan (TRP) potentially contributing to excitotoxicity via the release of quinolinic acid (QUIN) and 3-hydroxykynurenine (3HK). Despite the importance of excitotoxicity in the development of secondary brain damage, investigations on the KP in TBI are scarce. In this study, we comprehensively characterised changes in KP activation by measuring numerous metabolites in cerebrospinal fluid (CSF) from TBI patients and assessing the expression of key KP enzymes in brain tissue from TBI victims. Acute QUIN levels were further correlated with outcome scores to explore its prognostic value in TBI recovery. METHODS: Twenty-eight patients with severe TBI (GCS ≤ 8, three patients had initial GCS = 9-10, but rapidly deteriorated to ≤8) were recruited. CSF was collected from admission to day 5 post-injury. TRP, kynurenine (KYN), kynurenic acid (KYNA), QUIN, anthranilic acid (AA) and 3-hydroxyanthranilic acid (3HAA) were measured in CSF. The Glasgow Outcome Scale Extended (GOSE) score was assessed at 6 months post-TBI. Post-mortem brains were obtained from the Australian Neurotrauma Tissue and Fluid Bank and used in qPCR for quantitating expression of KP enzymes (indoleamine 2,3-dioxygenase-1 (IDO1), kynurenase (KYNase), kynurenine amino transferase-II (KAT-II), kynurenine 3-monooxygenase (KMO), 3-hydroxyanthranilic acid oxygenase (3HAO) and quinolinic acid phosphoribosyl transferase (QPRTase) and IDO1 immunohistochemistry. RESULTS: In CSF, KYN, KYNA and QUIN were elevated whereas TRP, AA and 3HAA remained unchanged. The ratios of QUIN:KYN, QUIN:KYNA, KYNA:KYN and 3HAA:AA revealed that QUIN levels were significantly higher than KYN and KYNA, supporting increased neurotoxicity. Amplified IDO1 and KYNase mRNA expression was demonstrated on post-mortem brains, and enhanced IDO1 protein coincided with overt tissue damage. QUIN levels in CSF were significantly higher in patients with unfavourable outcome and inversely correlated with GOSE scores. CONCLUSION: TBI induced a striking activation of the KP pathway with sustained increase of QUIN. The exceeding production of QUIN together with increased IDO1 activation and mRNA expression in brain-injured areas suggests that TBI selectively induces a robust stimulation of the neurotoxic branch of the KP pathway. QUIN's detrimental roles are supported by its association to adverse outcome potentially becoming an early prognostic factor post-TBI.

Le sarcome de Ewing est la seconde tumeur pédiatrique de l’os la plus fréquente. Elle est caractérisée par une translocation chromosomique résultant à la fusion de EWSR1 avec un membre de la famille ETS. Chez 85% des patients, cette fusion conduit à l’expression de la protéine chimérique EWS-FLI1 qui est l’oncogène majeur de ce sarcome. Ce dernier agit principalement par son action transcriptionelle sur des cibles qui lui sont propres. Au niveau thérapeutique, le sarcome d’Ewing est traité par chimiothérapie, chirurgie locale et par radiothérapie. La survie à long terme des patients est de l’ordre de 70%, mais beaucoup plus basse pour les patients métastatiques et quasi nulle lors d’une récidive. Parmi maintes caractéristiques, certains cancers présentent une dérégulation énergétique. L’influence d’EWS-FLI1 sur cet aspect n’a fait l’objet d’aucune étude dans le contexte du sarcome d’Ewing. Nous avons donc étudié par profilage métabolomique des cellules de sarcome d’Ewing en présence ou en absence d’EWS-FLI1. En comparant ces deux conditions, des modulations du profil énergétique relatif au cycle de Krebs, des précurseurs de le glycosylation ainsi que des métabolites de la voie de la méthionine et du tryptophane ont été observés. En parallèle, grâce à un crible de banque de shRNAs réalisé dans des conditions expérimentales similaires à l’étude métabolomique (lignée d’Ewing avec ou sans EWS-FLI1), nous avons pu identifier des gènes présentant des caractéristiques « synthétique létales », c'est-à-dire tuant uniquement les cellules du sarcome d’Ewing en présence de son oncogène
Ewing sarcoma, the second most commonly occurring pediatric bone tumor, is most often characterized by a chromosomal translocation between EWSR1 and FLI1. The gene fusion EWS-FLI1 accounts for 85% of all Ewing sarcoma and is considered the major oncogene and master regulator of Ewing sarcoma. EWS-FLI1 is a transcriptional modulator of targets, both directly and indirectly. Ewing sarcoma is aggressively treated with chemotherapy, localized surgery and radiation and has an overall survival of about 70%, however, survival for metastasis or relapsed cases remains low. One of the cancer hallmarks, metabolic deregulation, is most likely partly dependent on EWS-FLI1 in Ewing sarcoma cells. In order to get a better understanding of Ewing sarcoma biology and oncogenesis, it might be of high interest to investigate the influence of EWS-FLI1 in Ewing sarcoma cells. We therefore performed a global metabolic profiling of Ewing sarcoma cells with or without inhibition of EWS-FLI1. Several changes in the energy metabolism were observed throughout this study; the observed changes were consistent with an energy profile that moved from a cancer cell energy metabolism towards the energy metabolism of a more normal cell upon EWS-FLI1 inhibition, primarily based on the TCA cycle. Levels of TCA intermediates, glycosylation precursors, methionine pathway metabolites and amino acids, especially changes in the tryptophan metabolic pathway, were altered upon EWS-FLI1 inhibition. Parallel to this study, we performed a high-throughput synthetic lethality screen, in order to not only identify essential genes for cell survival and proliferation, but also to identify new synthetic lethal targets that could specifically target Ewing sarcoma cells carrying the EWS-FLI1 fusion gene

Amino acid availability plays an important role in modulating the activity of T-cells. One of the pathways employed by T-cells to sense nutrient levels is the “mammalian target of rapamycin” (mTOR) pathway that is inhibited in response to nutrient depletion. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme along the tryptophan catabolising kynurenine pathway. T-cells are very sensitive to lack of this essential amino acid in their microenvironment and this confers strong immunomodulatory properties to cells expressing active IDO. It therefore has a significant physiological role as a homeostatic mechanism used in mammalian organisms to dampen excessive activation of the immune system but is also used as an immune evasion mechanism by many cancers. In this study, we investigated the IDO inhibitory properties and mechanism of action of the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) that potentially forms a negative feedback loop in the kynurenine pathway. We studied the molecule in enzymatic assays, in live cells and discovered that it inhibits IDO in an indirect way via the formation of hydrogen peroxide. Secondly, we looked at the effects of tryptophan and its metabolites on T-cell proliferation and mTOR activity, and discovered a metabolite that inhibits T-cell proliferation. Lastly we examined mechanisms of T-cell suppression employed by myeloid derived suppressor cells (MDSCs), focusing on their ability to deplete amino acids from their microenvironment. We were able to exclude tryptophan consumption as a suppressive mechanism and established that by manipulating extracellular concentrations of several amino acids other than arginine and cysteine – that are known to be utilised by MDSCs - we were able to reduce their inhibitory properties. In summary, we have described in detail how 3-HAA inhibits IDO in in vitro assays, outlined how some tryptophan metabolites can inhibit T-cell proliferation, and clarified aspects of suppressive mechanism employed by MDSCs.

"A thesis submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy".
Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007.
Bibliography: p. 243-277.
Introduction -- A convenient synthesis of 30HKG -- Facile synthesis of the UV filter compounds 30HKyn and AHBG -- Synthesis, identification and quantification of novel human lens metabolites -- Modification of bovine lens protein with UV filters and related metabolites -- Effect of UV light on UV filter-treated lens proteins -- Conclusions and future directions.
The kynurenine-based UV filters are unstable under physiological conditions and undergo side chain deamination, resulting in α,β-unsaturated carbonyl compounds. These compounds can react with free or protein bound nucleophiles in the lens via Michael addition. The key sites of the UV filters kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) modification in human lenses include cysteine (Cys), and to a lesser extent, lysine (Lys) and histidine (His) residues. Recent in vivo studies have revealed that 3-hydroxykynurenine-O-β-D-glucoside (3OHKG) binds to Cys residues of lens crystallins in older normal human lenses. As a result of this binding, human lens proteins become progressively modified by UV filters in an age-dependent manner, contributing to changes that occur with the development of age-related nuclear (ARN) cataract. Upon exposure to UV light, free UV filters are poor photosensitisers, however the role of protein-bound species is less clear. It has been recently demonstrated that Kyn, when bound to lens proteins, becomes more susceptible to photo-oxidation by UV light. Therefore, the investigation of 3OHKG binding to lens proteins, and the effect of UV light on proteins modified with 3OHKG and 3OHKyn, were major aims of this study. As a result of the role of these compounds as UV filters and their possible involvement in ARN cataract formation, it is crucial to understand the nature, concentration and modes of action of the UV filters and their metabolites present in the human lenses. Therefore, an additional aim was to investigate human lenses for the presence of novel kynurenine-based human lens metabolites and examine their reactivity.--As 3OHKG is not commercially available, to conduct protein binding studies, an initial aim of this study was to synthesise 3OHKG (Chapter 2). Through the expansion and optimisation of a literature procedure, 3OHKG was successfully synthesised using commercially available and inexpensive reagents, and applying green chemistry principles, where toxic and corrosive reagents were replaced with benign reagents and solvent-free and microwave chemistry was used. A detailed investigation of different reaction conditions was also conducted, resulting in either the improvement of reaction yields or reaction time compared to the literature method. Applying the same synthetic strategy, and using key precursors from the synthesis of 3OHKG, the UV filters 3OHKyn and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG), were also successfully synthesised (Chapter 3).
Chapter 4 describes the investigation of both normal and cataractous human lenses in an attempt to identify novel human lens metabolites derived from deaminated Kyn and 3OHKyn (Chapter 4, Part A). Initially, 4-(2-aminophenyl)-4-oxobutanoic acid (AHA), glutathionyl-kynurenine (GSH-Kyn), kynurenine yellow (Kyn yellow), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid (AHB), glutathionyl-3-hydroxykynurenine (GSH-3OHKyn) and 3-hydroxykynurenine yellow (3OHKyn yellow) were synthesised and human lenses were examined for their presence. AHA and AHB were synthesised from similar precursors to those used in the synthesis of 3OHKG, while the GSH adducts and yellow compounds were synthesised from Kyn and 3OHKyn via base induced deamination. Following isolation and structural elucidation, AHA, AHB and GSH-Kyn were confirmed as novel human lens metabolites. They were quantified in low pmol/mg lens (dry mass) levels in normal and cataractous lenses of all ages, while GSH-3OHKyn, Kyn yellow and 3OHKyn yellow were not detected. In contrast to AHA, the lens metabolites AHB, GSH-Kyn and GSH-3OHKyn were found to be unstable at physiological pH. The spectral properties of these compounds suggest that they may act as UV filters. --Chapter 4 (Part B) also describes the identification and characterisation of a novel human lens UV filter, cysteinyl-3-hydroxykynurenine -O-β-D-glucoside (Cys-3OHKG). An authentic standard was synthesised via Michael addition of cysteine to deaminated 3OHKG. Cys-3OHKG was detected in low pmol/mg lens (dry mass) levels in normal lenses only after the 5th decade of life and was absent in cataractous lenses. Cys-3OHKG showed rapid decomposition at physiological pH.
Chapter 5 describes the identification and quantification of amino acids involved in covalent binding of 3OHKG to lens proteins. Model studies with bovine lens proteins and 3OHKG at pH 7.2 and 9.5 were undertaken. The amino acid adducts were identified via total synthesis and spectral analysis, and subsequently quantified upon acid hydrolysis of the modified lens proteins. Under both pH conditions, 3OHKG was found to react with lens proteins predominantly via Cys residues with low levels of binding also detected at Lys residues. Comparative studies with Kyn (pH 9.5) and 3OHKyn (pH 7.2 and 9.5) resulted in modified lens proteins at Cys residues, with only minor modification at Lys residues at pH 9.5. The extent of modification was found to be significantly higher at pH 9.5 in all cases. His adducts were not identified. 3OHKG-, Kyn- and 3OHKyn-modified lens proteins were found to be coloured and fluorescent, resembling those of aged and ARN cataractous lenses. In contrast, AHB and AHA, which can not form α,β-unsaturated carbonyl compounds, resulted in non-covalent modification of lens proteins. AHB may contribute to lens colouration and fluorescence as further reactions of this material yielded species that have similar characteristics to those identified from 3OHKyn modification. These species are postulated to arise via auto-oxidation of the o-aminophenol moiety present in both 3OHKyn and AHB.--In Chapter 6, the potential roles of 3OHKG and 3OHKyn, and the related species AHA and AHB, in generating reactive oxygen species and protein damage following illumination with UV light was examined. The UV filter compounds were examined in both their free and protein-bound forms. Kyn-modified proteins were used as a positive control. Exposure of these compounds to UV light (λ 305-385 nm) has been shown to generate H2O2 and protein-bound peroxides in a time-dependent manner, with shorter wavelengths generating more peroxides. The yields of peroxides were observed to be highly dependent on the nature of the UV filter compound and whether these species were free or protein bound, with much higher levels being detected with the bound species. Thus, protein-bound 3OHKyn yielded higher levels of peroxide than 3OHKG, with these levels, in turn, higher than for the free UV filter compounds. AHB-treated lens proteins resulted in formation of low but statistically significant levels of peroxides, while AHA-treated lens proteins resulted in insignificant peroxide formation. The consequences of these photochemical reactions have been examined by quantifying protein-bound tyrosine oxidation products (3,4-dihydroxyphenylalanine [DOPA], di-tyrosine [di-Tyr]) and protein cross-linking. 3OHKG-modified proteins gave elevated levels of di-Tyr, but not DOPA, whereas 3OHKyn-modified protein gave the inverse. DOPA formation was observed to be independent of illumination and most likely arose via o-aminophenol auto-oxidation. AHB- and AHA-treated lens proteins resulted in statistically insignificant di-Tyr formation, while a light independent increase in DOPA was observed for both samples. Both reducible (disulfide) and non-reducible cross-links were detected in modified proteins following illumination. These linkages were present at lower levels in modified, but non-illuminated proteins, and absent from unmodified protein samples.
This work has provided an optimised synthetic procedure for 3OHKG and other lens metabolites (Chapters 2 and 3). Four novel lens metabolites have been identified and quantified in normal and cataractous human lenses (Chapter 4). Subsequent experiments, described in Chapter 5, identified the major covalent binding sites of 3OHKG to lens proteins, while AHA and AHB showed non-covalent binding. Further work described in Chapter 6 showed that protein-bound 3OHKG, Kyn and 3OHKyn were better photosensitisers of oxidative damage than in their unbound state. Together, this research has provided strong evidence that post-translational modifications of lens proteins by kynurenine-based metabolites and their interaction with UV light appear, at least in part, responsible for the age-dependent colouration of human lenses and an elevated level of oxidative stress in older lenses. These processes may contribute to the progression of ARN cataract.
Mode of access: World Wide Web.
xxxix, 308 p. ill. (some col.)

Le métabolisme du tryptophane (Trp) a été investigué dans différentes pathologies, incluant les maladies cardiovasculaires, la cancérologie et les maladies neurodégénératives. Le Trp, acide aminé essentiel, est catabolisé en périphérie et au niveau central selon 2 voies : celle des kynurénines, quantitativement majoritaire et impliquée dans les cardiopathies et la tolérance immune, et celle de la sérotonine, connue pour son implication dans la dépression et le sommeil. Dans le cadre de ce travail, nous nous sommes intéressés à l’implication de cette voie métabolique dans la phénylcétonurie (PCU) et les maladies cardiovasculaires.Dans la première partie de ce travail, nous avons mis au point une technique analytique de dosage de 9 métabolites de la voie du Trp, incluant ses 2 voies cataboliques. La méthode proposée, en HPLC-MS/MS, est fiable, reproductible, peu coûteuse, avec un processus pré-analytique simple et une durée d’analyse de 15 min. Ses critères sont conformes aux exigences du laboratoire selon les recommandations de la norme NF EN ISO 15189.Dans la deuxième partie, nous nous sommes intéressés au métabolisme du Trp dans la phénylcétonurie (PCU ; OMIM 261600). Cette maladie, due à un déficit de la phénylalanine hydroxylase hépatique (PAH ; EC 1.14.16.1), entraine une accumulation de phénylalanine (Phe) associée à un déficit en tyrosine. Cette pathologie, causant principalement un profond retard mental (QI < 50), est traitable grâce à l’instauration le plus précocement possible d’un régime restreint en Phe.Plusieurs éléments rapprochent le métabolisme du Trp de celui de la Phe, que ce soient les transporteurs, cofacteurs et enzyme chaperone communs, ou les inhibitions de l’une des voies par des métabolites de l’autre voie. De plus, certains adultes atteints de PCU présentent des dysfonctions neuropsychologiques inexpliquées par leur concentration de Phe sanguine, ce qui pourrait être dû à des modifications de la voie métabolique du Trp, plusieurs kynurénines possédant des propriétés neuroactives.Nous avons mis en évidence une modification du métabolisme du Trp dans une population phénylcétonurique adulte française (n=151 patients), avec en particulier une diminution des concentrations sanguines de kynurénine (KYN) et d’acide 3-hydroxykynurénique (3HK) par rapport à notre population de référence (p-value < 0.0001), ces résultats étant modulés par le type de régime suivi.La troisième partie de notre travail a consisté en l’évaluation du métabolisme du Trp dans le contexte des maladies cardiovasculaires. La prévention de ces pathologies est un enjeu de santé publique majeur, et se base sur la maitrise des facteurs de risque, dont le syndrome d’apnées obstructives du sommeil (SAOS) qui touche 6 à 17% des adultes à travers le monde. Ce syndrome se caractérise par un collapsus pharyngé partiel ou total, une fragmentation du sommeil et une augmentation des efforts respiratoires et donc des séquences d’hypoxie – réoxygénations (hypoxie intermittente) responsables d’une inflammation de bas grade. Plusieurs études explorant les maladies cardiovasculaires et le métabolisme du Trp ont été publiées récemment, mais très peu apportaient un lien mécanistique. Nous présentons ici les prochaines études cliniques et pré-cliniques que nous souhaitons mener dans ce domaine, afin de caractériser les modifications du métabolisme du Trp dans le SAOS traité ou non, mais également lors de l’hypoxie intermittente (modèle animal du SAOS) ou lors d’un réentrainement à l’effort.Il est à présent évident que la voie métabolique du Trp est impliquée dans différentes conditions pathologiques. Nous avons mis en évidence lors de ce travail ses modifications dans la PCU. L’impact physiopathologique des modifications observées reste cependant à explorer dans de nombreuses pathologies
Tryptophan (Trp) metabolism was investigated in various pathology, including cardiovascular diseases, cancerology and neurodegenerative diseases. Trp, an essential aminoacid, is catabolized into two major pathways: kynurenine (KYN) pathway represents 98% of Trp catabolism and is involved in cardiopathy and immune tolerance, as serotonin (5HT) pathway is implicated in depression and sleep. In this work, we have studied Trp pathway in phenylketonuria (PKU) and cardiovascular diseases.Firstly, we developed a simple method for quantification of Trp and 8 of its metabolites, involved in both KYN and 5HT pathways, using liquid chromatography coupled to tandem mass spectrometry. This method, with a quick chromatographic runtime (15 min) and simple sample preparation, has been validated according to NF EN ISO 15189 criteria.Secondly, we explored Trp metabolism in phenylketonuria (OMIM 261600), a pathology caused by deficiency of phenylalanine hydroxylase enzyme (EC 1.14.16.1), that catalyses hydroxylation of phenylalanine (Phe) to tyrosine. Early low-Phe diet treatment, results in the prevention of severe mental retardation (IQ < 50) seen in untreated PKU patients.Many similarities between Trp and Phe metabolisms exist: transporters, cofactor and chaperone enzyme, and inhibition of one pathway by metabolites of the other pathway. As some of these metabolites have neuroactive properties, they should be considered in neurological impairment seen in this pathology and not totally explained by blood Phe concentrations.We assessed here a change of Trp metabolism in 151 adult PKU patients, with diminution of plasmatic concentrations of KYN and 3-hydroxykynurenic acid (3HK) in PKU patients compared to general population (P < .0001). These modifications were modified by diet type.In third line, we evaluated Trp metabolism in cardiovascular diseases. Prevention of these diseases representes a serious public health issue, based on diminution of co-morbidities like obstructive sleep apnoea (OSA). This syndrome affects 6-17% of adults worldwide, and is characterized by complete or partial pharyngeal collapse, sleep fragmentation and increased respiratory efforts, resulting in intermittent hypoxia and low-grade inflammation. Recent studies have explored Trp metabolism in cardiovascular diseases, but mainly in epidemiologic studies. We highlighted here clinical and preclinical studies that we will conducted in this field, in the way to characterized Trp modification in treated or untreated OSA, intermittent hypoxia (animal model of OSA) and exercise training.Trp metabolism is clearly involved in various pathological conditions. In this work, we have highlighted its modification in PKU. Physiopathological impact of these modifications have to been explored in various pathologies

Bien que l’effet psychotrope des antidépresseurs soit bien connu, afin de corriger les conséquences du stress et de renforcer la confiance en soi, de nombreux autres effets pharmacologiques, notamment périphériques, doivent encore être approfondis. Les antidépresseurs inhibiteurs de la recapture de la sérotonine (ISRS) peuvent avoir un effet bénéfique sur la performance physique en participant à une réparation et à une croissance plus rapides des muscles. Il a récemment été démontré que la sérotonine était impliquée dans la récupération de la force musculaire chez un modèle murin de myopathie de Duchenne (Gurel et al., 2015). Les antidépresseurs tels que les inhibiteurs sélectifs de la recapture de la sérotonine (ISRS) sont largement utilisés pour traiter divers troubles de la santé mentale, tels que la dépression modérée à sévère et l’anxiété. Les deux symptômes contribuent à l’insomnie, à la perte d’appétit, au manque de motivation et à une fatigue physique accrue. Ces symptômes peuvent nuire aux performances physiques des athlètes, en particulier de ceux qui développent des habiletés et des techniques spécifiques à un sport, reçoivent des volumes d’entraînement plus importants à différentes intensités et participent à des compétitions plus fréquentes. Par conséquent, les athlètes peuvent utiliser des médicaments qui renforcent la motivation et / ou améliorent la condition physique générale en réduisant les symptômes dépressifs. L'utilisation d'antidépresseurs n'est pas encore interdite dans les sports d'élite. Des rapports récents sur le dopage associé aux ISRS montrent une tendance croissante de son utilisation chez les athlètes en bonne santé. La consommation d'antidépresseurs chez les athlètes a augmenté dans différents sports au cours de la dernière décennie, notamment les sports d'endurance.. Notre projet doit donc permettre de caractériser les conséquences d'un traitement chronique par ISRS sur les performances physiques chez la souris et de mettre en évidence le ou les mécanismes impliqués, en particulier la variation du shunt métabolique sérotonine / kynurénine, ainsi que les modifications de biomarqueurs, variations potentiellement utilisables chez l'homme dans la lutte contre le dopage.Nous aimerions élucider notre travail de recherche dans les articles suivants:Article 1: Nous avons étudié les effets de l'exercice et de la fluoxétine seuls ou en association avec un traitement prolongé à la fluoxétine (18 mg / kg / jour) et un exercice physique d'endurance (six semaines) chez la souris mâle BalbC / j, sur tapis roulant. Nous avons ensuite évalué l'activité neurocomportementale, les marqueurs musculaires du stress oxydatif et les modifications du métabolisme du tryptophane dans les tissus plasmatiques, musculaires et cérébraux des souris BalbC / J. En général, nous nous sommes concentrés sur la vitesse aérobie la plus élevée, le temps d’endurance jusqu’à l’épuisement, la force musculaire des membres antérieurs en saisissant un mesureur de force, des tests neurocomportementaux tels que le test en champ ouvert et élevé et le labyrinthe, l’activité enzymatique mitochondriale (activité du citrate synthase et du cytochrome C oxydase) dans le muscle gastrocnémien. , marqueur de stress oxydant tel que le test DHE (Dihydroéthidium) et DCF-DA (Dichlorofluorscine diacétate).Article 2: Nous avons étudié les effets de l’exercice et de la fluoxétine seule ou les effets combinés d’un traitement prolongé à la fluoxétine (18 mg / kg / jour) et d’un exercice d’endurance physique (six semaines) chez la souris mâle BalbC / j, sur tapis roulant
As much as the psychotropic effect of antidepressants is well known, correcting the consequences of stress and boosting self-confidence, so many other pharmacological effects, peripheral in particular, remain to be deepened. Serotonin reuptake inhibitor antidepressants (SSRIs) may have a beneficial effect on physical performance by participating in faster muscle repair and growth. It has recently been shown that serotonin was involved in the recovery of muscle strength in a mouse model of Duchenne myopathy (Gurel et al., 2015).Antidepressants such as selective serotonin reuptake inhibitors (SSRIs) are widely used to treat various mental health disorders, such as moderate-to-severe depression and anxiety. Both symptoms contribute to insomnia, loss of appetite, lack of motivation and increased physical fatigue. These symptoms can impair physical performances for athletes, more specifically for those who develop sport-specific skills and techniques, receive higher training volumes at various intensities, and participate in more frequent competitions. Therefore athletes may use drugs that enhance motivation and/or improve overall fitness by reducing depressive symptoms. The use of antidepressants is not yet forbidden in elite sports. Recent reports on doping associated with SSRIs show an increasing trend of its usage among healthy athletes. The antidepressants intake among athletes has increased in different sports over the last decade, especially endurance sports. The antidepressants Bupropion and Amineptine were removed from the list of banned substances.Our project must therefore make it possible to characterize the consequences of chronic treatment with SSRIs on the physical performance in mice and to highlight the mechanism (s) involved, in particular the variation of the serotonin / kynurenine metabolic shunt, as well as the modifications of biomarkers, potentially usable variations in humans in the fight against doping.We would like to elucidate our research work in the following articles:Article 1: We studied the effects of exercise and fluoxetine alone or in combination of long-term fluoxetine treatment (18mg/kg/day) and endurance physical exercise (six weeks) in male balbC/j mice, on animal treadmill. Subsequently we evaluated neurobehavioral activity, muscle markers of oxidative stress, and changes in tryptophan metabolism in plasma, muscle and brain tissues in the BalbC/J mice. Generally we focused on the highest aerobic velocity, endurance time until exhaustion, forelimb muscle strength by gripping strength meter, neurobehavioral tests such as open field and elevated plus maze test, mitochondrial enzyme activity (Citrate synthase and cytochrome-C oxidase activity) in gastrocnemius muscle, oxidative stress marker such as DHE (Dihydroethidium) and DCF-DA (Dichlorofluorscine di-acetate)test.Article 2: We studied the effects of exercise and fluoxetine alone or combinative effects of long-term fluoxetine treatment (18mg/kg/day) and endurance physical exercise (six weeks) in male balbC/j mice, on animal treadmill. After the mentioned exercise protocol we focused on changes in tryptophan (TRP) metabolism in plasma, muscle and brain tissues in the BalbC/J mice. To confirm the metabolomic, we also studied the KP related enzyme related genes and proteins by the modern required materials and methods. We correlated the result of article1 with the metabolites level of kynurenine pathway of tryptophan metabolism. We studied the expression of transcriptor factor PGC1α level in muscle which is induced by physical exercise(Agudelo et al., 2014). PGC1α subsequently induce the expression of kynurenine aminotransferase 1 and 2 (KAT1 and KAT2) in skeletal muscles, which convert kynurenine (KYN) to kynurenic acid (KYNA). Conversion of kynurenine to kynurenic acid decrease the level of kynurenine and quinolinic acid an NMDA receptor agonist and a neurotoxic compound

Early life experiences play a key role in brain function and behaviour. Maternal separation produced negative early life experiences that induce emotional alterations. Contrary, the communal nest has been proposed as a protective model that may reduce the vulnerability of individuals to suffer psychiatric disorders. Moreover, early-life stress enhances the vulnerability to develop substance use disorders, principally during adolescence. Hence, depressive states are associated with drug use disorders and abuse vulnerability since depressed patients could consume drugs to alleviate their symptoms. Our results lead us to propose that detrimental early life events such as maternal neglect reproduces most of the behavioural and neurochemical alterations associated with emotional disorders in mice. In addition, maternal separation could be useful to study the comorbidity between depression and substance use disorder since induces alterations in emotional and cocaine addictive behaviours as well as in the dopaminergic system. However, in our conditions, we cannot confirm the protective role of communal nest due to the behavioural alterations found in these mice, probably because this breeding condition involves energetic cost and increases the competition for food in pups.
Las experiencias tempranas tienen un papel importante en el desarrollo cerebral y las respuestas emocionales. La separación maternal supone un factor de riesgo de desarrollar enfermedades psiquiátricas, mientras que el modelo de nido en comuna se ha propuesto como una condición protectora. Además, el estrés crónico durante la infancia aumenta la vulnerabilidad a desarrollar trastornos por uso de sustancias, principalmente durante la adolescencia. Por ello, los estados depresivos están asociados con una mayor vulnerabilidad para el uso de drogas puesto que los pacientes depresivos podrían consumir drogas para aliviar sus síntomas. Nuestros resultados demuestran que las experiencias adversas durante la infancia reproducen la mayoría de las alteraciones comportamentales y neuroquímicas relacionadas con depresión en el ratón. Además, la separación maternal puede considerarse como un modelo conveniente para el estudio de la comorbilidad psiquiátrica entre depresión y el trastorno por uso de drogas, ya que reproduce alteraciones emocionales y motivacionales en el animal de experimentación, así como en el sistema dopaminérgico. Sin embargo, no podemos confirmar el papel protector del nido en comuna debido a las alteraciones comportamentales que hemos encontrado en nuestros estudios, probablemente debido a que dicho modelo origina costes energéticos y aumenta la competencia por la comida entre las crías.

Triptofano (TRP), um amino ácido essencial, é metabolizado por duas vias principais, a via das quinureninas e a via serotonérgica. Em ambas as vias há a possibilidade de formação de compostos ativos no sistema imune que se caracterizam pelas ações imunossupressoras e indutoras de tolerância. Na via serotonérgica há a formação de serotonina (5-HT) e em alguns tecidos de melatonina (MEL). Este composto pode ainda ser oxidado por ação de peroxidases aos seus produtos de abertura de anel indólico o AFMK (N1-acetil-n2-formil-5-metoxiquinuramina) e AMK (N1-acetil-5-metoxiquinuramina). Já na via das quinureninas, o TRP é diretamente metabolizado à N-formilquinurenina (NFK) e este é rapidamente deformilado a quinurenina (QUIN). Neste projeto avaliamos qual o efeito do choque endotóxico induzido por injeção endovenosa de LPS (1 mg/kg) sobre a biodisponibilidade de TRP e formação de seu metabólito QUIN. Este estudo foi realizado em condições controle e na vigência de sobrecarga de TRP (administração subcutânea de 0,8 mg/kg). Utilizamos ratos machos Wistar com 30 dias separados em quatro grupos: GI (controle), GII (LPS), GIII (TRP) e GIV (TRP+LPS). TRP (0,8 mg/Kg) foi injetado por via subcutânea nos tempos 0 e 2 horas. Quando injetado, LPS (1 mg/kg) foi administrado por via intravenosa no tempo 2 horas. Após 1 hora da última administração, sangue e cérebro foram coletados. O cérebro foi seccionado em três regiões: cerebelo, córtex e mesencéfalo, os quais foram processados para obtenção de homogenatos. Tanto os homogenatos quanto o soro foram tratados com acetona para extração de TRP e seus metabólitos. A análise destes compostos foi realizada por cromatografia líquida de alta eficiência (HPLC). A administração de TRP elevou significativamente a sua concentração no soro e no SNC. Quando da administração de LPS no grupo que já havia recebido sobrecarga de TRP (GIV) houve uma marcada elevação de TRP e de QUIN séricos e das regiões do SNC, especialmente na região do córtex. Concluímos que na vigência de choque endotóxico há um aumento da biodisponibilidade de TRP, tanto no soro como no SNC e que há um aumento da metabolização deste pela rota das quinureninas, possivelmente via IDO. Estes resultados contribuem para a compreensão da toxicidade de TRP, especialmente relevante no caso em que haja um choque endotóxico concomitante e evidencia o córtex como uma região mais susceptível para os efeitos tóxicos do TRP.
Tryptophan (TRP) is an essential amino acid, metabolized by two main paths; the kynurenine and the serotonergic pathways. In both, there is the possibility of generation of biologic active compounds, especially on the immune system leading to immunosuppression and tolerance. In the serotonergic path there is the formation of serotonine (5-HT) and in some tissues of melatonine (MEL). The latter can be oxidized by the action of peroxidases to its indole ring opening product AFMK (N1-acetil-n2-formil-5-methoxikynuramine) and AMK (N1-acethyl-5-methoxykynuramine). In the kynurenine path, TRP is metabolized to N-formylkynurenine (NFK) that is deformilated to kynurenine (KYN). In this study we evaluated the effect of a endotoxic skock induced by an intravenous injection of LPS (1 mg/kg) on the bioavailability of TRP and formation of KYN. This study was carried out in control conditions and on TRP overload (subcutaneous administration of 0,8 mg/Kg). One month old male Wistar rats were divide in four groups: GI(control), GII(LPS), GIII(TRP) and GIV (TRP+LPS). TRP (0,8 mg/kg) was subcutaneously injected at zero and 2h times. When injected, LPS (1mg/kg) was intravenously administered at 2 h. After one hour from the last administration, blood and brain were collected. Brain is separated in cerebellum, midbrain and cortex and was lysed for the preparation of homogenates. Both, serum and homogenates were extracted in acetone; TRP and KYN were analyzed by HPLC. TRP overload caused a significant increase in its concentration in serum and brain. When LPS was administered in conjunction with TRP overload (GIV) there was a remarkable increase in TRP and KYN in serum and brain, especially in cortex. Our conclusion is that in the bioavailability of TRP, in serum and in brain, and its metabolization to kynurenine is increased by inflammation. IDO is probably involved in this condition. Our results contribute to the knowledge of TRP toxicity, particularly with a concomitant inflammation and demonstrate the cortex as a region of more susceptibility to TRP toxicity.

The essential amino acid tryptophan is an important substrate for the synthesis of serotonin, melatonin, tryptamine, proteins and the kynurenines. The aim of this study was to investigate tryptophan metabolism along the kynurenine pathway in a low income sub- Saharan HIV/AIDS patient population from the Gauteng Province of South Africa. The first objective was to develop and validate a novel gas chromatography mass spectrometry method to enable reliable quantification of tryptophan and metabolites of the kynurenine pathway in plasma. Validation parameters for the detection of tryptophan, kynurenine, quinolinic acid and nicotinamide conformed to international criteria for newly developed methods. The next objective of the study was to find an appropriate biomarker against which to express the results. Several substances previously described as indicators were assessed and compared, including plasma neopterin, procalcitonin, C-reactive protein, the cytokines IL-2, IL-4, IL-6, IL-10, TNF, and IFN-gamma, as well as factors routinely measured and elsewhere described as biomarkers in HIV, i.e., albumin, the albumin/globulin ratio, haemoglobin and red cell distribution width. Neopterin was shown to be superior as indicator of pro-inflammatory status, as indicator of the degree of immune deficiency, to predict disease progression, to distinguish between patients with and without tuberculosis co-infection and to reflect the success of highly active antiretroviral treatment (HAART). In the analyses of the kynurenine pathway metabolites, tryptophan levels were seen to be significantly lower (24.36 ± 4.14 vs. 43.57 ± 11.85 μmol/l; p<0.0001), while the activity of the enzyme, indoleamine 2,3 dioxygenase (IDO), (K/T:136.03 vs. 52.18; p<0.001), as well as kynurenine (3.21 ± 1.33 vs. 2.14 ± 0.45 μmol/l; p<0.001) and quinolinic acid (4.46 ± 2.32 vs. 0.25 ± 0.058 μmol/l; p<0.001) levels were significantly higher in the total patient group (n=105) than in the control group (n=60). Patients on HAART showed not only significantly higher CD4 counts (296.21 ± 195.50 vs. 170.05 ± 167.26 cells/μl; p=0.003), but also lower inflammatory activity (neopterin: 35.51 ± 35.70 vs. 66.63 ± 40.73 nmol/l; p<0.001 and IL-6: 9.56 ± 12.54 vs. 15.04 ± 19.34 pg/ml; p<0.05), lower IFN-γ (41.43 ± 14.14 vs. 53.68±34.39 pg/ml; p<0.05), higher tryptophan levels (25.13 ± 3.80 vs. 22.04 ± 4.32 μmol/l; p=0.033), lower kynurenine levels (3.08 ± 1.28 vs. 3.58 ± 1.42 μmol/l; p=0.144) and lower quinolinic acid levels (4.03 ± 2.04 vs. 5.77 ± 2.65μmol/l; p=0.072) than patients not on HAART. Tryptophan depletion and IDO activity, as well as the levels of kynurenine and quinolinic acid, were generally greater than in populations from developed countries. Indications are that this can be ascribed to higher levels of inflammatory activity at comparable levels of immune deficiency in the disadvantaged population of this study. The degree of tryptophan depletion and quinolinic acid accumulation found could negatively impact on the physical and neuropsychiatric wellness of the population. Correlations between quinolinic acid, and nicotinamide levels showed a significant contribution of kynurenine pathway metabolism to the plasma levels of nicotinamide. This de novo synthesis of nicotinamide could offer protection against niacin deficiency and NAD depletion in populations with inadequate dietary intake. This is the first study to assess plasma tryptophan, kynurenine, quinolinic acid and nicotinamide levels, as well as IDO activity, pro-inflammatory status and IFN-γ levels, simultaneously in one population and to compare it to that of HIV/AIDS patients in developed countries.
Thesis (PhD)--University of Pretoria, 2015.
tm2015
Physiology
PhD
Unrestricted