Les erreurs innées du métabolisme sont des maladies héréditaires pouvant affecter, entre autres, la synthèse et le transport des neurotransmetteurs. Le diagnostic de ces pathologies repose essentiellement sur l’analyse par chromatographie d’un fluide biologique [plasma, urine, liquide céphalorachidien (LCR)]. L’objectif de cette thèse était de développer des méthodes simples et rapides pour le diagnostique des troubles de la neurotransmission. Dans un premier temps, nous avons développé et validé le dosage simultané, en moins de 10 minutes, des métabolites de la dopamine, de la sérotonine et de la tétrahydrobioptérine (BH4) par chromatographie liquide ultra-haute pression (UHPLC) couplée à une détection séquentielle électrochimique et fluorimétrique. Cette méthode a été appliquée à l’analyse de 1372 échantillons de LCR, permettant ainsi d’établir les valeurs fréquentes de la population française. Afin de transposer la méthode précédente en UHPLC couplée à la spectrométrie de masse (MS), nous avons étudié les mécanismes d’auto- et d’électro- oxydation de la BH4, par mobilité ionique différentielle couplée à la MS haute résolution (résonance cyclonique ionique FTICR) et à la photodissociation Infra-rouge. Ce travail nous a permis d’isoler et de caractériser pour la première fois la qBH2, intermédiaire réactionnel fugace de la BH4, impliqué dans le mécanisme d’action de cette dernière. La méthode UHPLC-MS/MS développée, permet en outre le dosage simultané du 5-Méthyl-tétrahydrofolate. / Inborn errors of metabolism are inherited diseases that can alter the synthesis and transport of neurotransmitters. The diagnosis of these conditions is currently based on the chromatographic analysis of a biological fluid [plasma, urine, cerebrospinal fluid (CSF)]. The aim of this thesis was to develop simple and rapid methods for the diagnosis of neurotransmitter disorders. Firstly, we developed and validated the simultaneous determination of dopamine, serotonin, and tetrahydrobiopterin (BH4) metabolites by ultrahigh pressure liquid chromatography (UHPLC) coupled to sequential electrochemical and fluorimetric detection. This method was applied to the analysis of 1372 CSF samples, thus establishing the frequent ranges of the French population. In order to transpose the previous method into UHPLC coupled to mass spectrometry (MS), we studied the mechanisms of auto- and electro- oxidation of BH4, by Differential Ion Mobility coupled to high resolution MS (FTICR) in conjunction with Infra-Red photo-dissociation. This work allowed us to isolate and characterize qBH2, the transient reaction intermediate of BH4, involved in the mechanism of action of the latter. The proposed UHPLC-MS/MS method also allows the simultaneous determination of 5-methyltetrahydrofolate.

Inhibition of nitric oxide (NO) signalling may contribute to the pathogenesis of severe malaria. This thesis examines the impact of Plasmodium infection on three key determinants of nitric oxide synthase (NOS) biochemistry: substrate availability, substrate/inhibitor homeostasis and cofactor availability. Arginine, the NOS substrate, is depleted in human patients with severe Plasmodium falciparum malaria and mice infected with P. berghei ANKA. Using heavy isotope tracer infusions to quantify arginine metabolism in infected mice, we found no evidence of increased catabolism by the enzyme arginase, widely assumed to be responsible for arginine depletion. Genetic knock-out of parasite arginase had no effect on arginine depletion in mice. Instead, our findings link arginine depletion to decreased rates of arginine and citrulline appearance in the plasma of infected mice. Asymmetric dimethylarginine (ADMA) competes with arginine for binding to the NOS catalytic site. We observed elevation of the ADMA/arginine ratio in Gambian children with severe malaria, favouring NOS inhibition. In mice infected with P. berghei ANKA, we found evidence of degradation of dimethylarginine dimethylaminohydrolase 1 (DDAH1), the enzyme primarily responsible for ADMA metabolism. We also observed reduced DDAH activity and accumulation of intracellular ADMA in hepatic tissue of infected mice, suggesting that DDAH dysfunction could contribute to disruption of ADMA/arginine homeostasis. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. In P. berghei ANKA-infected mice, BH4 concentrations were decreased in plasma, erythrocytes and brain tissue, which could inhibit NO synthesis and promote NOS-dependent superoxide production. To reverse deficiencies of NOS substrate and cofactor availability, we infused P. berghei ANKA-infected mice with citrulline, an arginine precursor, and sepiapterin, a BH4 precursor. Restoration of systemic arginine and BH4 availability in infected mice improved whole blood nitrite concentrations, a biomarker of NO synthesis, but did not prevent onset of disease symptoms. These studies have identified biochemical disturbances that may contribute to severe malaria pathogenesis by inhibiting NO synthesis.

Les erreurs innées du métabolisme (EIM) constituent un large panel de désordres métaboliques héréditaires. Parmi les EIM, les anomalies de la neurotransmission peuvent affecter, entre autres, la synthèse ou le transport des neurotransmetteurs, notamment les amines biogènes (dopamine et sérotonine) et les folates. La reconnaissance de ces affections est d’une importance capitale pour le diagnostic et le traitement éventuel. L’analyse chimique du liquide céphalo-rachidien (LCR) est incontournable pour le diagnostic de ces pathologies. Or, les méthodes actuelles de dosage ne sont pas simples. Longues et fastidieuses, elles ont été, pendant longtemps, réservées aux laboratoires spécialisés. L’objectif de ce travail était de développer des méthodes simples et rapides de diagnostic des troubles de la neurotransmission et d’établir les valeurs normales fréquentes dans la population française. Le travail réalisé a permis, dans un premier temps, de développer une méthode de dosage directe, en une seule étape, de la tetrahydrobioptérine (BH4), cofacteur des hydroxylases intervenant dans la synthèse des amines biogènes, et des ptérines impliquées dans le cycle de synthèse et de dégradation de ce cofacteur, dans le LCR. Auparavant, le dosage de ces substances nécessitait au moins deux analyses chromatographiques précédées chacune d’une étape propre de préparation de l’échantillon. Par la suite, nous avons développé une méthode de diagnostic rapide, en moins de 10 minutes, par UHPLC (chromatographie liquide à ultra haute performance), couplée à une détection séquentielle par coulométrie et par fluorescence, des troubles dopaminergique et sérotoninergique. Cette méthode permet de doser simultanément, en une seule étape, tous les métabolites de la dopamine, de la sérotonine et de la noradrénaline, ainsi que les ptérines d’intérêt diagnostic, principalement la dihydroneoptérine (NH2) et la dihydrobioptérine (BH2). L’ensemble de ces explorations nécessitait, auparavant, la mise en œuvre d’au moins trois méthodes de dosage par HPLC (chromatographie liquide à haute performance), précédées chacune d’une étape propre de préparation de l’échantillon. Pour compléter l’exploration du métabolisme de la BH4 et du suivi thérapeutique des troubles de la neurotransmission, nous avons également proposé une méthode de dosage rapide, en une seule étape, par UHPLC, de tous les métabolites et de toutes les ptérines, incluant la BH4. Enfin, une méthode rapide de dosage (moins de 2 minutes), par UHPLC, du 5-méthyltetrahydrofolate dans le LCR a été développée, afin de compléter le diagnostic biologique de l’ensemble des troubles neurologiques visés. L’application des outils ainsi développés à plus de 1400 patients nous a permis d’établir des valeurs normales fréquentes dans la population française ainsi que de poser le diagnostic de quelques déficits enzymatiques. / Inborn errors of metabolism (IEM) consist of a wide range of hereditary metabolic disorders. Among IEM, neurotransmission anomaly can affect the synthesis or the transport of neurotransmitters, notably biogenic amines (dopamine and serotonin) and folates. Early diagnosis of such affections is of utmost importance especially as some of them can be treated effectively. Chemical analysis of cerebrospinal fluid (CSF) is essential for the diagnosis of neurotransmitter disorders; however, current quantitative methods are tedious and time consuming. For a long time the chemical diagnosis of neurotransmitter disorders has been available only in specialized laboratories. The purpose of this work was to develop simple and fast diagnosis methods of neurotransmitter disorders as well as to establish the reference values in French population. For this purpose, in a first step, we developed a single step direct method of simultaneous quantification of tetrahydrobiopterin (BH4), which is the main cofactor of the hydroxylases involved in biogenic amines syntheses, and the relevant reduced and oxidized forms of pterins involved in the cycle of synthesis – regeneration of BH4. Formerly, the quantification of those compounds required at least two chromatographic methods with two specific sample preparation procedures. Thereafter we developed a method of fast diagnosis in less than 10 minutes of dopaminergic and serotoninergic disorders using UHPLC (ultra high performance liquid chromatography) hyphenated to a sequential coulometric and fluorimetric detection. With only a simple filtration step as sample preparation procedure, this method enables the simultaneous quantification of all dopamine, serotonin and noradrenaline metabolites as well as dihydroneopterin (NH2) and dihydrobiopterin (BH2), the relevant pterin forms for the complete diagnosis. Formerly, at least three HPLC (high performance liquid chromatography) quantification methods preceeded by three tedious specific sample preparation procedures were required for such a diagnosis. To complete the investigation of BH4 metabolism and the follow up of neurotransmission disorders, we also developed a fast UHPLC method of simultaneous quantification of all the cited metabolites and pterins including BH4. In order to complete the rapid diagnosis of all targeted neurological disorders, we finally developed an UHPLC method of 5-methyltetrahydrofolate quantification in CSF. The application of these analytical tools in more than 1400 CSF samples, collected from patients followed in some Neurology centers located in several French areas covering nearly the entirety of the territory, allowed us to establish the reference values in French population as well as to diagnose several cases of enzymatic deficits.

The traditional treatment for phenylketonuria (PKU) is a phenylalanine (Phe)-restricted diet, supplemented with a Phe-free/low-Phe protein substitute. Pharmaceutical treatment with synthetic tetrahydrobiopterin (BH4), an enzyme cofactor, allows a patient subgroup to relax their diet. However, dietary protocols guiding the adjustments of protein equivalent intake from protein substitute with BH4 treatment are lacking. We systematically reviewed protein substitute usage with long-term BH4 therapy. Electronic databases were searched for articles published between January 2000 and March 2020. Eighteen studies (306 PKU patients) were eligible. Meta-analyses demonstrated a significant increase in Phe and natural protein intakes and a significant decrease in protein equivalent intake from protein substitute with cofactor therapy. Protein substitute could be discontinued in 51% of responsive patients, but was still required in 49%, despite improvement in Phe tolerance. Normal growth was maintained, but micronutrient deficiency was observed with BH4 treatment. A systematic protocol to increase natural protein intake while reducing protein substitute dose should be followed to ensure protein and micronutrient requirements are met and sustained. We propose recommendations to guide healthcare professionals when adjusting dietary prescriptions of PKU patients on BH4. Studies investigating new therapeutic options in PKU should systematically collect data on protein substitute and natural protein intakes, as well as other nutritional factors.

Le monoxyde d'azote est exclusivement synthétisé chez les mammifères par une famille d’hémoprotéines, les NO-Synthases. Le cœur de l’activité des NO-Synthases est l’activation de l’oxygène c'est-à-dire l’activation de l’intermédiaire réactionnel FeIIO2. Cette étape est contrôlée par la réactivité intrinsèque du fer, par les transferts de proton et les transferts d’électron. Elle doit être parfaitement maîtrisée car elle contrôle le chemin catalytique emprunté et la nature du produit final. Comprendre l’étape d’activation de l’oxygène est essentiel à la compréhension du rôle biologique et/ou pathologique de la NO-Synthase de mammifère. Cette question s'étend aux NO-Synthases bactériennes pour lesquelles on ne connait ni le mécanisme moléculaire ni la fonction biologique. Ce manuscrit propose une analyse approfondie de l’étape d’activation de l’oxygène de la NO-Synthase. Dans un premier temps, nous avons étudié l’influence de l’environnement proximal sur la réactivité intrinsèque du fer et l’activation de l’oxygène. Nous avons généré des protéines mutées qui modifient les propriétés électroniques de la liaison proximale de l’hème. Ces protéines mutées ont été caractérisées par différentes spectroscopies (résonance paramagnétique électronique, Raman de résonance). Dans un second temps nous avons directement étudié le complexe FeIIO2, en présence d’analogues de substrat, grâce à des analyses de cinétique rapide en flux continu et en flux arrêté (stopped-flow). Dans un troisième temps, le rôle du cofacteur tetrahydrobioptérine dans le transfert de proton et d’électron a été étudié par une méthode de piégeage à des temps très courts : le freeze-quench. L'ensemble de nos résultats montrent que l’activation de l’oxygène est régulée par les propriétés électro-donneuses du ligand proximal et par le réseau de liaisons H distal. Nous mettons en évidence des différences dans le rôle redox du cofacteur tetrahydrobioptérine entre la NO-Synthase de mammifère et la NO-Synthase bactérienne. La difficulté majeure pour comprendre l’étape d’activation de l’oxygène de la NO-Synthase réside dans la complexité et la rapidité de la réaction catalytique. Dans ce contexte, nous avons cherché à adapter une méthodologie qui a prouvé son efficacité dans le cas des cytochromes P450 : la cryo-réduction couplée à des sauts en température. / Nitric oxide is exclusively synthesized by NO-Synthases in mammals. The heart of the NO-synthase activity is oxygen activation, which corresponds to the activation of the FeIIO2 intermediate. This step depends on the heme electronic properties and on the electron and proton transfers. Oxygen activation has to be well mastered to control exactly the nature of the end-product. Understanding the oxygen activation step is necessary to better understand the biological/pathological role of the mammalian NO-Synthases. Furthermore, bacterial NO-Synthases function and oxygen activation mechanism are unknown. This PhD work proposes a deep analysis of the oxygen activation step in NO-Synthases. First, proximal environment has been studied with mutated proteins. These mutations impact the electronic properties of the heme proximal bond. Spectroscopic analyses of these mutants have been done by electron paramagnetic resonance and resonance Raman. Then, we have studied the FeIIO2 intermediate with substrate analogs which has necessitated continuous flow and stopped-flow analyses. Finally, the role of the tetrahydrobiopterin cofactor in the electron and proton transfer has been studied and clarified thanks to a very fast trapping method : the freeze-quench. Our results show that the oxygen activation step is elaborately controlled by the proximal bond electron donation and the distal H bond network. At the same time we show some differences between mammalian and bacterial NO-Synthases concerning the redox role of the tetrahydrobiopterin cofactor. The major obstacle to understand the oxygen activation step resides in the complexity of the active site chemistry and the rate of catalytic reactions. For this reason, we propose to adapt an already successful protocol to trap some intermediates in the cytochromes P450 mechanism : cryo-reduction coupled with temperature jumps.

Intense exercise is known to cause alterations in the psychophysiological status of an athlete. Monitoring the health and recovery of an athlete is imperative for the maintenance of performance and reduced fatigue and injury incidence. The physicality associated with select sports results in significant elevations and suppression of psychophysiological biomarkers that are often modulated by game-related impacts, intense training regimes and psychosocial aspects associated with the professional era. The aim of the studies outlined in this thesis were to determine the effectiveness of selected “stress” markers in several sports that result in significant “stress”, and quantify the level of acute and chronic “stress” following individual games and competitions to improve athlete management and recovery. Study one aimed at developing a new strong-cation exchange high performance liquid chromatography (SCX-HPLC) method for the detection and quantification of urinary pterins and creatinine in a body-building cohort completing high intensity resistance training. The method had an intra- and inter-assay variability of 3.04 % and 5.42 % respectively, with visibly clear peaks and no tailing. Urinary neopterin (NP) and 7,8-dihydroneopterin during a week of competitive natural body-building did not significantly change indicating no alteration in immune system function and oxidative stress. It did provide evidence for the use of specific gravity as a similarly reliable method for urine volume correction following exercise. Study two focused on a playoff game of elite amateur rugby. The time course changes of NP, cortisol, salivary immunoglobulin A (sIgA) and myoglobin in 11 elite amateur rugby players were measured up to 86 hours post-game. Cortisol increased 4-fold, myoglobin 2.85-fold, NP 1.75-fold and total NP 2.3-fold, all significant, whilst sIgA did not change. All markers returned to baseline within 17 hours providing valuable information for sample collection schedule optimization. Respiratory elastance was also measured by ventilation for the assessment of exercise induced lung inflammation/injury following the game (Chapter three). There was an increase in elastance in selected individuals that did not correlate with either global positioning system (GPS) or impact data. It was shown however, that a ventilator is capable of measuring respiratory changes in a conscious and healthy individual. Study three focused on the final three games of professional rugby in the 2013 ITM Cup. The acute and cumulative changes in the same four markers were analysed following three home games. There were significant increases in NP, total NP, cortisol and myoglobin along with significant suppression of sIgA (p < 0.05). Large intra- and inter-individual variation existed between players with changes associated with total impacts. Moreover, impact induced muscle damage may account for changes in oxidative status. Specific gravity (SG) was shown to be a more reliable marker for urine volume correction in comparison to creatinine; while some players showed signs of cumulative stress. Study four examined stress in a professional team throughout the 22 week 2014 Super 15 competition. Part one investigated changes in oxidative stress and muscle damage markers to solidify the muscle damage/oxidative status theory postulated in the previous study. Experimental evidence showed iron and myoglobin are separately capable of oxidizing 7,8-dihydroneopterin to NP in vitro. It was then identified that players who suffered the greatest muscle damage as a result of impacts also had the greatest change in oxidative status (NP). This evidence suggests rugby union induces significant alterations in oxidative status that may be exacerbated by the impact induced release of myoglobin. Part two measured urinary NT-proBNP during the last two consecutive home games to identify whether rugby union causes significant cardiovascular stress and if the pre to post-game change can be explained by GPS technology. Significant individualized elevations were observed in games one and two which did not correlate with any GPS measurements or impacts. Concentrations returned to normal ~ 36 hours post-game suggesting no permanent damage to cardiac muscle had occurred. The lack of correlation suggests GPS technology is not an accurate measure of cardiovascular stress in professional rugby union. Part three involved the measurement of cortisol, total NP and sIgA throughout the season to assess the degree of cumulative stress. Samples were taken at regular intervals ~ 36 hours post-game for 22 weeks. Extreme inter-individual variation was present. Select individuals showed continual elevation in immune system activation and psychophysiological stress, whilst others presented with a continual decline in immune system function. Collectively however, minor deviations from baseline in all markers were observed and participation in long distance travel did not significantly affect the psychophysiological status of the group. Together this suggests a season does not cause an accumulation in psychophysiological stress, although careful individual player analysis is warranted. Understanding rugby union positional demands is essential for training program specification and position specific development of players. Part four used GPS, video-analysis and biochemical analysis to identify positional demands in five regular season games. Forwards tended to be involved in more impacts and covered less distance, while backs covered more distance and carried the ball into contact more regularly. There was no difference in the psychophysiological status between positions indicating both aspects of stress (impacts and distance covered) may induce a similar response. Alternatively, individual biological variation may be solely responsible for this change suggesting careful consideration should be given when using traditional work-load measures such as GPS when quantifying “stress”. Part five assessed the effectiveness of varied recovery interventions. Total NP, cortisol, myoglobin and sIgA were measured pre- post- and ~ 36 hours post game to identify which intervention was most effective at returning players to a psychophysiological state that allowed for the resumption of normal training. Findings concluded the immediate post-game strategy employed by the team (cold bath, consumption of protein and carbohydrates, compression garments and eight hours sleep) seemed to provide the greatest psychophysiological improvement regardless of the “next-day” intervention. There was large inter-individual variation and players were still in a state of recovery ~ 36 hours post-game as indicated by the elevated total NP and sIgA concentrations. Study five had four aspects. Develop a new, cost-effective and simple reverse phase HPLC (RP-HPLC) method for the quantification of urinary myoglobin in a clinically relevant range, quantify the level of structural stress following a simulated mixed martial arts (MMA) contest, determine whether cold water immersion attenuates the level of inflammation and muscle damage following a contest, and whether this hypothesized attenuation may be explained by cryotherapy induced mononuclear cell activation suppression in vitro. The RP-HPLC method had an intra- and inter-assay variations from 0.32 - 2.94 %. Linearity was in the range of 5 – 1000 µg/mL which detected significant increases in urinary myoglobin following the MMA contest. Total NP was found to significantly increase following the contest and return to approximately pre-contest levels 24 hours later for the passive group only. Cold water immersion was further found to attenuate the total NP increase in the first two hours post-contest solidifying its use as a recovery technique following intense exercise, while cryotherapy significantly suppressed T-cell activation. This study provides a reliable and repeatable assay for muscle damage quantification in a clinically relevant range, evidence of the physicality associated with MMA, and indicates cold water immersion is a reliable recovery intervention that may impart its positive benefits through T-cell suppression. The data generated by these investigations highlights the necessity for individual physiological analysis. Group data often masks the extreme variation that exists in clinical and exercise trials where treatment and management of athletes is conducted for recovery and performance. Biochemical analysis provides an added sophistication of work-load and psychophysiological assessment that common technological methods cannot emulate. With a lack of correlation between the quantitative changes in specific non-overlapping biomarkers and GPS, video-analysis and questionnaires, it would seem pertinent to develop a non-invasive quantitative approach in elite sport to understand the level of exercise-induced psychophysiological stress for the precise management of athletes.