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Intensive care unit patients and chronic airway inflammatory disease are characterized by chronic systemic hypoxia and inflammation inducing a decrease in nitric oxide release due to impaired l-arginine (ARG) homeostasis. As ARG is synthesized from circulating l-citrulline (CIT), an alteration of CIT production in small intestine by ornithine carbamoyltransferase could be involved. Here, we posit that hypoxia and/or inflammation has effects on ornithine carbamoyltransferase regulation in enterocytes. A duodenal explant incubation model was used. Biopsy specimens taken from 25 selected patients were incubated for 6 h in 4 groups: control, inflammation, hypoxia, and hypoxia + inflammation. At the end of the incubation period, we measured CIT concentration in culture media, ornithine carbamoyltransferase activity, ornithine carbamoyltransferase protein and gene expression, protein expression of enzymes involved in the CIT production pathway, and expression of energy status proteins. Inflammation and/or hypoxia conditions did not affect CIT production. Ornithine carbamoyltransferase activity was increased in hypoxia conditions (p = 0.023). Expression of enzymes implicated in the CIT crossroads pathway and enzymes reflecting energy status variation was not affected by inflammation and hypoxia. Data sets were pooled to evaluate the variability of the four quartiles for each parameter. CIT production was found to increase over the quartiles whereas other parameters remained stable. Our results showed that intestinal CIT production is preserved during inflammation and/or hypoxia, thus confirming the significance of this metabolic pathway. This suggests that the CIT deficiency observed in clinical hypercatabolic states could be a consequence of high utilization for ARG synthesis.

Studies characterizing associations between phenols, phthalates and thyroid hormones during pregnancy produce inconsistent results. This divergence may be partly attributable to false positives due to multiple comparison testing of large numbers of chemicals, and measurement error as studies rely on small numbers of biospecimens despite high intra-individual variability in urinary chemical metabolite concentrations.This study employsA two-tiered approach was implemented:The ToxCast/Tox21 screening reduced the chemical set from 16 to 13 and the associated number of statistical comparisons by 19%. Parabens were negatively associated with free triiodothyronine (T3) and the T3/T4 (total thyroxine) ratio. Monobenzyl phthalate was positively associated with total T4 and negatively with the T3/T4 ratio. Effect modification by iodine status was detected for several compounds (among themFor these chemicals, screening for compounds with an increased likelihood for thyroid-related effects and relying on repeated urine samples to assess exposures improved the overall performance of multichemical analyses of thyroid disruption. This approach may improve future evaluations of human data for the thyroid pathway with implication for fetal health and may serve as a model for evaluating other toxicity outcomes. https://doi.org/10.1289/EHP10239.

Summary: Background & aims: The main role of the small intestine to absorb the bulk of dietary nutrients, including proteins and amino acids (AA). Enterocytes have long been considered a major player in amino acid homeostasis, especially the glutamine–citrulline–arginine crossroads. In vivo intestinal epithelium cell lines for studies of nitrogen metabolism have now reached their limitations. To push research forward, an interesting approach could be to use the explant incubation model of intestinal tissue, involving in vitro incubation of duodenal tissue biopsy specimens taken from human subjects. Methods: Eight duodenal biopsies were taken during endoscopy from patients without proven intestinal disease. Each biopsy was incubated in complete culture medium for 23 h then weighed, and cell viability was evaluated after 6 and 18 h. We then explored L-citrulline (L-CIT) production (reflecting metabolic activity) and the presence of the main enzymes involved in AA metabolism in the intestine (arginase II, glutaminase I, ornithine aminotransferase, and ornithine carbamoyltransferase). Results: Mean weight of biopsies was 12.36 ± 1.00 mg. Mortality was around 20% after 6 h and 50% after 18 h of incubation. CIT was amply produced by the biopsies, and enzymes implicated in intestinal AA metabolism were expressed in this model. Conclusions: This in vitro explant model of intestinal tissue emerges as a reliable model for conducting ex vivo investigations on AA metabolism. Keywords: Ornithine carbamoyltransferase, Amino acid, Enterocyte, Enzyme

International audience; Ornithine transcarbamylase (OTC; EC 2.1.3.3) is a ubiquitous enzyme found in almost all organisms, including vertebrates, microorganisms, and plants. Anabolic, mostly trimeric OTCs catalyze the production of L-citrulline from L-ornithine which is a part of the urea cycle. In eukaryotes, such OTC localizes to the mitochondrial matrix, partially bound to the mitochondrial inner membrane and part of channeling multi-enzyme assemblies. In mammals, mainly two organs express OTC: the liver, where it is an integral part of the urea cycle, and the intestine, where it synthesizes citrulline for export and plays a major role in amino acid homeostasis, particularly of L-glutamine and L-arginine. Here, we give an overview on OTC genes and proteins, their tissue distribution, regulation, and physiological function, emphasizing the importance of OTC and urea cycle enzymes for metabolic regulation in human health and disease. Finally, we summarize the current knowledge of OTC deficiency, a rare X-linked human genetic disorder, and its emerging role in various chronic pathologies.

Adult genetic disorders causing brain lesions have been mostly described as white matter vanishing diseases. We present here the investigations realized in patients referred for psychiatric disorder with magnetic resonance imaging showing atypical basal ganglia lesions. Genetic explorations of this family revealed a new hereditary disease linked to glutathione metabolism.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Amino acid chromatography allows the identification and quantification of more than forty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit amino acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Acylcarnitine profile allows the identification and quantification of more than thirty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit acylcarnitine profile. Validation parameters and recommendations are discussed in this specific framework.

Resume A l’heure ou la strategie de prevention est un axe majeur de la politique de Sante Publique et ou le depistage neonatal connait sa premiere reorganisation en France depuis 40 ans, il est interessant de visualiser ce programme national exhaustif avec le regard du biologiste, responsable de ces examens de biologie medicale (du pre au post-analytique) et confronte a la reglementation. Cette description de l’organisation du depistage sera rapidement caduque dans la mesure ou une reflexion est en cours avec la Haute Autorite de sante pour inclure de nouvelles pathologies dans le panel propose a chaque enfant.

Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive inborn error of metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1). If untreated, the disease leads to an important intellectual disability (IQ

Introduction et but de l’etude La relation entre systeme immunitaire et acides amines est connue depuis de nombreuses annees. Si l’apport proteique influence les fonctions immunitaires [1] , [2] il peut egalement favoriser le developpement des cellules cancereuses. Il est difficile de pouvoir predire quelle en sera la resultante. L’objectif de l’etude est donc d’evaluer les fonctions des cellules immunitaires dans un modele de rat cancereux sous chimiotherapie recevant des apports proteiques croissants [3] . Materiel et methodes Soixante-dix-huit rats femelles (Fisher 344) ont ete randomises en 6 groupes : un groupe controle (C) sain absolu (n = 8) et avec un regime standard (100 % des besoins proteiques) et 5 groupes avec cancer (tumeur du colon “Ward” injectee en sous cutane) a J-14, et 2 cycles de chimiotherapie (CPT-11 et 5-FU)(K) a J0/J1 et J7/J8, avec des regimes differents mais isocaloriques et des quantites croissantes en proteines (50 %, 75 %, 100 %, 150 %, 200 % des besoins en proteines) (n = 14 par groupe). A l’euthanasie (J9), les acides amines plasmatiques sont doses et le systeme immunitaire au niveau sanguin, splenique, des plaques de Peyer et de la tumeur sont analyses en cytometrie en flux. Le protocole a ete valide par le comite ethique (APAFIS#11492-2017112715178992). Resultats et analyse statistique Le cancer et la chimiotherapie induisent une perturbation des profils en acides amines, avec en particulier une baisse de l’argininemie (C = 146 μmol/L vs K = 125 μmol/L, p Tableau 1 ). Cependant les differents apports proteiques n’influencent ni la croissance tumorale, ni la biodisponibilite des acides amines ni le systeme immunitaire. (Analyses Statistiques : Shapiro test et ANOVA. En l’absence d’effet regime, les 5 groupes cancer + chimiotherapie ont ete pooles). Conclusion Ces premieres donnees suggerent que les variations d’apports proteiques sont sans consequences sur les fonctions immunitaires de rats cancereux sous chimiotherapie.

Many similarities between tryptophan (Trp) and phenylalanine (Phe) metabolisms exist. It is possible that a modification of Trp metabolism might be seen in phenylketonuria (PKU). As some of these metabolites have neuroactive properties, they should be consider in neurological impairment seen in this pathology and not totally explained by blood Phe concentrations. One hundred and fifty-one adult PKU patients (mean age 26.8 years) were included for this study. Plasma Trp, kynurenine (KYN), 3-hydroxykynurenic acid (3HK), and kynurenic acid (KA) were analyzed by liquid chromatography coupled with tandem mass spectrometry. KYN and 3HK were significantly lower in PKU patients compared to general population (P < .0001), and KA was significantly enhanced is this population (P = .009). Furthermore, 3HK concentration was significantly different between PKU patients underwent controlled low-Phe diet compared to PKU patients without this diet (P = .0016). In PKU patients with diet, taking AA substitute enable higher plasma 3HK concentration than without (P = .0008) but still not reaching general population level (P < .0001). Although further study has to be done, it is clear that Trp metabolism is modified in adult PKU patients. An exploration of complete Trp metabolism, and not only Trp concentration, is needed in PKU population, but also in other inborn error of metabolism treated with hypoprotidic diet.

Background: Exercise and citrulline (CIT) are both regulators of muscle protein metabolism. However, the combination of both has been under-studied yet may have synergistic effects on muscle metabolism and performance. Methods: Three-month-old healthy male rats were randomly assigned to be fed ad libitum for 4 weeks with either a citrulline-enriched diet (1 g·kg−1·day−1) (CIT) or an isonitrogenous standard diet (by addition of nonessential amino acid) (Ctrl) and trained (running on treadmill 5 days·week−1) (ex) or not. Maximal endurance activity and body composition were assessed, and muscle protein metabolism (protein synthesis, proteomic approach) and energy metabolism [energy expenditure, mitochondrial metabolism] were explored. Results: Body composition was affected by exercise but not by CIT supplementation. Endurance training was associated with a higher maximal endurance capacity than sedentary groups (P Conclusion: CIT supplementation and endurance training in healthy male rats modulates both muscle protein and energy metabolisms, with synergic effects on an array of parameters, including performance and protein synthesis.

Guanidinoacetate (GAA) et creatine (Cr) sont des marqueurs biochimiques des syndromes de deficit en Cr (SDC). Nous decrivons une methode de chromatographie liquide couplee a la spectrometrie de masse en tandem (LC/MSMS) pour la determination simultanee de GAA, Cr et creatinine (Crn). L’incorporation de Cr dans les fibroblastes pour la confirmation diagnostique du deficit en transporteur de la Cr est egalement etudiee. La separation chromatographique des trois composes butyles et l’utilisation d’etalons internes marques permet leur quantification par LC/MSMS en mode electrospray positif. La technique est lineaire de 0 a 600, 675 et 4 500 μmol/L et la limite de quantification est de 0,1 ; 0,04 et 0,9 μmol/L pour GAA, Cr et Crn respectivement. La repetabilite et la fidelite intermediaire presentent un coefficient de variation < 11 %. Les taux de recuperation des surcharges sont compris entre 83 et 109 %. Les valeurs de reference dans le liquide cephalorachidien de sujets ≥ 14 ans ont ete etablies pour GAA et Cr. Cinq lignees de fibroblastes ont ete utilisees pour l’etude de l’incorporation de Cr. Celle-ci augmente avec la concentration en Cr dans le milieu de culture et est diminuee par un inhibiteur du transporteur de la Cr (3-guanidinopropionate, 500 μmol/L) (96 h d’incubation ; [Cr dans le milieu] = 25 μmol/L ; p < 0,05). Le dosage de GAA, Cr et Crn par LC/MSMS pour le diagnostic de SDC propose est fiable et s’applique a plusieurs milieux biologiques. Nous renforcons egalement l’interet de l’etude d’incorporation de la Cr pour le diagnostic de deficits en transporteur de la Cr.

Introduction et but de l’etude Il est maintenant bien etabli que la citrulline (CIT) plasmatique est un excellent marqueur de la fonctionnalite intestinale, en particulier chez les patients de reanimation ou elle est generalement effondree [1] . Or, les mecanismes impliques ne sont pas connus. Nous proposons que l’hypoxie et l’inflammation observees dans ces situations pourraient impacter negativement la production de CIT. L’objectif de cette etude est donc de determiner, in vitro, si l’hypoxie et l’inflammation ont des effets sur la production de CIT par l’ornithine transcarbamylase (OTC). Ces effets ont ete evalues en utilisant un modele d’incubation d’explant duodenal humain [2] . Le protocole a ete approuve par le comite de protection des personnes du CHU de Grenoble. Materiel et methodes Huit biopsies duodenales ont ete prelevees chez des sujets apres information et obtention d’un consentement ecrit. Les biopsies ont ete incubees dans du milieu de culture complet pendant un temps court (6 h) ou long (18 h) selon quatre conditions : temoin, inflammation (melange de cytokines : IL-1β, 1 μg/L ; TNFα, 20 μg/L ; IFNγ, 10 μg/L), hypoxie (3 % d’O 2 ) et inflammation avec hypoxie. Apres mesure de la viabilite cellulaire, les taux de CIT liberee dans le milieu de culture pendant l’incubation ont ete doses par chromatographie d’echange d’ions. Le niveau d’activite de l’OTC a ete mesure par dosage enzymatique et l’expression proteique de cette enzyme par western blot . Resultats Les pourcentages de mort cellulaire a l’issue de la periode d’incubation ne differaient pas quelles que soient les conditions ( Tableau 1 ). La production intestinale de citrulline etait augmentee dans les conditions hypoxie et hypoxie avec inflammation apres 6 h d’incubation. Ces differences avaient disparu apres 18 h d’incubation. Cependant, cette augmentation de production de citrulline a 6 h ne s’accompagnait ni d’une modification de l’activite ni d’une modification de la quantite de proteine de l’OTC. Conclusion La production de citrulline par les enterocytes apparait augmentee en condition d’hypoxie avec ou sans inflammation sans que cela ne soit explique par une modification de l’activite enzymatique ou de l’expression proteique de l’OTC. Les mecanismes precis restent a determiner.

This work presents the performances of silicon micro-preconcentrators chips for breath sampling. The silicon chips were coupled to a handheld battery powered system for breath sampling and direct injection in a laboratory gas chromatography mass spectrometry system through thermal desorption (TD). Performances of micro-preconcentrators were first compared to commercial TD for benzene trapping. Similar chromatographic peaks after gas chromatographic separation were observed while the volume of sample needed was reduced by a factor of 5. Repeatability and day to day variability of the micro-preconcentrators were then studied for a 500 ppb synthetic model mixture injected three times a day four days in a row: 8% and 12% were measured respectively. Micro-preconcentrator to micro-preconcentrator variability was not significant compared to day to day variability. In addition, micro-preconcentrators were tested for breath samples collected in Tedlar® bags. Three analyses of the same breath sample displayed relative standard deviations values below 16% for eight of the ten most intense peaks. Finally, the performances of micro-preconcentrators for breath sampling on a single expiration were illustrated with the example of volatile tobacco markers tracking. The signals of three smoking markers in breath, benzene, 2,5-dimethylfuran, and toluene were studied. Concentrations of benzene and toluene were found to be 10 to 100 higher in the breath of smokers. 2,5-dimethylfuran was only found in the breath of smokers. The elimination kinetics of the markers were followed as well during 4 h: a fast decrease of the signal of the three markers in breath was observed 20 min after smoking in good agreement with what is described in the literature. Those results demonstrate the efficiency of silicon chips for breath sampling, compared to the state of the art techniques. Thanks to miniaturization and lower sample volumes needed, micro-preconcentrators could be in the future a key technology towards portable breath sampling and analysis.

Measurements of pyruvate and ketones bodies (acetoacetate and 3-hydroxybutyrate) are essential to the investigation of intermediary metabolism. Indeed, their blood levels reflect energy balance influenced by nutritional status. This balance can be disturbed in certain diseases such as diabetes and some inherited metabolic disorders. We have developed methods for assays on open automated biochemistry analyser, Konelab 20 XT (ThermoFischer, Whaltham USA), using kits marketed by Sobioda (Montbonnot, France) for pyruvate and Wako Chemicals GmbH (Neuss, Germany) for ketones, on deproteinised blood sample. We have validated the performance of these three quantitative methods using NF EN ISO 15189 (range B) standard criteria. We obtain satisfactory results concerning fidelity (precision measured as within and between batch CVs are respectively less than 7% and less than 6%), measuring ranges (from 7.7 to 228 μmol/L for pyruvate and from 22.6 to 650 μM for total ketone bodies), accuracy (10.4 μmol/L in physiological range for pyruvate and 7.1 μmol/L for 3-hydroxybutyrate) and comparing methods (versus manual assay with spectrophotometry on Uvikon XL). Establishment of reference ranges (35 to 74 μmol/L for pyruvate, less than 100 μM for 3-hydroxybutyrate and less than 44 μmol/L for acetoacetate) and reagents stability study (up to 12 weeks if frozen) have enabled us to finalize method validation and to add these assays to our routine laboratory repertoire.

Revue Neurologique - In Press.Proof corrected by the author Available online since mercredi 15 juillet 2015

Introduction et but de l’etude La citrulline et l’exercice sont connus pour etre des regulateurs positifs de l’homeostasie musculaire. Cependant, il n’existe a ce jour aucune donnee sur un possible effet synergique. Nous proposons d’evaluer l’effet combine de la citrulline et de l’exercice sur le metabolisme proteique musculaire et la performance en endurance chez le rat sain. Materiel et methodes Quarante rats Wistar adultes sains (3 mois) ont ete randomises en 4 groupes. Les animaux ont ete nourris ad libitum avec un regime standard avec ou sans citrulline (1 g/kg/j) et avec ou sans entrainement (ex) (5 j/7) pendant 4 semaines. L’entrainement a ete effectue sur tapis roulant de facon progressive afin d’atteindre une vitesse de 25 m/min pendant 1 heure. Avant l’euthanasie, tous les rats ont participe a un test maximal d’endurance jusqu’a epuisement. Le jour de l’euthanasie, le plasma a ete recueilli afin de doser les acides amines et le muscle plantaris a ete preleve afin de mesurer la synthese proteique musculaire (methode SUnSET), l’expression des proteines de la voie mTORC1 ( western blot ), ainsi que la taille des fibres musculaires (analyse histologique). Resultats Les resultats sont presentes dans le Tableau 1 . Par ailleurs, les modifications du metabolisme proteique et de la performance s’accompagnent de modifications dans la repartition des tailles des fibres musculaires ( p Conclusion La citrulline et l’exercice semblent avoir un effet synergique sur le muscle conduisant a une amelioration de la capacite d’endurance et de la synthese proteique musculaire avec une implication possible de la voie mTORC1.

Introduction Parmi tous les cas de douleurs abdominales, il faut savoir rester attentif aux signes atypiques pouvant reveler une maladie rare. Observation Une femme de 52 ans, aux antecedents d’hemiparesie neonatale et d’epilepsie partielle sous lamotrigine, phenytoine et carbamazepine, presentait une asthenie et des douleurs abdominales depuis 7 jours. Apres l’introduction d’un traitement antibiotique probabiliste par nitrofurantoine, la patiente developpa un tableau confusionnel. L’examen cytobacteriologique realise sur un echantillon de couleur rouge apres exposition a la lumiere n’avait pas mis en evidence d’infection. La TDM abdominale retrouvait une occlusion fonctionnelle responsable d’une dilatation caecale. Le trace EEG etait compatible avec une encephalopathie metabolique. Le bilan biochimique plasmatique (ionogramme, uree, TSH, ammoniaque) etait sans particularite. Devant un taux eleve de porphobilinogene (PBG) urinaire = 57 mmol/mol de creatinine, il avait ete alors evoque une porphyrie aigue et un traitement intraveineux par hemine humaine fut debute rapidement. Malgre une resolution rapide de l’etat confusionnel, la patiente developpa une paralysie peripherique du membre superieur droit. Discussion Les crises aigues de porphyrie secondaires sont rarement symptomatiques et peuvent survenir en l’absence d’antecedents familiaux, suite a l’accumulation de facteurs favorisants comme le jeune, l’infection et l’action de medicaments inducteurs enzymatiques hepatiques. Malgre une augmentation du PBG urinaire, l’activite enzymatique PBGD erythrocytaire et l’analyse moleculaire du gene HMBS etaient sans anomalie. Conclusion Une porphyrie aigue est a evoquer devant une crise neuro-viscerale, quelle qu’en soit l’etiologie. De nombreux medicaments sont contre-indiques mais l’hemine humaine est un traitement simple a administrer en urgence.

Vitamin B12 deficiency can lead to serious haematological and neurological signs in infants. The reported clinical cases of vitamin B12 deficiency were found in exclusively breast-fed infants whose asymptomatic mothers were diagnosed later with pernicious anaemia. For the infants, the diagnosis required urinary methylmalonic acid quantification (grossly elevated in these two cases) and treatment rapidly improved the clinical signs. These cases underline the serious consequences of vitamin B12 deficiency in infants and the helpful role of early methylmalonic acid quantification for diagnosis.

The Trima separator, manufactured by the Gambro, was introduced at the end of 1997 and the first separators were tested in France in early 1998. They are now routinely used on our Grenoble site for the collection of platelets and plasma. The aim of this paper is to evaluate the residual contamination of leucocytes of platelet products routinely collected on the Trima separator in a French blood transfusion center (ETS). Two separators are used at the fixed site and two separators are used for mobile collection (500 km/week). After a preliminary period of validation on site, 3237 plateletpheresis concentrates were collected by four separators qualified for fixed site or mobile unit use. An analysis taking into account the separator site (fixed or mobile) fails to reveal any significant difference for means or non-conformity percentages (data available).

Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.

An original oligonucleotide-array, coupled with SPR-imaging detection, has been developed to study biological interactions between DNA base lesions and DNA repair enzymes. This bioanalytical tool constitutes an efficient screening platform to quantify DNA repair activities and to search for new DNA repair inhibitors.

Mémoire de Diplôme d'Etudes Spécialisées (DES) tenant lieu de thèse d'exercice; Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequent fatty acid oxidation defect, with an estimated incidence of 1/15 000 in France. The clinical form consists of hypoglycemic non-ketotic episodes after a period of fasting or intercurrent illness, which can quickly degenerate into a coma or even death. These episodes can be avoided with preventive measures, which consist essentially of dietary guidelines. Newborn screening is a public health program tasked with preventing the often irreversible consequences of this type of disorder through early detection, therefore allowing preventive treatment to be put in place before symptoms appear. In the context of the expansion of the newborn screening program in France to include MCAD deficiency, we implemented the multiplex assay of octanoylcarnitine, decanoylcarnitine and also phenylalanine and tyrosine (for the screening of phenylketonuria) on dried blood spots. We verified the analytical method’s performance at the CHU Grenoble-Alpes newborn screening laboratory. We have therefore validated the performance of the Chromsystems’ MassChrom® kit on the tandem mass spectrometer Waters® Xevo® TQD in the work conditions of our laboratory according to NF EN ISO 15 189 requirements. Our laboratory is now ready to start the newborn screening of MCAD deficiency, expected to be on the 3rd trimester of 2020 nationwide. It is a stepping-stone for a future greater extension of newborn screening to other inborn errors of metabolism.; Le déficit en acyl-CoA déshydrogénase des acides gras à chaîne moyenne (MCAD) est l'anomalie de la bêta-oxydation la plus fréquente, avec une incidence estimée autour de 1/15 000 en France. Il se présente généralement sous forme d'épisodes hypoglycémiques non-cétosiques pouvant se compliquer par un coma voire un décès. Ces épisodes de décompensation peuvent être évités par la mise en place de mesures préventives sous forme de règles hygiéno-diététiques. Le dépistage néonatal est un programme de santé publique dont la vocation est de prévenir les conséquences souvent irréversibles de ce type de pathologie en détectant les malades avant l'apparition de symptômes en période néonatale, et en mettant en place d'emblée une prise en charge préventive. Ainsi, dans le contexte de l’extension du dépistage néonatal au déficit en MCAD en France, nous avons mis en place un dosage multiplex quantifiant l’octanoylcarnitine, la décanoylcarnitine et également la phénylalanine et la tyrosine (pour le dépistage de la phénylcétonurie) sur taches de sang total séché sur papier buvard et réalisé la vérification de méthode au laboratoire de dépistage néonatal du CHU Grenoble-Alpes. Nous avons ainsi pu démontrer la conformité des performances du kit MassChrom® de Chromsystems sur le spectromètre de masse en tandem (MS/MS) Waters® Xevo® TQD dans les conditions de travail du laboratoire selon la norme NF EN ISO 15189. Le laboratoire est désormais prêt à proposer le dépistage du déficit en MCAD prévu au 3e trimestre 2020 au niveau national. L’introduction de la MS/MS permettra ensuite une extension du dépistage néonatal à d'autres erreurs innées du métabolisme.

Mémoire de Diplôme d'Etudes Spécialisées (DES) tenant lieu de thèse d'exercice.; Phenylketonuria (PKU; OMIM 261600), an inborn error of amino acid metabolism, is diagnosed in approximately 1:16, 000 newborns in France. Since 1972, with the instauration of newborn screening, clinical evolution of phenylketonuric patients is almost identical than in healthy subjects. This treatable disease has a little known neurologic pathophysiology. Tryptophan (Trp), an essential amino acid, is degraded into two pathways: kynurenine pathway and serotonin pathway. Search about Trp metabolism had been prolific since last decades, and some features allowed us to hypothesize that PKU and Trp metabolism share an interconnection. We developed a quantification method of 9 Trp metabolism analytes using HPLC-MS/MS. The method was validated using NF EN ISO 15189 recommendations. A comparison of metabolites concentrations in phenylketonuria patients blood and reference population was performed. We highlight a significant diminution of Trp, kynurenine and 3-hydrokynurenine in non-treated adult phenylketonuria patients. The exploration of these metabolism modifications needs more studies in PKU, but also in other inborn or non-inborn pathologies. That will help us to understand the implication of Trp catabolism, by kynurenine pathway and serotonin pathway, in neurological symptoms of phenyketonuric patients.; La phénylcétonurie (PCU ; OMIM 261600) est une amino-acidopathie dont la prévalence est d’environ 1/16 000 bébés en France. Son évolution clinique s’est fortement améliorée depuis 1972, date à laquelle le dépistage systématique a été instauré en France. Cette pathologie est traitable, mais la physiopathologie neurologique n’est pas encore parfaitement comprise. Le tryptophane (Trp), acide aminé essentiel, possède une dégradation selon deux voies métaboliques principales : celle des kynurénines et celle de la sérotonine. La recherche sur le métabolisme du Trp a vu un grand essor au cours des dernières années, et certains éléments nous ont permis d’émettre l’hypothèse d’une relation entre la PCU et le métabolisme du Trp. Nous avons mis au point, en HPLC-MS/MS, une méthode de quantification de 9 molécules de la voie métabolique du Trp. Nous avons également validé ce dosage plasmatique selon la norme NF EN ISO 15189. Une comparaison des concentrations de ces différents métabolites dans un groupe de patients phénylcétonuriques par rapport à une population de référence a ensuite été effectuée, permettant la mise en évidence d’une diminution significative de Trp, kynurénine et 3-hydroxykynurénine dans notre population phénylcétonurique adulte non traitée. L’étude des modifications de ce métabolisme reste cependant à explorer plus largement dans la PCU, mais également dans d’autres pathologies, qu’elles soient héréditaires ou non. Nous espérons ainsi comprendre plus amplement l’implication du catabolisme du Trp, via la voie des kynurénines et de la sérotonine, dans les symptômes neurologiques des patients atteints de PCU.

Mémoire de DES (Diplôme d'Etudes Spécialisées) tenant lieu de thèse d'exercice; Mitochondrial fatty acid β-oxidation disorders are a group of inherited metabolic disorders. Nowadays, almost twenty disorders are known. These diseases can occur at any age and in various ways such as hypoglycemia, cardiomyopathy or rhabdomyolysis... The biochemical diagnosis is based on the acylcarnitine profiling studies. Indeed, carnitine is able to detoxify the acyl-coenzyme A which are accumulating in the mitochondria. They can thus be found as acylcarnitines, which pile up with β-oxidation disorders. We developed a method of acylcarnitine profiling in plasma by tandem mass spectrometry. Indeed today, mass spectrometry, thanks to its many advantages, has become a powerful and essential diagnostic tool for medical biology. Method criteria were analyzed (stability, analytical interferences, anticoagulant use, repeatability, reproducibility...) and biological validation limits were also studied. Thus, the Grenoble University Hospital is now able to offer a full check-up for intermediary metabolism and can respond to the emergency of such diagnosis.; Les anomalies de la β-oxydation mitochondriale des acides gras constituent un groupe à part entière des maladies héréditaires du métabolisme. On connaît environ un vingtaine de déficits qui peuvent se manifester à tout âge et de diverses manières : hypoglycémie, myocardiopathie ou épisodes de rhabdomyolyse... Leur diagnostic biochimique repose sur la détermination du profil des acylcarnitines. En effet, la carnitine permet de détoxifier les acyl-coenzyme A qui s'accumulent dans la mitochondrie, on les retrouve alors sous forme d'acylcarnitines qui s'accumulent en cas d'anomalies de la β-oxydation. Nous avons mis en place au laboratoire, la détermination du profil des acylcarnitines plasmatiques par spectrométrie de masse en tandem. En effet, grâce à ses nombreux avantages, la spectrométrie de masse est devenue un outil diagnostique puissant indispensable à la biologie médicale actuelle. Les critères de validation de la méthode ont été analysés (stabilité, interférences analytiques, anticoagulant utilisé, répétabilité, reproductibilité...) et les limites de la technique concernant la validation biologique des profils ont également été étudiées. Ainsi, le CHU de Grenoble est désormais en mesure de proposer un bilan complet du métabolisme intermédiaire (chromatographie des acides organiques urinaires, chromatographie des acides aminés plasmatiques, profil des acylcarnitines plasmatiques) et peut répondre à l'urgence diagnostique des pathologies héréditaires du métabolisme.