Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse. The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Comparison of cannabinoid concentrations in oral fluid and whole blood between occasional and regular cannabis smokers prior to and after smoking a cannabis joint.

A cross-over controlled administration study of smoked cannabis was carried out on occasional and heavy smokers. The participants smoked a joint (11 % Δ9-tetrahydrocannabinol (THC)) or a matching placebo on two different occasions. Whole blood (WB) and oral fluid (OF) samples were collected before and up to 3.5 h after smoking the joints. Pharmacokinetic analyses were obtained from these data. Questionnaires assessing the subjective effects were administered to the subjects during each session before and after the smoking time period. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) were analyzed in the blood by gas chromatography or liquid chromatography (LC)-tandem mass spectrometry (MS/MS). The determination of THC, THCCOOH, cannabinol (CBN), and Δ9-tetrahydrocannabinolic acid A (THC-A) was carried out on OF only using LC-MS/MS. In line with the widely accepted assumption that cannabis smoking results in a strong contamination of the oral cavity, we found that THC, and also THC-A, shows a sharp, high concentration peak just after smoking, with a rapid decrease in these levels within 3 h. No obvious differences were found between both groups concerning THC median maximum concentrations measured either in blood or in OF; these levels were equal to 1,338 and 1,041 μg/L in OF and to 82 and 94 μg/L in WB for occasional and heavy smokers, respectively. The initial WB THCCOOH concentration was much higher in regular smokers than in occasional users. Compared with the occasional smokers, the sensation of confusion felt by the regular smokers was much less while the feeling of intoxication remained almost unchanged.

Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents.

The study reports a set of forty proteinogenic histidine-containing dipeptides as potential carbonyl quenchers. The peptides were chosen to cover as exhaustively as possible the accessible chemical space, and their quenching activities toward 4-hydroxy-2-nonenal (HNE) and pyridoxal were evaluated by HPLC analyses. The peptides were capped at the C-terminus as methyl esters or amides to favor their resistance to proteolysis and diastereoisomeric pairs were considered to reveal the influence of configuration on quenching. On average, the examined dipeptides are less active than the parent compound carnosine (βAla + His) thus emphasizing the unfavorable effect of the shortening of the βAla residue as confirmed by the control dipeptide Gly-His. Nevertheless, some peptides show promising activities toward HNE combined with a remarkable selectivity. The results emphasize the beneficial role of aromatic and positively charged residues, while negatively charged and H-bonding side chains show a detrimental effect on quenching. As a trend, ester derivatives are slightly more active than amides while heterochiral peptides are more active than their homochiral diastereoisomer. Overall, the results reveal that quenching activity strongly depends on conformational effects and vicinal residues (as evidenced by the reported QSAR analysis), offering insightful clues for the design of improved carbonyl quenchers and to rationalize the specific reactivity of histidine residues within proteins.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB(2) knockout mice and were not prevented by CB(1/2) antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB(1/2) receptors.

Combining Internet Monitoring Processes, Packaging and Isotopic Analyses to Determine The Market Structure: Example of Gamma Butyrolactone

The Internet is becoming more and more popular among drug users. The use of websites and forums to obtain illicit drugs and relevant information about the means of consumption is a growing phenomenon mainly for new synthetic drugs. Gamma Butyrolactone (GBL), a chemical precursor of Gamma Hydroxy Butyric acid (GHB), is used as a "club drug" and also in drug facilitated sexual assaults. Its market takes place mainly on the Internet through online websites but the structure of the market remains unknown. This research aims to combine digital, physical and chemical information to help understand the distribution routes and the structure of the GBL market. Based on an Internet monitoring process, thirty-nine websites selling GBL, mainly in the Netherlands, were detected between January 2010 and December 2011. Seventeen websites were categorized into six groups based on digital traces (e.g. IP addresses and contact information). In parallel, twenty-five bulk GBL specimens were purchased from sixteen websites for packaging comparisons and carbon isotopic measurements. Packaging information showed a high correlation with digital data confirming the links previously established whereas chemical information revealed undetected links and provided complementary information. Indeed, while digital and packaging data give relevant information about the retailers, the supply routes and the distribution close to the consumer, the carbon isotopic data provides upstream information about the production level and in particular the synthesis pathways and the chemical precursors. A three-level structured market has been thereby identified with a production level mainly located in China and in Germany, an online distribution level mainly hosted in the Netherlands and the customers who order on the Internet.

A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

Probing functional groups at the gas-aerosol interface using heterogeneous titration reactions: a tool for predicting aerosol health effects?

The complex chemical and physical nature of combustion and secondary organic aerosols (SOAs) in general precludes the complete characterization of both bulk and interfacial components. The bulk composition reveals the history of the growth process and therefore the source region, whereas the interface controls--to a large extent--the interaction with gases, biological membranes, and solid supports. We summarize the development of a soft interrogation technique, using heterogeneous chemistry, for the interfacial functional groups of selected probe gases [N(CH(3))(3), NH(2)OH, CF(3)COOH, HCl, O(3), NO(2)] of different reactivity. The technique reveals the identity and density of surface functional groups. Examples include acidic and basic sites, olefinic and polycyclic aromatic hydrocarbon (PAH) sites, and partially and completely oxidized surface sites. We report on the surface composition and oxidation states of laboratory-generated aerosols and of aerosols sampled in several bus depots. In the latter case, the biomarker 8-hydroxy-2'-deoxyguanosine, signaling oxidative stress caused by aerosol exposure, was isolated. The increase in biomarker levels over a working day is correlated with the surface density N(i)(O3) of olefinic and/or PAH sites obtained from O(3) uptakes as well as with the initial uptake coefficient, γ(0), of five probe gases used in the field. This correlation with γ(0) suggests the idea of competing pathways occurring at the interface of the aerosol particles between the generation of reactive oxygen species (ROS) responsible for oxidative stress and cellular antioxidants.

Fast gas chromatography and negative-ion chemical ionization tandem mass spectrometry for forensic analysis of cannabinoids in whole blood.

The present work describes a fast gas chromatography/negative-ion chemical ionization tandem mass spectrometric assay (Fast GC/NICI-MS/MS) for analysis of tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) in whole blood. The cannabinoids were extracted from 500 microL of whole blood by a simple liquid-liquid extraction (LLE) and then derivatized by using trifluoroacetic anhydride (TFAA) and hexafluoro-2-propanol (HFIP) as fluorinated agents. Mass spectrometric detection of the analytes was performed in the selected reaction-monitoring mode on a triple quadrupole instrument after negative-ion chemical ionization. The assay was found to be linear in the concentration range of 0.5-20 ng/mL for THC and THC-OH, and of 2.5-100 ng/mL for THC-COOH. Repeatability and intermediate precision were found less than 12% for all concentrations tested. Under standard chromatographic conditions, the run cycle time would have been 15 min. By using fast conditions of separation, the assay analysis time has been reduced to 5 min, without compromising the chromatographic resolution. Finally, a simple approach for estimating the uncertainty measurement is presented.

In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule.

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that inactivates 11β-hydroxy glucocorticoids. High expression levels of 11β-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11β-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11β-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as alpha-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies.

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Investigation phytochimique d'une brosse à dents africaine Zanthoxylum zanthoxyloides (Lam.) Zepernick et Timler (Syn. Fagara zanthoxiloides L.) (Rutaceae)

Résumé: Dans le but de rechercher de nouveaux composés naturels à intérêt thérapeutique, les extraits dichlorométhanique et méthanolique de Zanthoxylum zanthoxyloides (Lam.) Zepernick et Timler (Syn. Fagara zanthoxyloides L.) (Rutaceae), une brosse à dents africaine ont été soumis à un criblage chimique et biologique. Un dépistage des activités: antifongiques contre le champignon phytopathogène Cladosporium cucumerinum et la levure commensale responsable de mycoses chez l'homme Candida albicans, antibactérienne contre la bactérie opportuniste Bacillus subtilis, larvicide contre le moustique vecteur de la fièvre jaune Aedes aegypti et molluscicide contre Biomphalaria glabrata, un escargot impliqué dans la transmission de la schistosomiase urinaire a été réalisé. Les propriétés antiradicalaires et inhibitrices de l'acétylcholinestérase de ces extraits ont aussi été dépistées. Sur la base des résultats obtenus lors de ce screening, l'investigation phytochimique de ces extraits a été entreprise. Elle a abouti à l'isolement de 14 composés, actifs pour la majorité contre Cladosporium cucumerinum et Bacillus subtilis, dont la structure a été établie au moyen de méthodes spectroscopiques (UV, MS, IR, 1H- et 13C-NMR). Des méthodes chimiques (hydrolyse, acétylation) ont été requises pour la confirmation de structures. L'extrait dichlorométhanique a fourni un nouveau composé, un dérivé du phényléthane, ainsi que dix composés connus, dont trois dérivés du phénylpropane, un lignane, un alcaloïde de la famille des benzophénanthridines, un triterpène, deux amides phénoliques et deux amides oléfmiques. L'extrait méthanolique a fourni un nouveau composé avec une fonction endoperoxyde, qui avait montré une activité inhibitrice modérée de l'acétylcholinestérase, ainsi que l'hespéridine et un dérivé de la chélérythrine. Par ailleurs, l'analyse LC/UV/APC1-MS de cet extrait a permis de détecter on-une sept produits connus. Parmi ces composés, se trouvent l'acide divanilloylquinique, la chélérythrine et quatre de ses dérivés: norchélérythrin.e, 6-(2-oxybutyl) dihydrochélérythrine, 6-hydroxy-dihydrochélérythrine et avicine, ainsi qu'une amide phénolique, l'amottianamide. La présence de ces dérivés de la chélérythrine a été mise en évidence dans deux autres espèces du même genre lors d'une étude LC/UV/APCI-MS comparative. Les activités fongicides contre Cladosporium cucumerinum et Candida albicans et bactéricides contre Bacillus subtilis et Streptococcus mutans ATCC 25175, mises en évidence sur plaque CCM et par les tests de dilution dans l'agar de ces composés, permettent de justifier l'utilisation de Zanthoxylum zanthoxyloides (Lam.) Zepemick et Timler comme brosse à dents africaine. Les techniques couplées de pointe utilisées dans cette étude ont montré leur apport inestimable dans le domaine de la recherche phytochimique et les applications futures dans le domaine de déréplication d'extraits bruts. Abstract: With the aim of discovering new natural therapeutics, the dichloromethane and methanol extracts of the African toothbrush tree Zanthoxylum zanthoxyloides (Lam.) Zepernick et Timler (Syn. Fagara zanthoxyloides L.) (Rutaceae), were submitted to biological and chemical assays. The former included: the antifimgal activities of the extracts against the phytopathogenic fungus Cladosporium cucumerinum, the commensal yeast which causes human mycoses Candida albicans, the bactericidal activity against the opportunistic bacteria Bacillus subtilis, the larvicidal activity against the yellow fever-transmitting mosquito Aedes aegypti and the molluscicidal effect on the snail Biomphalaria glabrata involved in the transmission of urinary schistosomiasis. The antiradical and acetylcholinesterase-inhibiting properties of these extracts were also investigated. On the basis of these results, a phytochemical investigation of the dichloromethane and methanol extracts of Zanthoxylum zanthoxyloides was undertaken. Their fractionation led to the isolation of 14 compounds, the majority of which were active against Cladosporium cucumerinum and Bacillus subtilis, whose structures were elucidated by spectroscopic techniques (UV, MS, IR, 1H- and 13C-NMR). Chemical methods (hydrolysis, acetylation) were performed to confirm the structures. The dichloromethane extract yielded a new phenylethane derivative, together with ten known compounds: three phenylpropane derivatives, a lignan, a benzophenanthridine alkaloid, a triterpene and four phenolic and olefinic amides. The methanol extract yielded a new compound with an endoperoxide moiety, which showed moderate acetylcholinesterase-inhibiting activity, together with hesperidin and a chelerythrine derivative. Seven more compounds were detected on-line by LC/UV/APCI-MS. Among the compounds detected were divanilloylquinic acid, chelerythrine and four chelerythrine derivatives: norchelerythrine, 6-(2-oxybuty1)-dihydrochelerythrine, 6-hydroxy dihydrochelerythrine and avicine, together with the phenolic amide amottianamide. Most of the chelerythrine derivatives were also found in two other Zanthoxylum species following LC/UV/APCI-MS analysis. The antifungal activities against Cladosporium cucumerinum and Candida albicans and antibacterial activities against Bacillus subtilis and Streptococcus mutans ATCC 25175, may explain the utilization in traditional medicine of the roots of this plant as a toothbrush. The advanced hyphenated techniques used in this study showed their inestimable contribution to the field of phytochemical research and applications in the field of dereplication of crude extracts.

Sphingomonas wittichii RW1 gene reporters interrogating the dibenzofuran metabolic network highlight conditions for early successful development in contaminated microcosms.

In order to better understand the fate and activity of bacteria introduced into contaminated material for the purpose of enhancing biodegradation rates, we constructed Sphingomonas wittichii RW1 variants with gene reporters interrogating dibenzofuran metabolic activity. Three potential promoters from the dibenzofuran metabolic network were selected and fused to the gene for enhanced green fluorescent protein (EGFP). The stability of the resulting genetic constructions in RW1 was examined, with plasmids based on the broad-host range vector pME6012 being the most reliable. One of the selected promoters, upstream of the gene Swit_4925 for a putative 2-hydroxy-2,4-pentadienoate hydratase, was inducible by growth on dibenzofuran. Sphingomonas wittichii RW1 equipped with the Swit_4925 promoter egfp fusion grew in a variety of non-sterile sandy microcosms contaminated with dibenzofuran and material from a former gasification site. The strain also grew in microcosms without added dibenzofuran but to a very limited extent, and EGFP expression indicated the formation of consistent small subpopulations of cells with an active inferred dibenzofuran metabolic network. Evidence was obtained for competition for dibenzofuran metabolites scavenged by resident bacteria in the gasification site material, which resulted in a more rapid decline of the RW1 population. Our results show the importance of low inoculation densities in order to observe the population development of the introduced bacteria and further illustrate that the limited availability of unique carbon substrate may be the most important factor impinging growth.

Neuropathogenesis in glutaric aciduria type I and methylmalonic aciduria

Glutaric aciduria type-I (GA-I) and methylmalonic aciduria (MMA-uria) are two neurometabolic diseases manifesting in neonatal period and early childhood. They belong to the group of organic acidurias and are caused by defects in the catabolism of amino acids, leading to massive accumulation of toxic metabolites in the body and severe brain injury. Therapeutic strategies are mainly based on reversing catabolic state during metabolic crisis and dietary protein restriction that both aim to prevent extra production of toxic metabolites. Specific and neuroprotective treatments are missing because the mechanisms of brain damage in these diseases are only poorly understood. The principal objective of my work was to develop in vitro models for both diseases aiming at elucidation of toxic effects of the main metabolites accumulating in GA-I (glutaric acid (GA) and 3-hydroxy glutaric acid (3-OHGA)) and MMA-uria (methylmalonic acid (MMA), propionic acid (PA) and 2-methylcitric acid (2-MCA)) on developing brain cells, and to study the cellular pathways targeted by these deleterious effects in order to find new therapeutic potentials. We used re-aggregated embryonic rat brain cells in organotypic 3D cultures, which were exposed to toxic metabolites at different developing stages of the cultures. In parallel, we studied the cellular localization of the defected enzyme in GA-I, glutaryl-CoA dehydrogenase (GCDH), in the brain and peripheral tissues of rats in adulthood and during embryonic development. GCDH expression: GCDH showed a strong neuronal expression in embryonic central and peripheral nervous system. In the adult brain, GCDH expression was exclusively neuronal with the strongest signal in cerebral cortex and Purkinje cells. GCDH expression was homogenous in embryonic peripheral organs with high levels in intestinal mucosa at late stages. Strong GCDH expression was also observed in liver and intestinal mucosa and with lower intensity in muscles, convoluted renal tubules and renal collecting tubes in adult peripheral organs. GA-I and MMA-uria in vitro models: 3-OHGA (for GA-I) and 2-MCA (for MMA-uria) showed the most deleterious effects at early stages of the cultures with morphological and biochemical alterations and induction of cell death. 3-OHGA and 2-MCA caused astrocytic cell suffering reflected by astrocytic fiber loss and swelling and retardation in oligodendrocytic maturation and/or differentiation. High ammonium increase concomitant with glutamine decrease was observed in these cultures. Neurons were not substantially affected. Our studies revealed that brain-cell generated ammonia may play a role in the neuropathogenesis of these diseases. Thus, developing neuroprotective strategies that target ammonium toxicity in the brain of GA-I and MMA-uria patients might be important according to our findings. -- L'acidurie glutarique de type I (GA-I) et l'acidurie méthylmalonique (MMA-urie) sont deux maladies neurométaboliques se manifestant durant la période néonatale ou la petite enfance, et qui appartiennent aux aciduries organiques. Elles sont causées par des défauts dans le catabolisme des acides aminés, conduisant à une accumulation des métabolites toxiques dans le corps et aussi des lésions cérébrales sévères. Le traitement est limité à une prise en charge d'urgence pendant la crise métabolique et à une diète restreinte en protéines naturelles. Des traitements spécifiques, neuroprotecteurs manquent principalement parce que les mécanismes conduisant aux lésions cérébrales dans ces maladies sont peu connus. L'objectif principal de mon travail était d'élucider les effets toxiques des métabolites accumulés dans GA-I (l'acide glutarique (GA) et l'acide 3-hydroxyglutarique (3-OHGA)) et MMA-uria (l'acide méthylmalonique (MMA), l'acide propionique (PA) et l'acide 2-méthylcitrique(2-MCA) sur les cellules du cerveau ainsi que les voies cellulaires impliquées, dans le but de trouver de potentielles nouvelles stratégies thérapeutiques. Nous avons utilisé un modèle in vitro de cultures 3D de cellules de cerveau d'embryons de rat (en développement) en les exposant aux métabolites toxiques à différents stades de développement des cultures. En parallèle, nous avons étudié la localisation cellulaire de l'enzyme déficiente dans GA-I, la CoA-glutarly déshydrogénase (GCDH), dans le cerveau et les organes périphériques des rats adultes et pendant le développement embryonnaire. L'expression de GCDH: GCDH a montré une expression neuronale forte dans le système nerveux chez l'embryon et le cerveau adulte. L'expression était homogène dans les organes périphériques avec une forte expression dans l'intestin. Les modèles in vitro de GA-I et MMA-uria : 3-OHGA en modèle GA-I et 2-MCA en modèle MMA-uria ont montré les effets délétères les plus importants avec des altérations morphologiques des cellules et biochimiques dans le milieu de culture et l'induction de mort cellulaire non-apoptotique (3-OHGA) ou apoptotique (2-MCA). 3-OHGA et 2-MCA ont provoqué une souffrance astrocytaire avec perte des fibres et gonflement et un retard de maturation et/ou de différentiation des oligodendrocytes. Une augmentation importante d'ammonium avec une diminution concomitante de glutamine a été observée dans les cultures. Les neurones n'étaient pas vraiment affectés. Nos études ont révélé que l'ammonium généré par les cellules cérébrales pourrait jouer un rôle dans la neuropathogenèse de ces deux maladies. Par conséquent, développer des stratégies neuroprotectrices ciblant la toxicité de l'ammonium dans le cerveau des patients atteints de GA-I ou MMA-urie pourrait être très important selon nos résultats.

Renal function can be impaired in children with primary hyperoxaluria type 3.

BACKGROUND: Primary hyperoxaluria type 3 (PH3) is characterized by mutations in the 4-hydroxy-2-oxoglutarate aldolase (HOGA1) gene. PH3 patients are believed to present with a less severe phenotype than those with PH1 and PH2, but the clinical characteristics of PH3 patients have yet to be defined in sufficient detail. The aim of this study was to report our experience with PH3. METHODS: Genetic analysis of HOGA1 was performed in patients with a high clinical suspicion of PH after the presence of mutations in the alanine-glyoxylate aminotransferase gene had been ruled out. Clinical, biochemical and genetic data of the seven patients identified with HOGA1 mutations were subsequently retrospectively reviewed. RESULTS: Among the seven patients identified with HOGA1 mutations the median onset of clinical symptoms was 1.8 (range 0.4-9.8) years. Five patients initially presented with urolithiasis, and two other patients presented with urinary tract infection. All patients experienced persistent hyperoxaluria. Seven mutations were found in HOGA1, including two previously unreported ones, c.834 + 1G > T and c.3G > A. At last follow-up, two patients had impaired renal function based on estimated glomerular filtration rates (GFRs) of 77 and 83 mL/min per 1.73 m(2), respectively. CONCLUSIONS: We found that the GFR was significantly impaired in two of our seven patients with PH3 diagnosed during childhood. This finding is in contrast to the early-impaired renal function in PH1 and PH2 and appears to refute to preliminary reassuring data on renal function in PH3.

In Vivo Profiling and Distribution of Known and Novel Phase I and Phase II Metabolites of Efavirenz in Plasma, Urine, and Cerebrospinal Fluid.

Efavirenz (EFV) is principally metabolized by CYP2B6 to 8-hydroxy-efavirenz (8OH-EFV) and to a lesser extent by CYP2A6 to 7-hydroxy-efavirenz (7OH-EFV). So far, most metabolite profile analyses have been restricted to 8OH-EFV, 7OH-EFV, and EFV-N-glucuronide, even though these metabolites represent a minor percentage of EFV metabolites present in vivo. We have performed a quantitative phase I and II metabolite profile analysis by tandem mass spectrometry of plasma, cerebrospinal fluid (CSF), and urine samples in 71 human immunodeficiency virus patients taking efavirenz, prior to and after enzymatic (glucuronidase and sulfatase) hydrolysis. We have shown that phase II metabolites constitute the major part of the known circulating efavirenz species in humans. The 8OH-EFV-glucuronide (gln) and 8OH-EFV-sulfate (identified for the first time) in humans were found to be 64- and 7-fold higher than the parent 8OH-EFV, respectively. In individuals (n = 67) genotyped for CYP2B6, 2A6, and CYP3A metabolic pathways, 8OH-EFV/EFV ratios in plasma were an index of CYP2B6 phenotypic activity (P < 0.0001), which was also reflected by phase II metabolites 8OH-EFV-glucuronide/EFV and 8OH-EFV-sulfate/EFV ratios. Neither EFV nor 8OH-EFV, nor any other considered metabolites in plasma were associated with an increased risk of central nervous system (CNS) toxicity. In CSF, 8OH-EFV levels were not influenced by CYP2B6 genotypes and did not predict CNS toxicity. The phase II metabolites 8OH-EFV-gln, 8OH-EFV-sulfate, and 7OH-EFV-gln were present in CSF at 2- to 9-fold higher concentrations than 8OH-EFV. The potential contribution of known and previously unreported EFV metabolites in CSF to the neuropsychological effects of efavirenz needs to be further examined in larger cohort studies.