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Background: Cannabis use is a component risk factor for the manifestation of schizophrenia. The biological effects of cannabis include effects on epigenetic systems, immunological parameters, in addition to changes in cannabinoid receptors 1 and 2, that may be associated with this risk. However, there has been limited study of the effects of smoked cannabis on these biological effects in human peripheral blood cells. We analyzed the effects of two concentrations of tetrahydrocannabinol (THC) vs. placebo in lymphocytes of a subset of participants who enrolled in a double-blind study of the effects of cannabis on driving performance (outcome not the focus of this study). Methods: Twenty four participants who regularly use cannabis participated in an experiment in which they smoked cannabis cigarettes (5.9 or 13.4% THC) or placebo (0.02%) ad libitum. Blood samples were drawn at baseline and several times after smoking. Lymphocytes were separated and stored at -80°C for further analysis. Samples were analyzed for mRNA content for cannabinoid receptors 1 (CB1) and 2 (CB2), methylation and demethylating enzymes (DNMT, TET), glucocorticoid receptor (NRC3) and immunological markers (IL1B, TNFα) by qPCR using TaqMan probes. The results were correlated with THC whole blood levels during the course of the day, as well as THCCOOH baseline levels. Statistical analyses used analysis of variance and covariance and t-tests, or non-parametric equivalents for those values which were not normally distributed. Results: There were no differences in background baseline characteristics of the participants except that the higher concentration THC group was older than the low concentration and placebo groups, and the low concentration THC group had higher baseline CB2 mRNA levels. Both the 5.9 and 13.4% THC groups showed increased THC blood levels that then decreased toward baseline within the first hour. However, there were no significant differences between THC blood levels between the 5.9 and 13.4% groups at any time point. At the 4-h time point after drug administration the 13.4% THC group had higher CB2 (P = 0.021) and DNMT3A (P = 0.027) mRNA levels than the placebo group. DNMT1 mRNA levels showed a trend in the same direction (P = 0.056). The higher 13.4% THC group had significantly increased CB2 mRNA levels than the 5.9% concentration group at several post drug administration time points and showed trends for difference in effects for between 5.9 and 13.4% THC groups for other mRNAs. TET3 mRNA levels were higher in the 13.4% THC group at 55 min post-cannabis ingestion. When the high and lower concentration THC groups were combined, none of the differences in mRNA levels from placebo remained statistically significant. Changes in THC blood levels were not related to changes in mRNA levels. Conclusion: Over the time course of this study, CB2 mRNA increased in blood lymphocytes in the high concentration THC group but were not accompanied by changes in immunological markers. The changes in DNMT and TET mRNAs suggest potential epigenetic effects of THC in human lymphocytes. Increases in DNMT methylating enzymes have been linked to some of the pathophysiological processes in schizophrenia and, therefore, should be further explored in a larger sample population, as one of the potential mechanisms linking cannabis use as a trigger for schizophrenia in vulnerable individuals. Since the two THC groups did not differ in post-smoking blood THC concentrations, the relationship between lymphocytic changes and the THC content of the cigarettes remains to be determined.
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Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show that cyclic GMP-selective phosphodiesterase 9A inhibition (PDE9-I) in both male and ovariectomized female mice suppresses preestablished severe diet-induced obesity/CMS with or without superimposed mild cardiac pressure load. PDE9-I reduces total body, inguinal, hepatic, and myocardial fat; stimulates mitochondrial activity in brown and white fat; and improves CMS, without significantly altering activity or food intake. PDE9 localized at mitochondria, and its inhibition in vitro stimulated lipolysis in a PPARα-dependent manner and increased mitochondrial respiration in both adipocytes and myocytes. PPARα upregulation was required to achieve the lipolytic, antiobesity, and metabolic effects of PDE9-I. All these PDE9-I-induced changes were not observed in obese/CMS nonovariectomized females, indicating a strong sexual dimorphism. We found that PPARα chromatin binding was reoriented away from fat metabolism-regulating genes when stimulated in the presence of coactivated estrogen receptor-α, and this may underlie the dimorphism. These findings have translational relevance given that PDE9-I is already being studied in humans for indications including heart failure, and efficacy against obesity/CMS would enhance its therapeutic utility.
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3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Tecido Adiposo/embriologia , Síndrome Metabólica/enzimologia , Obesidade/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Feminino , Masculino , Síndrome Metabólica/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Obesidade/genética , PPAR alfa/genética , PPAR alfa/metabolismoRESUMO
OBJECTIVES: We sought to evaluate the relevance of pediatric dairy fat recommendations for children at risk for nonalcoholic fatty liver disease (NAFLD) by studying the association between dairy fat intake and the amount of liver fat. The effects of dairy fat may be mediated by odd chain fatty acids (OCFA), such as pentadecanoic acid (C15:0), and monomethyl branched chain fatty acids (BCFA), such as iso-heptadecanoic acid (iso-C17:0). Therefore, we also evaluated the association between plasma levels of OCFA and BCFA with the amount of liver fat. METHODS: Observational, cross-sectional, community-based sample of 237 children ages 8 to 17. Dairy fat intake was assessed by 3 24-hour dietary recalls. Plasma fatty acids were measured by gas chromatography-mass spectrometry. Main outcome was hepatic steatosis measured by whole liver magnetic resonance imaging proton density fat fraction (MRI-PDFF). RESULTS: Median dairy fat intake was 10.6âgrams/day (range 0.0--44.5âg/day). Median liver MRI-PDFF was 4.5% (range 0.9%-45.1%). Dairy fat intake was inversely correlated with liver MRI-PDFF (râ=â-0.162; Pâ=â.012). In multivariable log linear regression, plasma C15:0 and iso-C17:0 were inverse predictors of liver MRI-PDFF (Bâ=â-0.247, Pâ=â0.048; and Bâ=â-0.234, Pâ=â0.009). CONCLUSIONS: Dairy fat intake, plasma C15:0, and plasma iso-C17:0 were inversely correlated with hepatic steatosis in children. These hypothesis-generating findings should be tested through clinical trials to better inform dietary guidelines.
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Ácidos Graxos , Hepatopatia Gordurosa não Alcoólica , Adolescente , Criança , Estudos Transversais , Humanos , Fígado/diagnóstico por imagem , Imageamento por Ressonância Magnética , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagemRESUMO
Anaplerotic odd-chain fatty acid supplementation has been suggested as an approach to replenish citric acid cycle intermediate (CACi) pools and facilitate adenosine triphosphate (ATP) production in subjects with long-chain fatty acid oxidation disorders, but the evidence that cellular CACi depletion exists and that repletion occurs following anaplerotic substrate supplementation is limited. We exercised very long-chain acyl-CoA dehydrogenase-deficient (VLCAD-/-) and wild-type (WT) mice to exhaustion and collected cardiac tissue for measurement of CACi by targeted metabolomics. In a second experimental group, VLCAD-/- and WT mice that had been fed chow prepared with either medium-chain triglyceride (MCT) oil or triheptanoin for 4 weeks were exercised for 60 minutes. VLCAD-/- mice exhibited lower succinate in cardiac muscle at exhaustion than WT mice suggesting lower CACi in VLCAD-/- with prolonged exercise. In mice fed either MCT or triheptanoin, succinate and malate were greater in VLCAD-/- mice fed triheptanoin compared to VLCAD-/- animals fed MCT but lower than WT mice fed triheptanoin. Long-chain odd acylcarnitines such as C19 were elevated in VLCAD-/- and WT mice fed triheptanoin suggesting some elongation of the heptanoate, but it is unknown what proportion of heptanoate was oxidized vs elongated. Prolonged exercise was associated with decreased cardiac muscle succinate in VLCAD-/- mice in comparison to WT mice. VLCAD-/- fed triheptanoin had increased succinate compared to VLCAD-/- mice fed MCT but lower than WT mice fed triheptanoin. Cardiac CACi were higher following dietary ingestion of an anaplerotic substrate, triheptanoin, in comparison to MCT.
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Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Síndrome Congênita de Insuficiência da Medula Óssea/dietoterapia , Síndrome Congênita de Insuficiência da Medula Óssea/metabolismo , Erros Inatos do Metabolismo Lipídico/dietoterapia , Erros Inatos do Metabolismo Lipídico/metabolismo , Doenças Mitocondriais/dietoterapia , Doenças Mitocondriais/metabolismo , Doenças Musculares/dietoterapia , Doenças Musculares/metabolismo , Triglicerídeos/administração & dosagem , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Animais , Ciclo do Ácido Cítrico , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Gorduras na Dieta/administração & dosagem , Ácidos Graxos/metabolismo , Feminino , Erros Inatos do Metabolismo Lipídico/genética , Fígado/metabolismo , Masculino , Camundongos , Doenças Mitocondriais/genética , Doenças Musculares/genética , Miocárdio/metabolismo , Oxirredução , Triglicerídeos/químicaRESUMO
Objective Orotic aciduria and deficiency of uridine monophosphate synthetase have been observed in a patient, studied over 10 years, who had no megaloblastic anemia. Excretion of orotic acid and orotidine were 8.24 and 0.52 mmol/mol of creatinine. The ratio of 15.85 differed appreciably from that of 6 patients reported with no megaloblastic anemia. Methods The analysis of orotidine by gas chromotography mass spectrometry was conducted. Conclusion Patients with orotic aciduria with and without megaloblastic anemia cannot be distinguished by ratio of orotic acid to orotidine.
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Orotato Fosforribosiltransferase/deficiência , Ácido Orótico/urina , Orotidina-5'-Fosfato Descarboxilase/deficiência , Erros Inatos do Metabolismo da Purina-Pirimidina/urina , Uridina/análogos & derivados , Criança , Feminino , Humanos , Orotato Fosforribosiltransferase/efeitos dos fármacos , Orotato Fosforribosiltransferase/urina , Orotidina-5'-Fosfato Descarboxilase/efeitos dos fármacos , Orotidina-5'-Fosfato Descarboxilase/urina , Uridina/uso terapêutico , Uridina/urina , Adulto JovemRESUMO
BACKGROUND: Cystine determination is a critical biochemical test for the diagnosis and therapeutic monitoring of the lysosomal storage disease cystinosis. The classical mixed-leukocyte cystine assay requires prompt specialized recovery/isolation following blood drawing, providing cystine concentrations normalized to total protein from assorted types of white blood cells, each with varying cystine content. METHODS: We present a new workflow for cystine determination using immunomagnetic granulocyte purification, and new reference ranges established from 47 patient and 27 obligate heterozygote samples assayed. Samples were collected in acid-citrate dextrose tubes and their stability was proven to allow for overnight shipping before analysis. Cystine was quantified by LC-MS/MS. RESULTS: The new method was reproducible (<15% root mean square error) and specific, assaying purified granulocytes from blood samples that no longer required immediate preparation and therefore allowing for up to 30 h before processing. There was a nearly a 2-fold increase in the therapeutic target (1.9 nmol half-cystine/mg protein) range, established using distributions of patient, obligate heterozygote, and control samples. The 2.5-97.5 percentile ranges (-2 SD to +2 SD around mean) for these cohorts were 0.67-6.05 nmol/mg protein for patients, 0.33-1.35 nmol/mg protein for obligate heterozygotes, and 0.09-0.35 nmol/mg protein for controls. CONCLUSIONS: The intracellular cystine determination method using immunopurified granulocytes followed by LC-MS/MS analysis improves the inherent variability of mixed leukocyte analysis and eliminates the need for immediate sample preparation following blood draw.
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Cistina/sangue , Cistinose/sangue , Cistinose/diagnóstico , Granulócitos/patologia , Separação Imunomagnética , Adolescente , Adulto , Criança , Pré-Escolar , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Epidermal keratinocytes participate in immune defense through their capacity to recognize danger, trigger inflammation, and resist infection. However, normal skin immune function must tolerate contact with an abundant community of commensal microbes without inflammation. We hypothesized that microbial environmental conditions dictate the production of molecules that influence epigenetic events and cause keratinocytes to break innate immune tolerance. Propionibacterium acnes, a commensal skin bacterium, produced the short-chain fatty acids (SCFAs) propionate and valerate when provided a lipid source in hypoxic growth conditions, and these SCFAs inhibited histone deacetylase (HDAC) activity. Inhibition of HDAC activity in keratinocytes promoted cytokine expression in response to Toll-like receptor (TLR) ligands for TLR2 or TLR3. This response was opposite to the action of HDAC inhibition on production of inflammatory cytokines by monocytes and involved HDAC8 and HDAC9 because small interfering RNA silencing of these HDACs recapitulated the activity of SCFAs. Analysis of cytokine expression in mice confirmed the response of the epidermis where application of SCFA on the skin surface promoted cytokine expression, whereas subcutaneous administration was inhibitory. These findings show that the products of commensal microbes made under specific conditions will inhibit HDAC activity and break tolerance of the epidermis to inflammatory stimuli.
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Many human skin diseases, such as seborrheic dermatitis, potentially occur due to the over-growth of fungi. It remains a challenge to develop fungicides with a lower risk of generating resistant fungi and non-specifically killing commensal microbes. Our probiotic approaches using a selective fermentation initiator of skin commensal bacteria, fermentation metabolites or their derivatives provide novel therapeutics to rein in the over-growth of fungi. Staphylococcus lugdunensis (S. lugdunensis) bacteria and Candida parapsilosis (C. parapsilosis) fungi coexist in the scalp microbiome. S. lugdunensis interfered with the growth of C. parapsilosis via fermentation. A methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) (mPEG-PCL) copolymer functioned as a selective fermentation initiator of S. lugdunensis, selectively triggering the S. lugdunensis fermentation to produce acetic and isovaleric acids. The acetic acid and its pro-drug diethyleneglycol diacetate (Ac-DEG-Ac) effectively suppressed the growth of C. parapsilosis in vitro and impeded the fungal expansion in the human dandruff. We demonstrate for the first time that S. lugdunensis is a skin probiotic bacterium that can exploit mPEG-PCL to yield fungicidal short-chain fatty acids (SCFAs). The concept of bacterial fermentation as a part of skin immunity to re-balance the dysbiotic microbiome warrants a novel avenue for studying the probiotic function of the skin microbiome in promoting health.
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Alkaptonuria is an autosomal recessive disease involving a deficiency of the enzyme homogentisate dioxygenase, which is involved in the tyrosine degradation pathway. The enzymatic deficiency results in high concentrations of homogentisic acid (HGA), which results in orthopedic and cardiac complications, among other symptoms. Nitisinone (NTBC) has been shown to effectively treat alkaptonuria by blocking the conversion of 4-hydroxyphenylpyruvate to HGA, but there have been concerns that using doses higher than about 2 mg/day could cause excessively high levels of tyrosine, resulting in crystal deposition and corneal pathology. We have enrolled seven patients in a study to determine whether higher doses of NTBC were effective at further reducing HGA levels while maintaining tyrosine at acceptable levels. Patients were given varying doses of NTBC (ranging from 2 to 8 mg/day) over the course of between 0.5 and 3.5 years. Urine HGA, plasma tyrosine levels, and plasma NTBC were then measured longitudinally at various doses. We found that tyrosine concentrations plateaued and did not reach significantly higher levels as NTBC doses were increased above 2 mg/day, while a significant drop in HGA continued from 2 to 4 mg/day, with no significant changes at higher doses. We also demonstrated using untargeted metabolomics that elevations in tyrosine from treatment resulted in proportional elevations in alternative tyrosine metabolic products, that of N-acetyltyrosine and γ-glutamyltyrosine.
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The drug nitisinone (NTBC) is used to treat tyrosinemia type I, and more recently has been also used for the treatment of another disorder of tyrosine metabolism, alkaptonuria. While studying the dose effects of NTBC treatment on alkaptonuria, untargeted metabolomics revealed perturbations in a completely separate pathway, that of tryptophan metabolism. Significant elevations in several indolic compounds associated with the indolepyruvate pathway of tryptophan metabolism were present in NTBC-treated patient sera and correlated with elevations of an intermediate of tyrosine metabolism. Indolic compounds of this pathway have long been associated with commensal bacterial and plant metabolism. These exogenous sources of indoles have been more recently implicated in affecting mammalian cell function and disease. We studied the correlation of these indolic compounds in other disorders of tyrosine metabolism including tyrosinemia types I and II as well as transient tyrosinemia, and demonstrated that 4-hydroxyphenylpyruvate (4-HPP) was directly responsible for the promotion of this pathway. We then investigated the regulation of the indolepyruvate pathway and the role of 4-HPP further in both mammalian cells and intestinal microbial cultures. We demonstrated that several of the indolic products, including indolepyruvate and indolelactate, were in fact generated by human cell metabolism, while the downstream indole metabolite, indolecarboxaldehyde, was produced exclusively by microbial cultures of human gut flora. This study describes a symbiotic perturbation in host and microbiome tryptophan metabolism in response to elevations related to defects of tyrosine metabolism and concomitant drug treatment.
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Alcaptonúria/metabolismo , Cicloexanonas/uso terapêutico , Microbioma Gastrointestinal/fisiologia , Indóis/metabolismo , Nitrobenzoatos/uso terapêutico , Triptofano/metabolismo , Tirosina/metabolismo , Tirosinemias/metabolismo , Aldeídos/metabolismo , Alcaptonúria/sangue , Alcaptonúria/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Indóis/sangue , Espectrometria de Massas , Metabolômica , Ácidos Fenilpirúvicos/metabolismo , Simbiose , Tirosinemias/sangue , Tirosinemias/tratamento farmacológicoRESUMO
Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC-HRMS. This dual-PFP LC-MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC-HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.
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Cysteamine is approved for the treatment of cystinosis and is being evaluated for Huntington's disease and non-alcoholic fatty liver disease. Little is known about the bioavailability and biodistribution of the drug. The aim was to determine plasma, cerebrospinal fluid (CSF), and tissue (liver, kidney, muscle) cysteamine levels following intraduodenal delivery of the drug in rats pretreated and naïve to cysteamine and to estimate the hepatic first-pass effect on cysteamine. Healthy male rats (n = 66) underwent intraduodenal and portal (PV) or jugular (JVC) venous catheterization. Half were pretreated with cysteamine, and half were naïve. Following intraduodenal cysteamine (20 mg/kg), serial blood samples were collected from the PV or the JVC. Animals were sacrificed at specific time points, and CSF and tissue were collected. Cysteamine levels were determined in plasma, CSF, and tissue. The Cmax was achieved in 5-10 min from PV and 5-22.5 min from JVC. The PV-Cmax (P = 0.08), PV-AUC0-t (P = 0.16), JVC-Cmax (P = 0.02) and JVC-AUC0-t (P = 0.03) were higher in naive than in pretreated animals. Plasma cysteamine levels returned to baseline in ≤120 min. The hepatic first-pass effect was estimated at 40%. Peak tissue and CSF cysteamine levels occurred ≤22.5 min, but returned to baseline levels ≤180 min. There was no difference in CSF and tissue cysteamine levels between naïve and pretreated groups, although cysteamine was more rapidly cleared in the pretreated group. Cysteamine is rapidly absorbed from the small intestine, undergoes significant hepatic first-pass metabolism, crosses the blood brain barrier, and is almost undetectable in plasma, CSF, and body tissues 2 h after ingestion. Sustained-release cysteamine may provide prolonged tissue exposure.
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Cisteamina/administração & dosagem , Cisteamina/farmacocinética , Duodeno/metabolismo , Animais , Disponibilidade Biológica , Cateteres de Demora , Cisteamina/sangue , Cisteamina/líquido cefalorraquidiano , Absorção Intestinal , Rim/metabolismo , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Músculo Esquelético/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
The advent of model-enabled workflows in systems biology allows for the integration of experimental data types with genome-scale models to discover new features of biology. This work demonstrates such a workflow, aimed at establishing a metabolomics platform applied to study the differences in metabolomes between anaerobic and aerobic growth of Escherichia coli. Constraint-based modeling was utilized to deduce a target list of compounds for downstream method development. An analytical and experimental methodology was developed and tailored to the compound chemistry and growth conditions of interest. This included the construction of a rapid sampling apparatus for use with anaerobic cultures. The resulting genome-scale data sets for anaerobic and aerobic growth were validated by comparison to previous small-scale studies comparing growth of E. coli under the same conditions. The metabolomics data were then integrated with the E. coli genome-scale metabolic model (GEM) via a sensitivity analysis that utilized reaction thermodynamics to reconcile simulated growth rates and reaction directionalities. This analysis highlighted several optimal network usage inconsistencies, including the incorrect use of the beta-oxidation pathway for synthesis of fatty acids. This analysis also identified enzyme promiscuity for the pykA gene, that is critical for anaerobic growth, and which has not been previously incorporated into metabolic models of E coli.
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Escherichia coli/metabolismo , Metabolômica/métodos , Modelos Biológicos , Aerobiose/fisiologia , Anaerobiose/fisiologia , Bioengenharia , Reatores Biológicos/microbiologia , Redes e Vias Metabólicas/fisiologia , TermodinâmicaRESUMO
Therapy to slow the relentless expansion of interstitial extracellular matrix that leads to renal functional decline in patients with CKD is currently lacking. Because chronic kidney injury increases tissue oxidative stress, we evaluated the antifibrotic efficacy of cysteamine bitartrate, an antioxidant therapy for patients with nephropathic cystinosis, in a mouse model of unilateral ureteral obstruction. Fresh cysteamine (600 mg/kg) was added to drinking water daily beginning on the day of surgery, and outcomes were assessed on days 7, 14, and 21 after surgery. Plasma cysteamine levels showed diurnal variation, with peak levels similar to those observed in patients with cystinosis. In cysteamine-treated mice, fibrosis severity decreased significantly at 14 and 21 days after unilateral ureteral obstruction, and renal oxidized protein levels decreased at each time point, suggesting reduced oxidative stress. Consistent with these results, treatment of cultured macrophages with cysteamine reduced cellular generation of reactive oxygen species. Furthermore, treatment with cysteamine reduced α-smooth muscle actin-positive interstitial myofibroblast proliferation and mRNA levels of extracellular matrix proteins in mice and attenuated myofibroblast differentiation and proliferation in vitro, but did not augment TGF-ß signaling. In a study of renal ischemia reperfusion, cysteamine therapy initiated 10 days after injury and continued for 14 days decreased renal fibrosis by 40%. Taken together, these data suggest previously unrecognized antifibrotic actions of cysteamine via TGF-ß-independent mechanisms that include oxidative stress reduction and attenuation of the myofibroblast response to kidney injury and support further investigation into the potential benefit of cysteamine therapy in the treatment of CKD.
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Antioxidantes/uso terapêutico , Cisteamina/uso terapêutico , Miofibroblastos/efeitos dos fármacos , Insuficiência Renal Crônica/tratamento farmacológico , Actinas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Fibrose , Proteínas de Ligação ao GTP/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Transglutaminases/metabolismo , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
Diabetic kidney disease is the leading cause of ESRD, but few biomarkers of diabetic kidney disease are available. This study used gas chromatography-mass spectrometry to quantify 94 urine metabolites in screening and validation cohorts of patients with diabetes mellitus (DM) and CKD(DM+CKD), in patients with DM without CKD (DM-CKD), and in healthy controls. Compared with levels in healthy controls, 13 metabolites were significantly reduced in the DM+CKD cohorts (P≤0.001), and 12 of the 13 remained significant when compared with the DM-CKD cohort. Many of the differentially expressed metabolites were water-soluble organic anions. Notably, organic anion transporter-1 (OAT1) knockout mice expressed a similar pattern of reduced levels of urinary organic acids, and human kidney tissue from patients with diabetic nephropathy demonstrated lower gene expression of OAT1 and OAT3. Analysis of bioinformatics data indicated that 12 of the 13 differentially expressed metabolites are linked to mitochondrial metabolism and suggested global suppression of mitochondrial activity in diabetic kidney disease. Supporting this analysis, human diabetic kidney sections expressed less mitochondrial protein, urine exosomes from patients with diabetes and CKD had less mitochondrial DNA, and kidney tissues from patients with diabetic kidney disease had lower gene expression of PGC1α (a master regulator of mitochondrial biogenesis). We conclude that urine metabolomics is a reliable source for biomarkers of diabetic complications, and our data suggest that renal organic ion transport and mitochondrial function are dysregulated in diabetic kidney disease.
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Nefropatias Diabéticas/metabolismo , Metabolômica/métodos , Doenças Mitocondriais/etiologia , Adulto , Idoso , Feminino , Taxa de Filtração Glomerular , Humanos , Transporte de Íons , Masculino , Pessoa de Meia-Idade , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Insuficiência Renal Crônica/metabolismo , Fatores de Transcrição/genéticaRESUMO
INTRODUCTION: Ubiquinone (UQ) is a redox active lipid that transfers electrons from complex I or II to complex III in the electron transport chain (ETC). The long-lived Caenorhabditis elegans mutant clk-1 is unable to synthesize its native ubiquinone, and accumulates high amounts of its precursor, 5-demethoxyubiquinone-9 (DMQ(9)). In clk-1, complexes I-III activity is inhibited while complexes II-III activity is normal. We asked whether the complexes I-III defect in clk-1 was caused by: (1) a defect in the ETC; (2) an inhibitory effect of DMQ(9); or (3) a decreased amount of ubiquinone. METHODS: We extracted the endogenous quinones from wildtype (N2) and clk-1 mitochondria, replenished them with exogenous ubiquinones, and measured ETC activities. RESULTS: Replenishment of extracted mutant and wildtype mitochondria resulted in equal enzymatic activities for complexes I-III and II-III ETC assays. Blue native gels showed that supercomplex formation was indistinguishable between clk-1 and N2. The addition of a pentane extract from clk-1 mitochondria containing DMQ(9) to wildtype mitochondria specifically inhibited complexes I-III activity. UQ in clk-1 mitochondria was oxidized compared to N2. DISCUSSION: Our results show that no measurable intrinsic ETC defect exists in clk-1 mitochondria. The data indicate that DMQ(9) specifically inhibits electron transfer from complex I to ubiquinone.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Longevidade/fisiologia , Mitocôndrias/metabolismo , Mutação , Ubiquinona/análogos & derivados , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/genética , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Cysteamine bitartrate is taken lifelong, every 6 h and for the treatment of cystinosis. Recent studies using cysteamine for for other diseases such as neurodegenerative disorders adopt the same dosing regimen for cysteamine. Regular cysteamine bitartrate (Cystagon) may cause upper gastrointestinal symptoms in some patients. WHAT THIS STUDY ADDS: This is the only study that provides pharmacokinetic data for cysteamine delivered in an enteric-release preparation in normal subjects. EC-cysteamine is very well tolerated and does not cause increased gastrin concentrations, even at relatively high doses. EC-cysteamine at the higher dose results in better drug uptake as measured by Cmax and AUC and is more likely to be effective. AIMS: Cysteamine bitartrate (Cystagon) is the approved treatment for cystinosis. Poor compliance and patient outcome may occur because the drug needs to be taken every 6 h and in some patients causes gastrointestinal symptoms due to hypergastrinaemia. A formulation of cysteamine requiring twice daily ingestion would improve the quality of life for these patients. This study compares the pharmacokinetics and gastrin production following cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated in normal healthy subjects. METHODS: Enteric-coated cysteamine was prepared. Following single doses of cysteamine bitartrate non-enteric-coated 450 mg and cysteamine bitartrate enteric-coated 450 mg and 900 mg, serial plasma cysteamine and gastrin concentrations were measured. Two subjects also received cysteamine bitartrate non-enteric-coated 900 mg. Gastrointestinal (GI) symptoms were recorded. RESULTS: Six healthy adults (mean age 20.7 years, range 18-24 years; mean weight 59.3 kg) received drug. All post-dose gastrin concentrations were within the normal range (<100 pg ml(-1)). The tmax following cysteamine bitartrate non-enteric-coated (mean and SD is 75+/-19 min) was shorter than cysteamine bitartrate enteric-coated (220+/-74 min) (P=0.001), but only the Cmax and AUC estimates following 900 mg cysteamine bitartrate enteric-coated were significantly greater than any of the other preparations or doses (P<0.05). One patient had GI symptoms following both 900 mg cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated. CONCLUSION: Although patient numbers were low, single high doses of cysteamine bitartrate enteric-coated were better tolerated than similar doses of cysteamine bitartrate non-enteric-coated in the healthy subjects and all had normal gastrin concentrations. The delayed tmax following cysteamine bitartrate enteric-coated suggested that the cysteamine was released enterically.
Assuntos
Cisteamina/administração & dosagem , Cistinose/tratamento farmacológico , Gastrinas/sangue , Protetores contra Radiação/administração & dosagem , Adolescente , Área Sob a Curva , Cisteamina/farmacocinética , Feminino , Humanos , Absorção Intestinal/fisiologia , Masculino , Protetores contra Radiação/farmacocinética , Comprimidos com Revestimento Entérico , Adulto JovemRESUMO
OBJECTIVE: Cystinosis causes renal and other organ failure. Treatment with 6-hourly cysteamine bitartrate (Cystagon, Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A recent study showed that an enteric-release cysteamine required less frequent daily dosing. This report describes the long-term use of enteric-coated (EC) cysteamine bitartrate (Cystagon) in children with cystinosis. STUDY DESIGN: After a pharmacokinetic and pharmacodynamic study of EC-cysteamine in children with cystinosis, 5 patients remained on twice-daily treatment. White blood cell cystine levels were measured 12 hours after ingestion every 4 to 8 weeks. These levels were then compared with the patient's previous 6-h post-dose levels taken while on regular cysteamine bitartrate before entering the study. Blood chemistry was also measured. RESULTS: Five children with cystinosis (mean age, 9 years; range, 8 to 17 years) who previously took cysteamine bitartrate (mean dose, 47 mg/kg body wt), received EC-cysteamine for 10 to 27 months (mean dose, 25 mg/kg body wt) and had mean white blood cell cystine levels of 0.77 and 0.71 nmol half-cystine/mg protein, respectively. During the study period, patients maintained adequate growth and there was no significant deterioration in renal or thyroid function. Two children were required to restart acid suppression after 6 months on EC-cysteamine therapy. CONCLUSIONS: Long-term, twice-daily EC-cysteamine, given at approximately 60% of the previous daily dose of cysteamine bitartrate, was effective at maintaining white blood cell cystine levels within a satisfactory range. There was no significant deterioration in renal or thyroid function.
Assuntos
Cisteamina/administração & dosagem , Cistinose/tratamento farmacológico , Adolescente , Criança , Creatinina/sangue , Cistina/sangue , Cistinose/sangue , Esquema de Medicação , Feminino , Humanos , Leucócitos/metabolismo , Masculino , Comprimidos com Revestimento EntéricoRESUMO
OBJECTIVE: Cystinosis causes renal and other organ failure. Regular 6-hourly cysteamine bitartrate (Cystagon; Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A formulation of cysteamine requiring less frequent dosing may improve compliance and possibly patient outcome. METHODS: Enteric-release cysteamine was prepared. For a period of 1 month, patients received their regular cysteamine dose every 6 hours (stage I). The patients then underwent pharmacokinetic and pharmacodynamic studies following washout periods using single-doses of cysteamine and enteric-release cysteamine (stage II). Finally, the patients commenced regular enteric-release cysteamine therapy (stage III). Weekly trough white blood cell (WBC) cystine levels were recorded. RESULTS: Seven children with cystinosis (mean age, 11.8 years; range, 8-17 years) who received cysteamine and enteric-release cysteamine (mean dose, 45 and 28.8 mg/kg body weight/day, respectively) had mean WBC cystine levels of 0.7+/-0.3 and 0.41+/-0.22 nmol half-cystine/mg protein in study stages I and III, respectively. Study stage II showed that the mean time (T(max)) to reach the maximum plasma cysteamine level (C(max)) was longer for enteric-release cysteamine than for cysteamine (176 minutes vs 60 minutes; P=.001), but the mean C(max) at the same dose was similar. Mean serum gastrin levels were similar after ingestion of cysteamine and enteric-release cysteamine. CONCLUSIONS: Twelve-hour enteric-release cysteamine, given at approximately 60% of the previous daily dose of cysteamine, was effective in maintaining trough WBC cystine levels within a satisfactory range.