RESUMEN
Thiamine metabolism dysfunction syndrome (THMD) comprises a group of clinically and genetically heterogeneous encephalopathies with autosomal recessive inheritance. Four genes, SLC19A3, SLC25A19, SLC19A2, and TPK1, are associated with this disorder. This study aimed to explore the clinical, biochemical and molecular characteristics of seven Chinese patients with THMD. Targeted next-generation sequencing of mitochondrial DNA and nuclear DNA was used to identify the causative mutations. The patients presented with subacute encephalopathy between the ages of 1-27 months. Brain magnetic resonance imaging (MRI) revealed abnormalities in the basal ganglia, indicating Leigh syndrome. Urine α-ketoglutarate in five patients was elevated. In four patients, five novel mutations (c.1276_1278delTAC, c.265A > C, c.197T > C, c.850T > C, whole gene deletion) were found in SLC19A3, which is associated with THMD2. In two patients, four novel mutations (c.194C > T, c.454C > A, c.481G > A, and c.550G > C) were identified in SLC25A19, supporting a diagnosis of THMD4. In one patient, two novel mutations (c.395T > C and c.614-1G > A) were detected in TPK1, which is indicative of THMD5. The patients received thiamine, biotin, and symptomatic therapy, upon which six patients demonstrated clinical improvement. Our findings expanded the phenotypic and genotypic spectrum of THMD, with eleven novel mutations identified in seven Chinese patients. Early diagnosis and treatment have a significant impact on prognosis.
Asunto(s)
Encefalopatías/genética , ADN Mitocondrial/genética , Enfermedad de Leigh/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Tiamina Pirofosfoquinasa/genética , Tiamina/metabolismo , Pueblo Asiatico , Biotina/uso terapéutico , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Encefalopatías/diagnóstico , Encefalopatías/diagnóstico por imagen , Encefalopatías/fisiopatología , Preescolar , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Ácidos Cetoglutáricos/orina , Enfermedad de Leigh/diagnóstico , Enfermedad de Leigh/diagnóstico por imagen , Enfermedad de Leigh/fisiopatología , Imagen por Resonancia Magnética , Masculino , Enfermedades Metabólicas/diagnóstico , Enfermedades Metabólicas/diagnóstico por imagen , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/fisiopatología , Tiamina/genética , Tiamina/uso terapéuticoRESUMEN
OBJECTIVE: SLC13A3 encodes the plasma membrane Na+ /dicarboxylate cotransporter 3, which imports inside the cell 4 to 6 carbon dicarboxylates as well as N-acetylaspartate (NAA). SLC13A3 is mainly expressed in kidney, in astrocytes, and in the choroid plexus. We describe two unrelated patients presenting with acute, reversible (and recurrent in one) neurological deterioration during a febrile illness. Both patients exhibited a reversible leukoencephalopathy and a urinary excretion of α-ketoglutarate (αKG) that was markedly increased and persisted over time. In one patient, increased concentrations of cerebrospinal fluid NAA and dicarboxylates (including αKG) were observed. Extensive workup was unsuccessful, and a genetic cause was suspected. METHODS: Whole exome sequencing (WES) was performed. Our teams were connected through GeneMatcher. RESULTS: WES analysis revealed variants in SLC13A3. A homozygous missense mutation (p.Ala254Asp) was found in the first patient. The second patient was heterozygous for another missense mutation (p.Gly548Ser) and an intronic mutation affecting splicing as demonstrated by reverse transcriptase polymerase chain reaction performed in muscle tissue (c.1016 + 3A > G). Mutations and segregation were confirmed by Sanger sequencing. Functional studies performed on HEK293T cells transiently transfected with wild-type and mutant SLC13A3 indicated that the missense mutations caused a marked reduction in the capacity to transport αKG, succinate, and NAA. INTERPRETATION: SLC13A3 deficiency causes acute and reversible leukoencephalopathy with marked accumulation of αKG. Urine organic acids (especially αKG and NAA) and SLC13A3 mutations should be screened in patients presenting with unexplained reversible leukoencephalopathy, for which SLC13A3 deficiency is a novel differential diagnosis. ANN NEUROL 2019;85:385-395.
Asunto(s)
Ácido Aspártico/análogos & derivados , Ácidos Cetoglutáricos/metabolismo , Leucoencefalopatías/genética , Simportadores/genética , Adolescente , Ácido Aspártico/líquido cefalorraquídeo , Ácido Aspártico/metabolismo , Preescolar , Femenino , Células HEK293 , Humanos , Ácidos Cetoglutáricos/líquido cefalorraquídeo , Ácidos Cetoglutáricos/orina , Leucoencefalopatías/metabolismo , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Mutación Missense , Linaje , Infecciones del Sistema Respiratorio , Ácido Succínico/metabolismo , Simportadores/metabolismo , Tonsilitis , Secuenciación del ExomaRESUMEN
BACKGROUND: Primary hyperoxaluria type 3 (PH3) is a recently described cause of childhood renal calculi. It results from mutations in the HOGA1 gene and most cases have been diagnosed after clinical ascertainment, exclusion of other genetic hyperoxalurias and mutation testing. Metabolite testing has not been widely applied but holds promise for the rapid screening and diagnosis of patients who are not specifically suspected to have PH3. CASE-DIAGNOSIS/TREATMENT: Two cases presented with renal calculi. Urine metabolite testing by tandem mass spectrometry was performed as part of the routine diagnostic work-up for this condition. Both had significantly increased levels of the PH3 urine marker 4-hydroxyglutamate and related metabolites. The diagnosis of PH3 was confirmed by the finding of bi-allelic damaging HOGA1 mutations. CONCLUSIONS: Urine screening by tandem mass spectrometry is a rapid, high-throughput test that can detect PH3 cases that may otherwise not be diagnosed.
Asunto(s)
Glutamatos/orina , Hiperoxaluria Primaria/diagnóstico , Ácidos Cetoglutáricos/orina , Cálculos Renales/etiología , Oxalatos/orina , Adolescente , Femenino , Glutamatos/metabolismo , Humanos , Hiperoxaluria Primaria/complicaciones , Hiperoxaluria Primaria/genética , Hiperoxaluria Primaria/orina , Lactante , Ácidos Cetoglutáricos/metabolismo , Cálculos Renales/terapia , Cálculos Renales/orina , Litotricia , Masculino , Metabolómica/métodos , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Recurrencia , Espectrometría de Masas en TándemRESUMEN
Considering the pharmacological effects of chiral drugs, enantiopure drugs may differ from their racemic mixture formulation in efficacy, potency, or adverse effects. Levomethorphan (LVM) and Dextromethorphan (DXM) act on the central nervous system and exhibit different pharmacological features. LVM, the l-stereoisomer of methorphan, shows many similarities to opiates such as heroin, morphine and codeine, including the potential for addiction, while the d-stereoisomer, DXM, does not have the same opioid effect. In the present study, NMR-based metabolomics were performed on the urine of rats treated with these stereoisomers, and showed significant differences in metabolic profiles. In urine within 24 h after treatment of these samples, levels of citrate, 2-oxoglutarate, creatine, and dimethylglycine were higher in LVM-treated rats than in DXM-treated rats. While urinary levels of hippurate and creatinine gradually increased over 72 h in DXM-treated rats, these metabolites were decreased in the urine by 48-72 h after treatment with LVM. The levels of these changed metabolites may provide the first evidence for different cellular responses to the metabolism of stereoisomers.
Asunto(s)
Drogas Ilícitas/metabolismo , Drogas Ilícitas/farmacología , Espectroscopía de Resonancia Magnética , Animales , Ácido Cítrico/orina , Creatina/orina , Creatinina/orina , Dextrometorfano/química , Dextrometorfano/metabolismo , Dextrometorfano/farmacología , Hipuratos/orina , Drogas Ilícitas/química , Ácidos Cetoglutáricos/orina , Masculino , Metabolómica , Ratas , Sarcosina/análogos & derivados , Sarcosina/orina , Estereoisomerismo , Factores de TiempoRESUMEN
Current biomarkers of renal disease in systemic vasculitis lack predictive value and are insensitive to early damage. To identify novel biomarkers of renal vasculitis flare, we analysed the longitudinal urinary metabolomic profile of a rat model of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Wistar-Kyoto (WKY) rats were immunised with human myeloperoxidase (MPO). Urine was obtained at regular intervals for 181 days, after which relapse was induced by re-challenge with MPO. Urinary metabolites were assessed in an unbiased fashion using nuclear magnetic resonance (NMR) spectroscopy, and analysed using partial least squares discriminant analysis (PLS-DA) and partial least squares regression (PLS-R). At 56 days post-immunisation, we found that rats with vasculitis had a significantly different urinary metabolite profile than control animals; the observed PLS-DA clusters dissipated between 56 and 181 days, and re-emerged with relapse. The metabolites most altered in rats with active or relapsing vasculitis were trimethylamine N-oxide (TMAO), citrate and 2-oxoglutarate. Myo-inositol was also moderately predictive. The key urine metabolites identified in rats were confirmed in a large cohort of patients using liquid chromatography-mass spectrometry (LC-MS). Hypocitraturia and elevated urinary myo-inositol remained associated with active disease, with the urine myo-inositol:citrate ratio being tightly correlated with active renal vasculitis.
Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/orina , Enfermedades Renales/orina , Metabolómica/métodos , Peroxidasa/administración & dosificación , Animales , Ácido Cítrico/orina , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunización , Ácidos Cetoglutáricos/orina , Análisis de los Mínimos Cuadrados , Masculino , Metilaminas/orina , Peroxidasa/inmunología , Ratas , Ratas Endogámicas WKY , RecurrenciaRESUMEN
B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B1 and B6 and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH2PO4 (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B1 deficiency increased urinary levels of all 2-oxo acids, while vitamin B6 deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.
Asunto(s)
Cromatografía Líquida de Alta Presión/normas , Ácido Pantoténico/orina , Deficiencia de Tiamina/orina , Tiamina/orina , Deficiencia de Vitamina B 6/orina , Vitamina B 6/orina , Adipatos/orina , Animales , Biomarcadores/orina , Hemiterpenos , Cetoácidos/orina , Ácidos Cetoglutáricos/orina , Masculino , Ácido Oxaloacético/orina , Ácido Pantoténico/deficiencia , Fenilendiaminas/química , Ácido Pirúvico/orina , Ratas , Ratas Wistar , Espectrometría de FluorescenciaRESUMEN
INTRODUCTION: Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria. METHODS: In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death. RESULTS: Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean ± SD) was elevated in severe malaria (8.2 mEq/L ± 4.5) and severe sepsis (8.6 mEq/L ± 7.7) compared with uncomplicated malaria (6.0 mEq/L ± 5.1) and encephalopathy (6.6 mEq/L ± 4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and ß-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6-7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5-7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81). CONCLUSIONS: Newly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.
Asunto(s)
Malaria Falciparum/etiología , Ácido 3-Hidroxibutírico/sangre , Acidosis/complicaciones , Adolescente , Adulto , Anciano , Cromatografía Liquida , Femenino , Humanos , Hidroxibutiratos/sangre , Ácidos Cetoglutáricos/orina , Ácido Láctico/sangre , Malaria Falciparum/sangre , Malaria Falciparum/metabolismo , Malaria Falciparum/mortalidad , Malaria Falciparum/orina , Masculino , Malonatos/orina , Espectrometría de Masas , Ácido Metilmalónico/orina , Persona de Mediana Edad , Fenilpropionatos/sangre , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Metabolomics GC-MS samples involve high complexity data that must be effectively resolved to produce chemically meaningful results. Multivariate curve resolution-alternating least squares (MCR-ALS) is the most frequently reported technique for that purpose. More recently, independent component analysis (ICA) has been reported as an alternative to MCR. Those algorithms attempt to infer a model describing the observed data and, therefore, the least squares regression used in MCR assumes that the data is a linear combination of that model. However, due to the high complexity of real data, the construction of a model to describe optimally the observed data is a critical step and these algorithms should prevent the influence from outlier data. This study proves independent component regression (ICR) as an alternative for GC-MS compound identification. Both ICR and MCR though require least squares regression to correctly resolve the mixtures. In this paper, a novel orthogonal signal deconvolution (OSD) approach is introduced, which uses principal component analysis to determine the compound spectra. The study includes a compound identification comparison between the results by ICA-OSD, MCR-OSD, ICR and MCR-ALS using pure standards and human serum samples. Results shows that ICR may be used as an alternative to multivariate curve methods, as ICR efficiency is comparable to MCR-ALS. Also, the study demonstrates that the proposed OSD approach achieves greater spectral resolution accuracy than the traditional least squares approach when compounds elute under undue interference of biological matrices.
Asunto(s)
Metaboloma , Algoritmos , Aminoácidos/sangre , Ácido Cítrico/sangre , Ácido Cítrico/orina , Cromatografía de Gases y Espectrometría de Masas , Humanos , Inositol/sangre , Inositol/orina , Ácidos Cetoglutáricos/sangre , Ácidos Cetoglutáricos/orina , Análisis de los Mínimos Cuadrados , Análisis de Componente Principal , Urea/sangre , Urea/orinaRESUMEN
BACKGROUND: A simple and sensitive hollow fiber-liquid phase microextraction with in situ derivatization method was developed for the determination of α-ketoglutaric (α-KG) and pyruvic acids (PA) in small-volume urine samples. 2,4,6-trichloro phenyl hydrazine was used as derivatization agent. RESULTS: Under the optimum extraction conditions, enrichment factors of 742 and 400 for α-KG and PA, respectively, were achieved. Calibration curves were linear over the range 1 to 1000 ng/ml (r(2) ≥ 0.998). Detection and quantitation limits were 0.03 and 0.02, and 0.10 and 0.05 ng/ml for α-KG and PA, respectively. CONCLUSION: The concentrations in diabetic II and liver cancer samples were significantly lower than those from healthy people, showing their potential as biomarkers for these diseases.
Asunto(s)
Biomarcadores de Tumor/orina , Diabetes Mellitus Tipo 2/orina , Ácidos Cetoglutáricos/orina , Neoplasias Hepáticas/orina , Ácido Pirúvico/orina , Urinálisis/métodos , Biomarcadores de Tumor/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Humanos , Ácidos Cetoglutáricos/aislamiento & purificación , Microextracción en Fase Líquida , Ácido Pirúvico/aislamiento & purificaciónRESUMEN
UNLABELLED: There is no clinically applicable biomarker for surveillance of hepatocellular carcinoma (HCC), because the sensitivity of serum alpha-fetoprotein (AFP) is too low for this purpose. Here, we determined the diagnostic performance of a panel of urinary metabolites of HCC patients from West Africa. Urine samples were collected from Nigerian and Gambian patients recruited on the case-control platform of the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) program. Urinary proton nuclear magnetic resonance ((1) H-NMR) spectroscopy was used to metabolically phenotype 290 subjects: 63 with HCC; 32 with cirrhosis (Cir); 107 with noncirrhotic liver disease (DC); and 88 normal control (NC) healthy volunteers. Urine samples from a further cohort of 463 subjects (141 HCC, 56 Cir, 178 DC, and 88 NC) were analyzed, the results of which validated the initial cohort. The urinary metabotype of patients with HCC was distinct from those with Cir, DC, and NC with areas under the receiver operating characteristic (AUROC) curves of 0.86 (0.78-0.94), 0.93 (0.89-0.97), and 0.89 (0.80-0.98) in the training set and 0.81 (0.73-0.89), 0.96 (0.94-0.99), and 0.90 (0.85-0.96), respectively, in the validation cohort. A urinary metabolite panel, comprising inosine, indole-3-acetate, galactose, and an N-acetylated amino acid (NAA), showed a high sensitivity (86.9% [75.8-94.2]) and specificity (90.3% [74.2-98.0]) in the discrimination of HCC from cirrhosis, a finding that was corroborated in a validation cohort (AUROC: urinary panel = 0.72; AFP = 0.58). Metabolites that were significantly increased in urine of HCC patients, and which correlated with clinical stage of HCC, were NAA, dimethylglycine, 1-methylnicotinamide, methionine, acetylcarnitine, 2-oxoglutarate, choline, and creatine. CONCLUSION: The urinary metabotyping of this West African cohort identified and validated a metabolite panel that diagnostically outperforms serum AFP.
Asunto(s)
Biomarcadores de Tumor/orina , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Metionina/orina , Niacinamida/análogos & derivados , Sarcosina/análogos & derivados , alfa-Fetoproteínas/orina , Acetilcarnitina/orina , Adolescente , Adulto , África Occidental/epidemiología , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/epidemiología , Carcinoma Hepatocelular/orina , Estudios de Casos y Controles , Colina/orina , Creatina/orina , Femenino , Humanos , Ácidos Cetoglutáricos/orina , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/orina , Masculino , Persona de Mediana Edad , Niacinamida/orina , Fenotipo , Reproducibilidad de los Resultados , Sarcosina/orina , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L - Glutamine + α - keto acid + H2O â α - ketoglutarate + L - amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance.
Asunto(s)
Amoníaco/metabolismo , Encefalopatía Hepática/metabolismo , Hiperamonemia/metabolismo , Ácidos Cetoglutáricos/metabolismo , Trastornos Innatos del Ciclo de la Urea/metabolismo , Amidohidrolasas/metabolismo , Aminohidrolasas/metabolismo , Animales , Biomarcadores , Carbono/metabolismo , Glutamina/metabolismo , Encefalopatía Hepática/etiología , Humanos , Hiperamonemia/clasificación , Hiperamonemia/diagnóstico , Cetoácidos/metabolismo , Ácidos Cetoglutáricos/líquido cefalorraquídeo , Ácidos Cetoglutáricos/orina , Quinurenina/metabolismo , Hepatopatías/líquido cefalorraquídeo , Mamíferos/metabolismo , Metionina/metabolismo , Nitrógeno/metabolismo , Azufre/metabolismo , Transaminasas/metabolismo , Transaminasas/fisiologíaRESUMEN
Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle's loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A-non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl(-)-dependent HCO(3)(-) secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1(-/-) mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1(-/-) mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO(3)(-) secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.
Asunto(s)
Equilibrio Ácido-Base , Ácidos Cetoglutáricos/orina , Túbulos Renales Colectores/fisiología , Comunicación Paracrina , Animales , Bicarbonatos/metabolismo , Técnicas In Vitro , Ácidos Cetoglutáricos/sangre , Masculino , Ratones , Ratones Noqueados , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Cloruro de Sodio/metabolismoRESUMEN
Intrarenal control mechanisms play an important role in the maintenance of body fluid and electrolyte balance and pH homeostasis. Recent discoveries of new ion transport and regulatory pathways in the distal nephron and collecting duct system have helped to better our understanding of these critical kidney functions and identified new potential therapeutic targets and approaches. In this issue of the JCI, Tokonami et al. report on the function of an exciting new paracrine mediator, the mitochondrial the citric acid(TCA) cycle intermediate α-ketoglutarate (αKG), which via its OXGR1 receptor plays an unexpected, nontraditional role in the adaptive regulation of renal HCO(3â») secretion and salt reabsorption.
Asunto(s)
Equilibrio Ácido-Base , Ácidos Cetoglutáricos/orina , Túbulos Renales Colectores/fisiología , Comunicación Paracrina , Animales , MasculinoRESUMEN
A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of α-ketoglutaric acid (α-KG), L-carnitine (L-CAR) and acetyl-L-carnitine (acetyl-L-CAR) in human urine as potential biomarkers of cardiovascular disease. The separation was performed using an isocratic elution of 0.1% formic acid in water and acetonitrile (97:3, v/v) on an Acclaim 120 C8 column (150 mm × 4.6 mm, 3.0 µm). The flow rate of the mobile phase was 1.2 mL/min and the total assay run time was 3 min. Detection was performed on a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode via an electrospray ionization (ESI) source in positive and negative ion modes. This method covered a linearity range of 0.1-500 ng/mL for L-CAR and acetyl-L-CAR and 1-1000 ng/mL for α-KG with lower limits of quantification (LLOQ) of 0.08 ng/mL for L-CAR, 0.04 ng/mL for acetyl-L-CAR and 0.8 ng/mL for α-KG. The intra-day and inter-day precision and accuracy of the quality control samples exhibited relative standard deviations of less than 5.54% and relative error values from -5.95% to 3.11%. Analyte stability was evaluated under various sample preparation, analysis and storage conditions and varied from -9.89% to -0.47%. A two-step solid-phase extraction (SPE) procedure using silica gel and quaternary amine cartridges was used for urine sample cleanup. The average recoveries for all analyzed compounds were better than 86.64% at three concentrations. The method was successfully applied for the quantitation of α-KG, L-CAR and acetyl-L-CAR in human urine samples.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Insuficiencia Cardíaca/orina , Espectrometría de Masas en Tándem/métodos , Acetilcarnitina/química , Acetilcarnitina/orina , Biomarcadores/química , Biomarcadores/orina , Carnitina/química , Carnitina/orina , Estudios de Casos y Controles , Estabilidad de Medicamentos , Humanos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/orina , Modelos Lineales , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Extracción en Fase Sólida , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
A rapid, highly sensitive, and selective method was applied in a non-invasive way to investigate the antidepressant action of Xiaoyaosan (XYS) using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and chemometrics. Many significantly altered metabolites were used to explain the mechanism. Venlafaxine HCl and fluoxetine HCl were used as chemical positive control drugs with a relatively clear mechanism of action to evaluate the efficiency and to predict the mechanism of action of XYS. Urine obtained from rats subjected to chronic unpredictable mild stress (CUMS) was analyzed by UPLC-MS. Distinct changes in the pattern of metabolites in the rat urine after CUMS production and drug intervention were observed using partial least squares-discriminant analysis. The results of behavioral tests and multivariate analysis showed that CUMS was successfully reproduced, and a moderate-dose XYS produced significant therapeutic effects in the rodent model, equivalent to those of the positive control drugs, venlafaxine HCl and fluoxetine HCl. Metabolites with significant changes induced by CUMS were identified, and 17 biomarker candidates for stress and drug intervention were identified. The therapeutic effect of XYS on depression may involve regulation of the dysfunctions of energy metabolism, amino acid metabolism, and gut microflora changes. Metabonomic methods are valuable tools for measuring efficacy and mechanisms of action in the study of traditional Chinese medicines.
Asunto(s)
Antidepresivos/uso terapéutico , Medicamentos Herbarios Chinos/uso terapéutico , Tracto Gastrointestinal/microbiología , Redes y Vías Metabólicas/efectos de los fármacos , Microbiota/efectos de los fármacos , Fitoterapia , Animales , Antidepresivos/orina , Benzoatos/orina , Biomarcadores/orina , Hidrocarburos Aromáticos con Puentes/orina , Catequina/orina , Chalcona/análogos & derivados , Chalcona/orina , Cromatografía Liquida , Ácido Cítrico/orina , Ciclo del Ácido Cítrico/efectos de los fármacos , Ácidos Cumáricos/orina , Creatina Quinasa/efectos de los fármacos , Creatina Quinasa/orina , Creatinina/orina , Ciclohexanoles/uso terapéutico , Medicamentos Herbarios Chinos/análisis , Flavanonas/orina , Fluoxetina/uso terapéutico , Ácido Gálico/orina , Glucósidos/orina , Glicina/análogos & derivados , Glicina/efectos de los fármacos , Glicina/orina , Hipuratos/orina , Ácidos Cetoglutáricos/orina , Ácido Quinurénico/orina , Masculino , Espectrometría de Masas , Metabolómica , Monoterpenos , Extractos Vegetales/uso terapéutico , Ratas , Ratas Sprague-Dawley , Estrés Psicológico/tratamiento farmacológico , Triptófano/efectos de los fármacos , Triptófano/orina , Tirosina/efectos de los fármacos , Tirosina/orina , Clorhidrato de VenlafaxinaRESUMEN
To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.
Asunto(s)
Biomarcadores/orina , Medicamentos Herbarios Chinos/farmacología , Enfermedades Renales/orina , Morinda/química , Deficiencia Yang/orina , Alanina/orina , Animales , Betaína/orina , Ácido Cítrico/orina , Medicamentos Herbarios Chinos/aislamiento & purificación , Hidrocortisona , Ácidos Cetoglutáricos/orina , Enfermedades Renales/inducido químicamente , Ácido Láctico/orina , Espectroscopía de Resonancia Magnética , Masculino , Metabolómica/métodos , Plantas Medicinales/química , Análisis de Componente Principal , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Sarcosina/orina , Ácido Succínico/orina , Taurina/orina , Deficiencia Yang/inducido químicamenteRESUMEN
Pantothenic acid (PaA) is involved in the metabolism of amino acids as well as fatty acid. We investigated the systemic metabolism of amino acids in PaA-deficient rats. For this purpose, urine samples were collected and 2-oxo acids and L-tryptophan (L-Trp) and its metabolites including nicotinamide were measured. Group 1 was freely fed a conventional chemically-defined complete diet and used as an ad lib-fed control, which group was used for showing reference values. Group 2 was freely fed the complete diet without PaA (PaA-free diet) and used as a PaA-deficient group. Group 3 was fed the complete diet, but the daily food amount was equal to the amount of the PaA-deficient group and used as a pair-fed control group. All rats were orally administered 100 mg of L-Trp/kg body weight at 09:00 on day 34 of the experiment and the following 24-h urine samples were collected. The urinary excretion of the sum of pyruvic acid and oxaloacetic acid was higher in rats fed the PaA-free diets than in the rats fed pair-fed the complete diet. PaA deficiency elicited the increased urinary excretion of anthranilic acid and kynurenic acid, while the urinary excretion of xanthurenic acid decreased. The urinary excretion of L-Trp itself, 3-hydroxyanthranilic acid, and quinolinic acid revealed no differences between the rats fed the PaA-free and pair-fed the complete diets. PaA deficiency elicited the increased excretion of N(1)-methylnicotinamide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-methyl-4-pyridone-3-carboxamide. These findings suggest that PaA deficiency disturbs the amino acid catabolism.
Asunto(s)
Adipatos/orina , Ácidos Cetoglutáricos/orina , Niacinamida/orina , Ácido Oxaloacético/orina , Ácido Pantoténico/deficiencia , Ácido Pirúvico/orina , Adipatos/química , Animales , Modelos Animales de Enfermedad , Ácidos Cetoglutáricos/química , Masculino , Ácido Oxaloacético/química , Ácido Pantoténico/administración & dosificación , Ácido Pantoténico/orina , Ácido Pirúvico/química , Ratas , Ratas Wistar , Triptófano/administración & dosificación , Triptófano/orinaRESUMEN
AIM: The purpose of this research was to use metabolomics to investigate the cystic phenotype in the Lewis polycystic kidney rat. METHODS: Spot urine samples were collected from four male Lewis control and five male Lewis polycystic kidney rats aged 5 weeks, before kidney function was significantly impaired. Metabolites were extracted from urine and analysed using gas chromatography-mass spectrometry. Principal component analysis was used to determine key metabolites contributing to the variance observed between sample groups. RESULTS: With the development of a metabolomics method to analyse Lewis and Lewis polycystic kidney rat urine, 2-ketoglutaric acid, allantoin, uric acid and hippuric acid were identified as potential biomarkers of cystic disease in the rat model. CONCLUSION: The findings of this study demonstrate the potential of metabolomics to further investigate kidney disease.