RESUMEN
Concurrent measurement of tyrosine, tryptophan and their metabolites, and other co-factors could help to diagnose and better understand a wide range of metabolic and neurological disorders. The two metabolic pathways are closely related to each other through co-factors, regulator molecules and enzymes. By using high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry, we present a robust, selective and comprehensive method to determine 30 molecules within 20 min using a Waters Atlantis dC18. The method was validated according to the guideline of European Medicines Agency on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all >0.998. Intra-day and inter-day accuracy were between 80-119% and 81-117%, precision 1-19% respectively. The method was applied to measure TYR, TRP and their metabolites, and other neurologically important molecules in human serum and CSF samples. The assay can facilitate the diagnosis and is suitable for determination of reference values in clinical laboratories.
Asunto(s)
Biomarcadores/análisis , Pruebas de Química Clínica/métodos , Triptófano/análisis , Tirosina/análisis , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Cromatografía Líquida de Alta Presión , Pruebas de Química Clínica/normas , Humanos , Redes y Vías Metabólicas , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray , Triptófano/sangre , Triptófano/líquido cefalorraquídeo , Tirosina/sangre , Tirosina/líquido cefalorraquídeoRESUMEN
Capillary electrophoresis (CE) is a well-established and one of the most powerful separation techniques in the field of chiral separations. Its hyphenation with mass spectrometry (MS) combines both the high separation efficiency and low sample consumption of CE and the high sensitivity and structural information of MS. Thus, the outstanding chiral resolution power of CE along with the MS advantages makes CE-MS a perfect combination to achieve sensitive enantioseparations. This chapter describes three representative examples of different approaches used in the chiral analysis of amino acids in biological fluids by CE-MS. The first methodology uses the partial filling technique to avoid the entry of cyclodextrins in the MS source. The second method shows the possibility to carry out the direct coupling EKC-MS even when a relative high concentration of a native cyclodextrin is used as chiral selector. The last example illustrates an alternative strategy based on the formation of stable diastereomers between an enantiomerically pure chiral reagent and the amino acids enantiomers which can be separated in an achiral environment.
Asunto(s)
Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Animales , Cromatografía Capilar Electrocinética Micelar , Fenilalanina/sangre , Fenilalanina/líquido cefalorraquídeo , Ratas , Estereoisomerismo , Tirosina/sangre , Tirosina/líquido cefalorraquídeoRESUMEN
Huntington's disease (HD) is a severe neurological disease leading to psychiatric symptoms, motor impairment and cognitive decline. The disease is caused by a CAG expansion in the huntingtin (HTT) gene, but how this translates into the clinical phenotype of HD remains elusive. Using liquid chromatography mass spectrometry, we analyzed the metabolome of cerebrospinal fluid (CSF) from premanifest and manifest HD subjects as well as control subjects. Inter-group differences revealed that the tyrosine metabolism, including tyrosine, thyroxine, L-DOPA and dopamine, was significantly altered in manifest compared with premanifest HD. These metabolites demonstrated moderate to strong associations to measures of disease severity and symptoms. Thyroxine and dopamine also correlated with the five year risk of onset in premanifest HD subjects. The phenylalanine and the purine metabolisms were also significantly altered, but associated less to disease severity. Decreased levels of lumichrome were commonly found in mutated HTT carriers and the levels correlated with the five year risk of disease onset in premanifest carriers. These biochemical findings demonstrates that the CSF metabolome can be used to characterize molecular pathogenesis occurring in HD, which may be essential for future development of novel HD therapies.
Asunto(s)
Enfermedad de Huntington/líquido cefalorraquídeo , Fenilalanina/líquido cefalorraquídeo , Tirosina/líquido cefalorraquídeo , Adulto , Anciano , Biomarcadores/líquido cefalorraquídeo , Dopamina/líquido cefalorraquídeo , Femenino , Humanos , Enfermedad de Huntington/patología , Levodopa/líquido cefalorraquídeo , Masculino , Persona de Mediana Edad , Tiroxina/líquido cefalorraquídeoRESUMEN
UNLABELLED: Low-grade gliomas (LGGs) may harbor malignant foci, which are characterized by increased tumor cellularity and angiogenesis. We used diffusion-weighted MR imaging (apparent diffusion coefficient [ADC]) and PET with the amino acid O-(2-(18)F-fluorethyl)-L-tyrosine ((18)F-FET) to search for focal changes of diffusion (ADC) and amino acid uptake and to investigate whether focal changes in these parameters colocalize within LGGs. METHODS: We retrospectively selected 18 patients with nonenhancing LGG. All patients had undergone (18)F-FET PET and MR imaging for preoperative evaluation or for therapy monitoring in recurrent or progressive LGG. Region-of-interest analysis was performed to compare (18)F-FET uptake and ADC values in areas with focal intratumoral maximum metabolic activity and diffusion restriction and between tumor and normal brain. (18)F-FET uptake was normalized to the mean cerebellar uptake (ratio). ADC values were also compared with the (18)F-FET uptake on a voxel-by-voxel basis across the whole tumor. RESULTS: The mean focal maximum (mean ± SD, 1.69 ± 0.85) and global (18)F-FET uptake in tumors (1.14 ± 0.41) exceeded that of normal cortex (0.85 ± 0.09) and cerebrospinal fluid (0.82 ± 0.20). ADC values in the area with most restricted diffusion (1.07 ± 0.22 × 10(-3) mm(2)/s) and in the whole tumor (1.38 ± 0.27 × 10(-3) mm(2)/s) were in the range between normal cortex (0.73 ± 0.06 × 10(-3) mm(2)/s) and cerebrospinal fluid (2.84 ± 0.09 × 10(-3) mm(2)/s). (18)F-FET uptake did not correlate with corresponding (colocalizing) ADC values, either in the area with focal maximum metabolic activity or in the area with most restricted diffusion or in the whole tumor. CONCLUSION: There is no congruency between (18)F-FET uptake and diffusivity in nonenhancing LGG. Diffusion restriction in these tumors most likely represents changes in brain and tumor cell densities as well as alteration of water distribution and is probably not directly correlated with the density of tumor cells.
Asunto(s)
Aminoácidos/metabolismo , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/patología , Imagen de Difusión por Resonancia Magnética/métodos , Glioma/diagnóstico por imagen , Glioma/patología , Tomografía de Emisión de Positrones/métodos , Adulto , Anciano , Neoplasias Cerebelosas/diagnóstico por imagen , Cerebelo/diagnóstico por imagen , Difusión , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico por imagen , Radiofármacos/líquido cefalorraquídeo , Estudios Retrospectivos , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeoRESUMEN
Forensic biomarkers are needed in sudden infant death syndrome (SIDS) to help identify this group among other sudden unexpected deaths in infancy. Previously, we reported multiple serotonergic (5-HT) abnormalities in nuclei of the medulla oblongata that help mediate protective responses to homeostatic stressors. As a first step toward their assessment as forensic biomarkers of medullary pathology, here we test the hypothesis that 5-HT-related measures are abnormal in the cerebrospinal fluid (CSF) of SIDS infants compared with those of autopsy controls. Levels of CSF 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), the degradative products of 5-HT and dopamine, respectively, were measured by high-performance liquid chromatography in 52 SIDS and 29 non-SIDS autopsy cases. Tryptophan (Trp) and tyrosine (Tyr), the substrates of 5-HT and dopamine, respectively, were also measured. There were no significant differences in 5-HIAA, Trp, HVA, or Tyr levels between the SIDS and non-SIDS groups. These data preclude the use of 5-HIAA, HVA, Trp, or Tyr measurements as CSF autopsy biomarkers of 5-HT medullary pathology in infants who have died suddenly and unexpectedly. They do, however, provide important information about monoaminergic measurements in human CSF at autopsy and their developmental profile in infancy that is applicable to multiple pediatric disorders beyond SIDS.
Asunto(s)
Ácido Hidroxiindolacético/líquido cefalorraquídeo , Serotonina/líquido cefalorraquídeo , Muerte Súbita del Lactante/líquido cefalorraquídeo , Análisis de Varianza , Cromatografía Líquida de Alta Presión , Bases de Datos Factuales/estadística & datos numéricos , Femenino , Ácido Homovanílico/líquido cefalorraquídeo , Humanos , Lactante , Masculino , Muerte Súbita del Lactante/patología , Triptófano/líquido cefalorraquídeo , Tirosina/líquido cefalorraquídeoRESUMEN
OBJECTIVE: Psychomotor impairment has been described in hypertyrosinemia types II and III (HT III). Only recently cognitive deficits have also been reported in hypertyrosinemia type I (HT I). The pathogenic mechanisms responsible are unknown. Since implementation of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC, Nitisinone (Swedish Orphan International)) in the treatment of HT I, plasma tyrosine elevation is a common finding as known from the other hypertyrosinemias. PATIENTS AND METHODS: With elevated tyrosine as suspected pathogenic factor in the development of cognitive deficits, we here investigated tyrosine in the cerebrospinal fluid (CSF) and serotonergic and dopaminergic neurotransmitter levels in three patients with HT I during long-term treatment with Nitisinone. In addition, Nitisinone concentrations in plasma and CSF were measured. We also assessed psychomotor and cognitive development by standardized test systems and brain morphology by magnetic resonance imaging. RESULTS: All patients presented with high tyrosine concentrations in CSF correlating with increased plasma tyrosine levels and a reduced CSF serotonin turnover. MRI revealed no structural abnormalities in the brain. All patients presented with either impaired cognitive development or behavioural abnormalities. CONCLUSIONS: We here outline the need to further study the exact pathogenic mechanisms responsible for the neurotransmitter changes observed in HT type I in order to possibly prevent cognitive dysfunction. Nitisinone has significantly improved outcome and quality of life in HT type I; however, it is also accompanied by elevated plasma and CSF tyrosine. Further studies are essential to identify the necessary dietary tyrosine restriction and the optimal Nitisinone dose.
Asunto(s)
Ciclohexanonas/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Nitrobenzoatos/uso terapéutico , Serotonina/metabolismo , Tirosina/líquido cefalorraquídeo , Tirosinemias/tratamiento farmacológico , Tirosinemias/enzimología , Encéfalo/anatomía & histología , Niño , Ciclohexanonas/sangre , Ciclohexanonas/líquido cefalorraquídeo , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/líquido cefalorraquídeo , Humanos , Lactante , Masculino , Nitrobenzoatos/sangre , Nitrobenzoatos/líquido cefalorraquídeo , Desempeño Psicomotor , Serotonina/análisis , Tirosina/sangreRESUMEN
BACKGROUND: Cerebral vasospasm is a significant cause of morbidity in patients after aneurysmal subarachnoid hemorrhage (aSAH). There are few effective treatments. The search for new treatments has focused predominantly on dilating cerebral blood vessels. Growing evidence supports a role for inflammation in its pathogenesis but no potential target for intervention has emerged. METHODS: CSF and clinical information from patients with aSAH were collected. Additionally, tyrosine modifications by stable isotope dilution HPLC with online tandem mass spectrometry were quantified in CSF samples. RESULTS: We report an association between neutrophil accumulation in the cerebrospinal fluid of patients with aSAH and the development of vasospasm. In particular, CSF neutrophil content of >62% on the third day after aSAH is an independent predictor of the later development of vasospasm (OR 6.8, 95% CI 2.0-23.3, P = 0.002). Further, activity of myeloperoxidase and NADPH oxidase is elevated in aSAH suggesting a role for modification of CSF proteins by reactive oxidant species. CONCLUSIONS: Neutrophil percentage is an independent predictor of vasospasm in aSAH patients, days prior to its onset suggesting a role of neutrophils in vasospasm. The activity of neutrophil enzymes is also increased suggesting a mechanism for blood vessel damage. Inflammation mediated by neutrophils is a potential target for therapies in vasospasm. More study is necessary to determine the mechanism by which neutrophils damage cerebral blood vessels.
Asunto(s)
Neutrófilos/metabolismo , Hemorragia Subaracnoidea/líquido cefalorraquídeo , Hemorragia Subaracnoidea/fisiopatología , Vasoespasmo Intracraneal/líquido cefalorraquídeo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Especies Reactivas de Oxígeno/líquido cefalorraquídeo , Hemorragia Subaracnoidea/complicaciones , Espectrometría de Masas en Tándem , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeo , Vasoespasmo Intracraneal/complicacionesRESUMEN
This study is to investigate the influence and mechanism of action of asymmetrical dimethylarginine (ADMA) and the induced oxidative stress level on Alzheimer's disease (AD) incidence. ADMA concentration, nitric oxide, Abeta(40)/Abeta(42) ratio, inducible NO synthase (iNOS) activity and the concentrations of the induced free radicals including malondialdehyde (MDA), 3-nitrotyrosine (3-NT) and peroxynitrite (ONOO-) in the cerebrospinal fluid (CSF) from 34 neurologically normal controls and 37 AD patients were quantitatively determined and statistically compared. The results showed that the ADMA concentration significantly decreased in AD patients, and it showed negative correlation with the NO, iNOS activity, and showed positive correlation with MMSE score. ADMA concentration was negatively correlated with Abeta(40)/Abeta(42) ratio (P<0.01) with the observation that Abeta(40)/Abeta(42) ratio increased while ADMA level decreased in CSF in AD patients. The concentration levels of MDA, 3-NT and ROS significantly increased compared with the control with all the P values less than 0.05. These findings suggested that the ADMA disorder and the oxidative damage effect of the induced free radicals in CSF of AD patients are an important mechanism of AD incidence, and their joint regulation may provide new idea for the prevention and clinical treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Arginina/análogos & derivados , Estrés Oxidativo , Anciano , Péptidos beta-Amiloides/líquido cefalorraquídeo , Arginina/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Malondialdehído/líquido cefalorraquídeo , Persona de Mediana Edad , Óxido Nítrico/líquido cefalorraquídeo , Óxido Nítrico Sintasa de Tipo II/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Ácido Peroxinitroso/líquido cefalorraquídeo , Especies Reactivas de Oxígeno/líquido cefalorraquídeo , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeoRESUMEN
The goal of our study was whether free radicals contribute to the pathogenesis of the lacunar stroke to investigate the day after hospitalization, the concentrations of 3-nitrotyrosine and tyrosine in the cerebrospinal fluid (CSF) from living patients. The subjects included 20 living patients with lacunar stroke and 20 controls. The NIH stroke scale score was used to assess the severity of the stroke, including that the patients were mild cases. There was no expansion of the infarct lesion in the brain, as assessed by CT on the day following admission. The concentration of 3-nitrotyrosine was significantly higher in patients with lacunar stroke. In contrast, the concentration of tyrosine did not differ between the two groups. Furthermore, the 3-nitrotyrosine/tyrosine ratio was significantly higher in patients with lacunar stroke than in controls. Our results show that free radicals are produced in the CSF of lacunar stroke patients and that nitration of neuronal proteines is enhanced under this condition. These obsetvations suggest that lacunar stroke patients should be treated with edaravon, which is a free radical scavenger usually prescribed for cases of major strokes, as it will likely improve the prognosis of these patients.
Asunto(s)
Infarto Encefálico/líquido cefalorraquídeo , Tirosina/análogos & derivados , Anciano , Recuento de Células , Femenino , Humanos , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Tirosina/líquido cefalorraquídeoRESUMEN
OBJECTIVES: Many studies have shown differences in carbonylation and nitration of individual proteins in brain and body fluids of Alzheimer's disease (AD) patients. Therefore, we wanted to examine whether total levels of these oxidative stress markers of proteins were altered in AD. PATIENTS AND METHODS: Total levels of carbonyls and nitrotyrosine in cerebrospinal fluid, serum and plasma were measured in 22 AD patients and 18 age-matched controls using commercially available enzyme immunoassay kits. RESULTS: Protein carbonylation in cerebrospinal fluid did not differ between AD patients and controls but was decreased in APOE epsilon4 carriers as compared with non-carriers. Serum but not plasma levels of carbonyls tended to be decreased in AD patients as compared with aged controls. Nitrotyrosine concentrations did not differ between the groups. Surrogate cerebrospinal fluid markers for AD, beta-amyloid (1-42) and tau, correlated with blood carbonyl and nitrotyrosine levels. CONCLUSIONS: According to these preliminary data, changes in oxidative metabolism related to the pathogenesis of AD cannot be detected as increased cerebrospinal fluid, serum or plasma protein carbonylation or nitration.
Asunto(s)
Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Proteínas Sanguíneas/metabolismo , Proteínas del Líquido Cefalorraquídeo/metabolismo , Estrés Oxidativo , Precursor de Proteína beta-Amiloide/sangre , Precursor de Proteína beta-Amiloide/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Compuestos Nitrosos , Oxidación-Reducción , Carbonilación Proteica , Valores de Referencia , Tirosina/análogos & derivados , Tirosina/sangre , Tirosina/líquido cefalorraquídeoRESUMEN
To evaluate the possible role of central free amino compounds in pediatric opsoclonus-myoclonus syndrome (OMS), 21 cerebrospinal fluid (CSF) amino compounds were measured by an amino acid analyzer or mass spectroscopy in 74 anesthetized children, 54 with OMS and 20 age-matched neurological controls. In OMS, only phosphoethanolamine was increased compared to controls; OMS severity and duration had significant converse effects on alanine and phosphoethanolamine. In contrast, corticotropin (ACTH) treatment was associated with increased alanine and phenylalanine, and decreased taurine compared to controls and untreated OMS, and increased glutamine, lysine, ornithine, and tyrosine compared to untreated OMS. Other than low taurine, these effects were not found with corticosteroid treatment, and non-steroidogenic immunotherapy had no effect. The ACTH dose-association was most apparent for alanine and phosphoethanolamine, but lysine and ornithine were also higher in the high-dose ACTH group. There were no significant disease- or treatment-associated perturbations in GABA, glycine, or other amino acids. These data suggest a unique pattern of ACTH effects on non-neurotransmitter CSF amino compounds, for the most part not shared by steroids.
Asunto(s)
Hormona Adrenocorticotrópica/uso terapéutico , Aminoácidos/líquido cefalorraquídeo , Inmunoterapia/métodos , Síndrome de Opsoclonía-Mioclonía/tratamiento farmacológico , Hormona Adrenocorticotrópica/farmacología , Alanina/líquido cefalorraquídeo , Alanina/metabolismo , Aminoácidos/metabolismo , Análisis de Varianza , Niño , Preescolar , Cromatografía de Gases/métodos , Corticosterona/farmacología , Corticosterona/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Glutamina/líquido cefalorraquídeo , Glutamina/metabolismo , Humanos , Lisina/líquido cefalorraquídeo , Lisina/metabolismo , Masculino , Espectrometría de Masas/métodos , Síndrome de Opsoclonía-Mioclonía/líquido cefalorraquídeo , Síndrome de Opsoclonía-Mioclonía/metabolismo , Ornitina/líquido cefalorraquídeo , Ornitina/metabolismo , Fenilalanina/líquido cefalorraquídeo , Fenilalanina/metabolismo , Índice de Severidad de la Enfermedad , Taurina/líquido cefalorraquídeo , Taurina/metabolismo , Tirosina/líquido cefalorraquídeo , Tirosina/metabolismoRESUMEN
BACKGROUND: The prevalence of HIV-associated neurocognitive disorders is increasing as HIV-infected individuals are living longer. The clinical manifestations of the syndrome also continue to evolve under the influence of antiretroviral drugs and comorbidities such as drugs of abuse. However, there are no surrogate markers for the disease, either to identify it de novo or to track its progression, and there is no proven treatment with the exception of antiretroviral drugs. METHODS: Levels of nitric oxide, nitrate, and 3-nitrotyrosine (3-NT)-modified proteins were measured in the CSF of 46 patients with HIV infection stratified according to their neurocognitive status and history of IV drug use (IVD). The 3-NT-modified proteins were isolated and identified by tandem mass spectrometry, and the functional consequence of 3-NT modification of L-prostaglandin D synthase (L-PGDS), the most abundant protein, was determined. RESULTS: 3-NT-modified proteins were significantly elevated in patients with HIV infection who had progressive neurocognitive decline over the next 6 months and in patients with a history of IVD. Thirteen different proteins with 3-NT modification were identified in the CSF of these patients. L-PGDS was the most abundant. 3-NT modification of this protein resulted in loss of its enzymatic activity. CONCLUSIONS: There is increased nitrosative stress in CSF of HIV-infected patients with active dementia and in patients with a history of IV drug use, measurement of which may serve as a surrogate marker for these patients. Nitrosative stress may also have important functional consequences and may impact the pathogenesis of HIV-associated neurocognitive disorders.
Asunto(s)
Complejo SIDA Demencia/líquido cefalorraquídeo , Complejo SIDA Demencia/diagnóstico , Oxidorreductasas Intramoleculares/líquido cefalorraquídeo , Lipocalinas/líquido cefalorraquídeo , Nitratos/líquido cefalorraquídeo , Óxido Nítrico/líquido cefalorraquídeo , Estrés Oxidativo , Complejo SIDA Demencia/fisiopatología , Adulto , Biomarcadores/análisis , Biomarcadores/líquido cefalorraquídeo , Encéfalo/enzimología , Encéfalo/fisiopatología , Estudios de Cohortes , Progresión de la Enfermedad , Regulación hacia Abajo/fisiología , Activación Enzimática/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nitrosación , Valor Predictivo de las Pruebas , Abuso de Sustancias por Vía Intravenosa/líquido cefalorraquídeo , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeo , Regulación hacia Arriba/fisiologíaRESUMEN
Different lines of evidence suggest that nitric oxide (NO) plays a key role in the pathogenesis of inflammatory neuropathies; however, it is still unclear which structures in the peripheral nerve are the primary targets of NO-mediated nerve injury. To address this issue, we determined the expression of NO metabolites in sural nerve biopsies and in cerebrospinal fluid from patients with inflammatory neuropathies and studied the pathologic effects of NO in an in vitro model of myelinated Schwann cell-neuron cocultures. In cerebrospinal fluid samples, nitrite levels remained unaltered; however, nitrotyrosine, a marker for peroxynitrite formation, could be identified in nerve biopsies from patients with inflammatory neuropathies. In an in vitro model of Schwann cell neuron cocultures, high concentrations of NO induced robust demyelination, which was the result of NO-mediated axonal injury, whereas Schwann cell viability remained unaffected. These findings suggest that in contrast to Schwann cells, sensory neurons are the primary target of NO-mediated cytotoxicity and the loss of myelin is the result of selective damage to axons rather than a direct harmful effect to Schwann cells. Our findings imply that NO contributes to the pathologic changes seen in the inflamed peripheral nervous system, which is characterized by the features of axonal injury and subsequent myelin degradation, previously described as Wallerian-like degeneration.
Asunto(s)
Óxido Nítrico/metabolismo , Polirradiculoneuropatía/enzimología , Polirradiculoneuropatía/patología , Nervio Sural/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo/métodos , Medios de Cultivo Condicionados/farmacología , Óxidos N-Cíclicos/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Depuradores de Radicales Libres/farmacología , Ganglios Espinales/citología , Humanos , Imidazoles/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/química , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/toxicidad , Compuestos Nitrosos/toxicidad , Ratas , Células de Schwann/química , Nervio Sural/patología , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeoRESUMEN
Long-term or high-dose L-DOPA therapy in patients with Parkinson's disease (PD) may accelerate degeneration of dopaminergic neurons, possibly by increasing oxidative stress. To investigate the effects of cabergoline on peroxynitrite-mediated oxidative damage caused by L-DOPA, the concentration of 3-nitrotyrosine in cerebrospinal fluid (CSF) of 18 PD patients was compared with that in 20 normal controls. The concentration of 3-nitrotyrosine in patients following L-DOPA therapy was significantly higher than in untreated PD patients and controls. On the other hand, the concentration in PD patients after cabergoline therapy was significantly lower than in PD patients after L-DOPA therapy alone. These data suggest that cabergoline scavenges peroxynitrite induced by L-DOPA in patients with PD.
Asunto(s)
Antiparkinsonianos/efectos adversos , Ergolinas/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Levodopa/efectos adversos , Enfermedad de Parkinson/tratamiento farmacológico , Ácido Peroxinitroso/metabolismo , Anciano , Cabergolina , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Masculino , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/líquido cefalorraquídeo , Enfermedad de Parkinson/metabolismo , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeo , Tirosina/efectos de los fármacosRESUMEN
This work presents two liquid chromatography/tandem mass spectrometry (LC/MS/MS) acquisition modes: multiple reaction monitoring (MRM) and neutral loss scan (NL), for the analysis of 28 compounds in a mixture. This mixture includes 21 compounds related to the metabolism of three amino acids: tyrosine, tryptophan and glutamic acid, two pterins and five deuterated compounds used as internal standards. The identification of compounds is achieved using the retention times (RT) and the characteristic fragmentations of ionized compounds. The acquisition modes used for the detection of characteristic ions turned out to be complementary: the identification of expected compounds only is feasible by MRM while expected and unexpected compounds are detected by NL. In the first part of this work, the fragmentations characterizing each molecule of interest are described. These fragmentations are used in the second part for the detection by MRM and NL of selected compounds in mixture with and without biological fluids. Any preliminary extraction precedes the analysis of compounds in biological fluids.
Asunto(s)
Neurotransmisores/análisis , Líquido Amniótico/química , Catecolaminas/análisis , Catecolaminas/líquido cefalorraquídeo , Catecolaminas/orina , Cromatografía Líquida de Alta Presión , Deuterio , Humanos , Indoles/análisis , Indoles/líquido cefalorraquídeo , Indoles/orina , Neurotransmisores/líquido cefalorraquídeo , Neurotransmisores/orina , Pterinas/análisis , Pterinas/líquido cefalorraquídeo , Pterinas/orina , Estándares de Referencia , Espectrometría de Masas en Tándem , Tirosina/análisis , Tirosina/líquido cefalorraquídeo , Tirosina/orina , Ácido gamma-Aminobutírico/análisis , Ácido gamma-Aminobutírico/líquido cefalorraquídeo , Ácido gamma-Aminobutírico/orinaRESUMEN
Chronic bilirubin encephalopathy, characterized clinically by extrapyramidal movement abnormalities, vertical gaze abnormalities, and hearing loss, results from neuronal injury after marked hyperbilirubinemia in term and preterm infants. In premature infants, bilirubin staining of specific brain structures has been described at autopsy after only moderate hyperbilirubinemia, but classic chronic bilirubin encephalopathy without marked hyperbilirubinemia has been reported only rarely. We report a case of a 7-year-old, former 29-weeks' gestation, gravely ill premature infant with a peak bilirubin level of 13.3 mg/dL in the neonatal period. We compare this case with a 12-year-old, former term infant with a peak bilirubin level of 49.4 mg/dL on day 10 of life. Both children have dystonia, athetosis, upward gaze palsy, and sensorineural hearing loss, with MRIs showing characteristic abnormal signal in the globus pallidus. We add previously unreported cerebrospinal fluid neurotransmitter levels that show a mild decrease in the dopamine metabolite homovanillic acid in the former premature infant only.
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Enfermedades del Prematuro/líquido cefalorraquídeo , Kernicterus/líquido cefalorraquídeo , Neurotransmisores/líquido cefalorraquídeo , Niño , Discapacidades del Desarrollo/etiología , Femenino , Estudios de Seguimiento , Globo Pálido/patología , Ácido Homovanílico/líquido cefalorraquídeo , Humanos , Ácido Hidroxiindolacético/líquido cefalorraquídeo , Hiperbilirrubinemia/líquido cefalorraquídeo , Recién Nacido , Recien Nacido Prematuro , Kernicterus/complicaciones , Kernicterus/patología , Imagen por Resonancia Magnética , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeoRESUMEN
Increased damage to proteins by glycation, oxidation and nitration has been implicated in neuronal cell death leading to Alzheimer's disease (AD). Protein glycation, oxidation and nitration adducts are consequently formed. Quantitative screening of these adducts in CSF may provide a biochemical indicator for the diagnosis of AD. To assess this, we measured 11 glycation adducts, three oxidation adducts and a nitration adduct, determining both protein adduct residues and free adducts, in CSF samples of age-matched normal healthy subjects (n = 18) and subjects with Alzheimer's disease (n = 32). In CSF protein, the concentrations of 3-nitrotyrosine, N(epsilon)-carboxymethyl-lysine, 3-deoxyglucosone-derived hydroimidazolone and N-formylkynurenine residues were increased in subjects with Alzheimer's disease. In CSF ultrafiltrate, the concentrations of 3-nitrotyrosine, methylglyoxal-derived hydroimidazolone and glyoxal-derived hydroimidazolone free adducts were also increased. The Mini-Mental State Examination (MMSE) score correlated negatively with 3-nitrotyrosine residue concentration (p < 0.05), and the negative correlation with fructosyl-lysine residues just failed to reach significance (p = 0.052). Multiple linear regression gave a regression model of the MMSE score on 3-nitrotyrosine, fructosyl-lysine and N(epsilon)-carboxyethyl-lysine residues with p-values of 0.021, 0.031 and 0.052, respectively. These findings indicate that protein glycation, oxidation and nitration adduct residues and free adducts were increased in the CSF of subjects with Alzheimer's disease. A combination of nitration and glycation adduct estimates of CSF may provide an indicator for the diagnosis of Alzheimer's disease.
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Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/metabolismo , Proteínas del Líquido Cefalorraquídeo/líquido cefalorraquídeo , Proteínas del Líquido Cefalorraquídeo/metabolismo , Trastornos del Conocimiento/líquido cefalorraquídeo , Lisina/análogos & derivados , Nitratos/metabolismo , Tirosina/análogos & derivados , Anciano , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquídeo , Trastornos del Conocimiento/diagnóstico , Trastornos del Conocimiento/etiología , Femenino , Glicosilación , Humanos , Lisina/líquido cefalorraquídeo , Masculino , Pruebas Neuropsicológicas , Compuestos Nitrosos/líquido cefalorraquídeo , Oxidación-Reducción , Valores de Referencia , Tirosina/líquido cefalorraquídeoRESUMEN
BACKGROUND: Deficiency of aromatic L-amino acid decarboxylase (AADC) is associated with severe developmental delay, oculogyric crises (OGC), and autonomic dysfunction. Treatment with dopamine agonists and MAO inhibitors is beneficial, yet long-term prognosis is unclear. OBJECTIVE: To delineate the clinical and molecular spectrum of AADC deficiency, its management, and long-term follow-up. RESULTS: The authors present six patients with AADC deficiency and review seven cases from the literature. All patients showed reduced catecholamine metabolites and elevation of 3-O-methyldopa in CSF. Residual plasma AADC activity ranged from undetectable to 8% of normal. Mutational spectrum was heterogeneous. All patients presented with hypotonia, hypokinesia, OGC, and signs of autonomic dysfunction since early life. Diurnal fluctuation or improvement of symptoms after sleep were noted in half of the patients. Treatment response was variable. Two groups of patients were detected: Group I (five males) responded to treatment and made developmental progress. Group II (one male, five females) responded poorly to treatment, and often developed drug-induced dyskinesias. CONCLUSIONS: The molecular and clinical spectrum of AADC deficiency is heterogeneous. Two groups, one with predominant male sex and favorable response to treatment, and the other with predominant female sex and poor response to treatment, can be discerned.
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Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Tirosina/análogos & derivados , Adolescente , Errores Innatos del Metabolismo de los Aminoácidos/genética , Descarboxilasas de Aminoácido-L-Aromático/sangre , Descarboxilasas de Aminoácido-L-Aromático/genética , Niño , Preescolar , Progresión de la Enfermedad , Agonistas de Dopamina/uso terapéutico , Femenino , Ácido Homovanílico/líquido cefalorraquídeo , Humanos , Ácido Hidroxiindolacético/líquido cefalorraquídeo , Lactante , Masculino , Inhibidores de la Monoaminooxidasa/uso terapéutico , Pronóstico , Factores Sexuales , Resultado del Tratamiento , Tirosina/líquido cefalorraquídeo , Vitamina B 6/uso terapéuticoRESUMEN
The mechanisms behind the degeneration of neurons in diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are not fully understood. However, oxidation of certain amino acid residues in proteins may contribute to cell injury and some of these oxidized amino acids may also be suitable as biomarkers for oxidative injury. Therefore, it is suggested that the reaction between peroxynitrite (ONOO(-)) and tyrosine in vivo can be monitored by monitoring the formation of 3-nitrotyrosine (3-NT). In this work, a newly developed gas chromatographic-mass spectrometric method was applied to human cerebrospinal fluid (CSF). The free 3-NT levels were determined in the CSF from 19 controls, 17 patients with AD and 14 patients with ALS. The levels of free 3-NT in the CSF were considerably lower than those previously reported. The majority of the patients with AD or ALS had free 3-NT levels in the same range as seen in the control individuals and only a few patients showed increased levels of free 3-NT.
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Enfermedad de Alzheimer/líquido cefalorraquídeo , Esclerosis Amiotrófica Lateral/líquido cefalorraquídeo , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquídeo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.