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1.
Pak J Pharm Sci ; 30(1): 135-142, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28603123

RESUMO

Nitrate is the stable product of nitric oxide, which is physiologically active radical, an immunomodulator and a neuromodulator; its quantification in biological fluids is important to study the physiological and biochemical nature. Therefore, the purpose of this study was to quantify nitrate in different biological fluids like serum, cerebrospinal fluid (CSF) and ascetic fluid (ASF) using HPLC technique. A new HPLC method for the estimation of nitrate in serum, CSF and ASF was developed using the mobile phase of 1.0mM each of Na2CO3 and NaHCO3 (1:1, v/v, pH 5 with H3PO4) at a flow rate of 1.0mLmin-1. Eluate was detected at 220nm with the retention time of nitrate 2.55 min. The LOD and LOQ values of nitrate were 0.03µgmL-1 and 0.098µgmL-1, respectively. Nitrate was eluted through SAX Hypersil column of 150 × 4.6mm, id, 5µm particle size. Run time was 10min. The method was validated according to the FDA guidelines and was found linear in the range of 0.39 to 50µgmL-1 and CV was <3%, within limits of FDA guidelines. The method was used successfully for the estimation of nitrate in biological fluids like serum, CSF and ASF of 20 patients each. This is an alternate and reproducible method for the detection of nitrates in biological fluids.


Assuntos
Líquido Ascítico/química , Cromatografia Líquida de Alta Pressão , Nitratos/sangue , Nitratos/líquido cefalorraquidiano , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Calibragem , Cromatografia Líquida de Alta Pressão/normas , Humanos , Limite de Detecção , Modelos Lineares , Padrões de Referência , Reprodutibilidade dos Testes
2.
J Chromatogr A ; 1447: 148-54, 2016 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-27108047

RESUMO

A new electrophoretic stacking method has been developed for the sensitive determination of nitrates in cerebrospinal fluid. 2M acetic acid was used as the BGE; inorganic anions were detected using a contactless conductivity detector and separation was carried out in an INST-coated capillary with inner diameter of 25µm. The sample of cerebrospinal fluid was injected in a large volume into the short end of the separation capillary (15cm) and separation first occurred in the isotachophoretic mode, where a long zone of the majority chloride migrates in the capillary and is followed by a concentrated zone of the unseparated nitrates. The sample zone passes to the end of the capillary where more than 99% of the chlorides are let out. Then the polarity of the voltage is switched and separation occurs in the zone electrophoresis mode, in which the nitrates are separated from the zone of chlorides. The time of switching the polarity is determined by the decrease in the electrophoretic current. Up to 99.95% of the original amount of chlorides present in the cerebrospinal fluid could be let out of the capillary by this technique, thus increasing the signal/noise ratio by up to 60-fold compared to classical electrophoretic separation.


Assuntos
Nitratos/líquido cefalorraquidiano , Adulto , Cloretos/líquido cefalorraquidiano , Eletroforese Capilar/métodos , Feminino , Humanos , Isotacoforese , Masculino , Pessoa de Meia-Idade , Adulto Jovem
3.
Amino Acids ; 47(9): 1837-45, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26055922

RESUMO

The pathogenic hallmarks of multiple sclerosis (MS) and neuromyelitis optica (NMO) are cellular and humoral inflammatory infiltrates and subsequent demyelination, or astrocytic cell death in NMO, respectively. These processes are accompanied by disruption of the blood-brain barrier as regularly observed by gadolinium enhancement on magnetic resonance imaging. The role of the L-arginine/nitric oxide (NO) pathway in the pathophysiology of neuroinflammatory diseases, such as MS and NMO, remains unclear. In the present study, we measured the concentrations of the nitric oxide (NO) metabolites nitrate and nitrite, the endogenous substrates of NO synthase (NOS) L-arginine (Arg) and L-homoarginine (hArg), and asymmetric dimethylarginine (ADMA), the endogenous inhibitor of NOS activity, in the serum and cerebrospinal fluid (CSF) of patients with MS, NMO or other neurologic diseases (OND). MS (551 ± 23 nM, P = 0.004) and NMO (608 ± 51 nM, P = 0.006) patients have higher ADMA concentrations in serum than healthy controls (HC; 430 ± 24 nM). For MS, this finding was confirmed in CSF (685 ± 100 nM in relapsing-remitting multiple sclerosis, RRMS; 597 ± 51 nM in secondary progressive multiple sclerosis, SPMS) compared with OND (514 ± 37 nM; P = 0.003). Serum concentrations of Arg (61.1 ± 9.7 vs. 63.6 ± 4.9 µM, P = 0.760), hArg (2.62 ± 0.26 vs. 2.52 ± 0.23 µM, P = 0.891), nitrate (38.1 ± 2.2 vs. 38.1 ± 3.0 µM) and nitrite (1.37 ± 0.09 vs. 1.55 ± 0.03 µM) did not differ between MS and OND. Also, CSF concentrations of hArg (0.685 ± 0.100 µM in RRMS, 0.597 ± 0.051 µM in SPMS, 0.514 ± 0.037 µM in OND), nitrate (11.3 ± 0.6 vs. 10.5 ± 0.3 µM) and nitrite (2.84 ± 0.32 vs. 2.41 ± 0.11 µM) did not differ between the groups. In NMO patients, however, serum Arg (117 ± 11 vs. 64 ± 4.9 µM, P = 0.004), nitrate (29 ± 2.1 vs. 38 ± 3 µM, P = 0.03), and nitrite (1.09 ± 0.02 vs. 1.55 ± 0.033 µM, P < 0.0001) were significantly different as compared to OND. Symmetric dimethylarginine (SDMA) concentration did not differ in serum between MS and HC (779 ± 43 vs. 755 ± 58 nM, P = 0.681) or in CSF between MS and OND patients (237 ± 11 vs. 230 ± 17 nM, P = 0.217). Our study suggests a potential role for ADMA and Arg in neuroinflammatory diseases with diverse functions in MS and NMO. Higher ADMA synthesis may explain reduced NO availability in NMO. hArg and SDMA seem not to play an important role in MS and NMO.


Assuntos
Arginina/análogos & derivados , Homoarginina , Esclerose Múltipla , Neuromielite Óptica , Nitratos , Nitritos , Adulto , Arginina/sangue , Arginina/líquido cefalorraquidiano , Feminino , Homoarginina/sangue , Homoarginina/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Neuromielite Óptica/sangue , Neuromielite Óptica/líquido cefalorraquidiano , Nitratos/sangue , Nitratos/líquido cefalorraquidiano , Nitritos/sangue , Nitritos/líquido cefalorraquidiano
4.
Methods Mol Biol ; 1274: 31-42, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25673480

RESUMO

Microchip capillary electrophoresis (MCE) is a relatively new analytical method requiring only small sample amounts, which is very favorable for the analysis of volume-limited biofluids. The practical use of MCE in bioanalysis is still restricted in terms of requirements for simplifying and/or concentrating sample pretreatment techniques. Here, we describe an MCE method for trace analysis of nitrite and nitrate, indicators of various neurological diseases, in cerebrospinal fluid (CSF). The complex CSF samples were simplified by solid-phase microextraction prior to an online combination of isotachophoresis with capillary zone electrophoresis performed on a microchip with coupled channels and a high-volume sample injection channel (9.9 µL). The method is suitable for rapid (total analysis time lasted 20 min), reproducible (0.6-2.4 % RSD for migration time), and sensitive (3-9 nM limits of detection) determinations of nitrite and nitrate in 15-50 times diluted CSF samples.


Assuntos
Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Nitratos/líquido cefalorraquidiano , Nitritos/líquido cefalorraquidiano , Humanos
5.
Artigo em Inglês | MEDLINE | ID: mdl-23727865

RESUMO

A new method for the determination of nitrite and nitrate, indicators of various neurological diseases (meningitis, multiple sclerosis, Parkinson's disease) in cerebrospinal fluid (CSF) on an electrophoresis chip was developed. An on-line combination of isotachophoresis (ITP) with capillary electrophoresis (CE) on a poly(methylmethacrylate) chip assembled with coupled separation channels (CC) and contact conductivity detectors was employed. ITP separations performed at low pH (3.6) in the first separation channel enabled a highly selective transfer of the analytes to the second CE stage working under micellar conditions implemented by zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)-propanesulfonate. The proposed method achieved low limits of detection varied from 0.2 to 0.4µgL(-1) when the sample volume injected onto the chip (9.9µl) was almost the same as the volume of both separation channels. Preferable working conditions on the CC chip (suppressed hydrodynamic and electroosmotic flow) contributed for reproducible migration velocities (intra-day reproducibility up to 2.1% RSD) and determinations of trace concentrations of nitrite and nitrate (intra-day precision up to 3.0% RSD). Huge amount of chloride present in CSF (approx. 4.5gL(-1)) was removed from analyzed CSF samples by microsolid phase extraction performed on silver-form resin prior to the ITP-CE analysis. Developed method provided fast (approx. 20min total analysis time) and reliable determinations of trace nitrite and nitrate and could be fully integrated into the analysis of CSF samples.


Assuntos
Eletroforese em Microchip/métodos , Nitratos/líquido cefalorraquidiano , Nitritos/líquido cefalorraquidiano , Microextração em Fase Sólida/métodos , Adolescente , Adulto , Biomarcadores/líquido cefalorraquidiano , Eletroforese em Microchip/instrumentação , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/líquido cefalorraquidiano , Reprodutibilidade dos Testes
6.
Einstein (Sao Paulo) ; 11(1): 88-94, 2013.
Artigo em Inglês, Português | MEDLINE | ID: mdl-23579750

RESUMO

OBJECTIVE: To determine the concentration of nitrate/nitrite in the cerebrospinal fluid and in the dorsal horn interstice of the L6-S1 spinal cord boundary in rats with or without cystitis induced by cyclophosphamide. METHODS: All experiments were conducted using Wistar female rats. A microdialysis probe was implanted in the subarachnoid space or in the spinal cord tissue at the L6-S1 segments (confirmed histologically). Two days later, the microdialysis probe was perfused with artificial cerebrospinal fluid, containing or not NG-monomethyl-L-arginine. Samples were collected every 15 minutes and kept at -20ºC. Nitrite/nitrate concentrations were determined by chemiluminescence. RESULTS: In normal animals, the mean values of nitrite/nitrate concentrations in the first microdialysate sample of the cerebrospinal fluid and of the spinal cord interstice were similar (482.5±90.2pmol/75µL, n=20, and 505.7±11.5pmol/75µL, n=6, respectively), whereas, in the samples from rats with cystitis, these values were significantly greater (955.5±66.3pmol/75µL, n=8, and 926.5±131.7pmol/75µL, n=11, respectively). In both groups, NG-monomethyl-L- arginine caused a significant reduction in the nitrite/nitrate concentration. Interestingly, the maximal reduction of nitrite/nitrate concentration caused by NG-monomethyl-L- arginine was no greater than 30% of the initial values. CONCLUSIONS: These results constitute the first demonstration that nitrite/nitrate concentrations in the cerebrospinal fluid and spinal cord interstice are elevated between 20- and 22 hours after cyclophosphamide-induced cystitis, and indicate that cystitis is associated with changes in the production of nitric oxide in the spinal cord segments, where most primary bladder afferents end.


Assuntos
Cistite/induzido quimicamente , Óxido Nítrico/metabolismo , Medula Espinal/química , Animais , Ciclofosfamida , Cistite/metabolismo , Cistite/patologia , Feminino , Região Lombossacral , Luminescência , Microdiálise , Nitratos/líquido cefalorraquidiano , Nitratos/metabolismo , Óxido Nítrico/líquido cefalorraquidiano , Óxido Nítrico Sintase/antagonistas & inibidores , Nitritos/líquido cefalorraquidiano , Nitritos/metabolismo , Ratos , Ratos Wistar , Valores de Referência , Medula Espinal/metabolismo , Fatores de Tempo , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , ômega-N-Metilarginina/farmacologia
7.
Pediatr Crit Care Med ; 14(4): 403-12, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23439461

RESUMO

OBJECTIVES: Pathological increases in asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, have been implicated in endothelial dysfunction and vascular diseases. Reduced nitric oxide early after traumatic brain injury may contribute to hypoperfusion. Currently, methods to quantify asymmetric dimethylarginine in the cerebrospinal fluid have not been fully explored. We aimed to develop and validate a method to determine asymmetric dimethylarginine in the cerebrospinal fluid of a pediatric traumatic brain injury population and to use this method to assess the effects of 1) traumatic brain injury and 2) therapeutic hypothermia on this mediator. DESIGN, SETTING, AND PATIENTS: An ancillary study to a prospective, phase II randomized clinical trial of early hypothermia in a tertiary care pediatric intensive care unit for children with Traumatic brain injury admitted to Children's Hospital of Pittsburgh. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A UPLC-MS/MS method was developed and validated to quantitate asymmetric dimethylarginine. A total of 56 samples collected over 3 days with injury onset were analyzed from the cerebrospinal fluid of consented therapeutic hypothermia (n = 9) and normothermia (n = 10) children. Children undergoing diagnostic lumbar puncture (n = 5) were enrolled as controls. Asymmetric dimethylarginine was present at a quantifiable level in all samples. Mean asymmetric dimethylarginine levels were significantly increased in normothermic Traumatic brain injury children compared with that in control (0.19 ± 0.08 µmol/L and 0.11 ± 0.02 µmol/L, respectively, p = 0.01), and hypothermic children had significantly reduced mean asymmetric dimethylarginine levels (0.11 ± 0.05 µmol/L) vs. normothermic (p = 0.03) measured on day 3. Patient demographics including age, gender, and nitric oxide levels (measured as nitrite and nitrate using liquid chromatography coupled with Griess reaction) did not significantly differ between normothermia and hypothermia groups. Also, nitric oxide levels did not correlate with asymmetric dimethylarginine concentrations. CONCLUSIONS: Asymmetric dimethylarginine levels were significantly increased in the cerebrospinal fluid of traumatic brain injury children. Early hypothermia attenuated this increase. The implications of attenuated asymmetric dimethylarginine on nitric oxide synthases activity and regional cerebral blood flow after traumatic brain injury by therapeutic hypothermia deserve future study.


Assuntos
Arginina/análogos & derivados , Lesões Encefálicas/líquido cefalorraquidiano , Lesões Encefálicas/terapia , Hipotermia Induzida , Adolescente , Arginina/líquido cefalorraquidiano , Estudos de Casos e Controles , Líquido Cefalorraquidiano/química , Criança , Pré-Escolar , Cromatografia Líquida , Feminino , Humanos , Lactente , Masculino , Nitratos/líquido cefalorraquidiano , Nitritos/líquido cefalorraquidiano , Espectrometria de Massas em Tandem
8.
J Neurosurg ; 115(6): 1213-20, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21888479

RESUMO

OBJECT: Subarachnoid hemorrhage (SAH)-induced vasospasm is a significant underlying cause of aneurysm rupture-related morbidity and death. While long-term intravenous infusion of sodium nitrite (NaNO(2)) can prevent cerebral vasospasm after SAH, it is not known if the intravenous administration of this compound can reverse established SAH-induced vasospasm. To determine if the intravenous infusion of NaNO(2) can reverse established vasospasm, the authors infused primates with the compound after SAH-induced vasospasm was established. METHODS: Subarachnoid hemorrhage-induced vasospasm was created in 14 cynomolgus macaques via subarachnoid implantation of a 5-ml blood clot. On Day 7 after clot implantation, animals were randomized to either control (saline infusion, 5 monkeys) or treatment groups (intravenous NaNO(2) infusion at 300 µg/kg/hr for 3 hours [7 monkeys] or 8 hours [2 monkeys]). Arteriographic vessel diameter was blindly analyzed to determine the degree of vasospasm before, during, and after treatment. Nitric oxide metabolites (nitrite, nitrate, and S-nitrosothiols) were measured in whole blood and CSF. RESULTS: Moderate-to-severe vasospasm was present in all animals before treatment (control, 36.2% ± 8.8% [mean ± SD]; treatment, 45.5% ± 12.5%; p = 0.9). While saline infusion did not reduce vasospasm, NaNO(2) infusion significantly reduced the degree of vasospasm (26.9% ± 7.6%; p = 0.008). Reversal of the vasospasm lasted more than 2 hours after cessation of the infusion and could be maintained with a prolonged infusion. Nitrite (peak value, 3.7 ± 2.1 µmol/L), nitrate (18.2 ± 5.3 µmol/L), and S-nitrosothiols (33.4 ± 11.4 nmol/L) increased significantly in whole blood, and nitrite increased significantly in CSF. CONCLUSIONS: These findings indicate that the intravenous infusion of NaNO(2) can reverse SAH-induced vasospasm in primates. Further, these findings indicate that a similar treatment paradigm could be useful in reversing cerebral vasospasm after aneurysmal SAH.


Assuntos
Nitrito de Sódio/farmacologia , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/tratamento farmacológico , Vasoespasmo Intracraniano/etiologia , Animais , Angiografia Cerebral , Modelos Animais de Doenças , Infusões Intravenosas , Macaca fascicularis , Artéria Cerebral Média/diagnóstico por imagem , Artéria Cerebral Média/efeitos dos fármacos , Nitratos/sangue , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/sangue , Óxido Nítrico/líquido cefalorraquidiano , Nitritos/sangue , Nitritos/líquido cefalorraquidiano , S-Nitrosotióis/sangue , S-Nitrosotióis/líquido cefalorraquidiano , Hemorragia Subaracnóidea/metabolismo , Resultado do Tratamento , Vasoespasmo Intracraniano/diagnóstico por imagem , Vasoespasmo Intracraniano/metabolismo
9.
Cephalalgia ; 30(6): 696-702, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20511209

RESUMO

The pathophysiology of cluster headache (CH) is only partly understood. Nitric oxide (NO), a potent vasodilator, has been suggested to be involved, and increased plasma levels of nitrite, a stable product on NO degradation, have been identified in the active period and in remission. The aim of our study was to investigate the role of NO in CH by measuring its oxidation products, nitrite and nitrate, in the cerebrospinal fluid (CSF), a biological compartment closer to the supposed focus of the disorder. We collected CSF from 14 episodic CH patients. Lumbar puncture (LP) was performed at two occasions: in active period between headache attacks, and in remission, not earlier than three weeks after the last CH attack. Eleven healthy volunteers served as controls. To estimate NO production, we determined the levels of NO-oxidation end products (NOx), that is, the sum of nitrite and nitrate, by using capillary electrophoresis. CH patients in the active period had significantly increased NOx levels (mean 9.3, 95% confidence interval [CI] 8.5-10.1) compared with those in remission (mean 7.6, 95% CI 6.9-8.2; p < 001) and control subjects (mean 6.2, 95% CI 4.9-7.5; p < 0.001). CH patients also had statistically significant enhanced NOx levels in remission compared with those of control subjects (p = 0.034). CSF was also analysed with regard to inflammatory parameters and protein content. CSF showed signs of pleocytosis or oligoclonal bands or albumin increase in 43% of CH patients although these results were not conclusive. We suggest that CH patients have a generally raised NO tonus, both in the active period and in remission. We interpret these results as indications of a basal hyperfunction of the L-arginine-NO pathway, possibly as an expression of inflammatory activity, and sensitization of pain pathways. This is the first study analysing NOx in CSF in CH, and the results support NO involvement in the pathogenesis of CH.


Assuntos
Cefaleia Histamínica/líquido cefalorraquidiano , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/metabolismo , Nitritos/líquido cefalorraquidiano , Adulto , Eletroforese Capilar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
10.
Pediatr Neurol ; 41(3): 179-82, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19664532

RESUMO

Oxidative damage plays a role in neurodegenerative diseases. Levels of cerebrospinal fluid nitrite and nitrate levels (oxidation products that provide an indirect estimation of nitric oxide) were investigated in relation to clinical and laboratory features in subacute sclerosing panencephalitis (n = 47) and age-matched control (n = 43) groups. Significantly decreased levels of nitrite (median, 4.91 micromol/L) and nitrate (median, 6.14 micromol/L) were found in the patients. Nitrite and nitrate levels did not correlate with clinical or laboratory findings, except for presence of myoclonus. Cerebrospinal fluid nitrite levels of subacute sclerosing panencephalitis patients without myoclonic jerks were significantly higher than in those with myoclonus (median, 15.63 vs 4.34 micromol/L, respectively). The higher levels of nitrite in these patients can be explained by short disease duration and early stages of disease. Nitrate levels in subacute sclerosing panencephalitis patients with myoclonus (median, 9.26 micromol/L) were higher than in those without myoclonus (median, 4.25 micromol/L). Microbleeding resulting in conversion of nitrite to nitrate and increased production of superoxide can be suggested as possible mechanisms underlying these findings.


Assuntos
Mioclonia/líquido cefalorraquidiano , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/líquido cefalorraquidiano , Nitritos/líquido cefalorraquidiano , Panencefalite Esclerosante Subaguda/líquido cefalorraquidiano , Análise de Variância , Criança , Feminino , Febre/líquido cefalorraquidiano , Humanos , Imunoglobulinas/líquido cefalorraquidiano , Hipertensão Intracraniana/líquido cefalorraquidiano , Leucemia/líquido cefalorraquidiano , Masculino , Sarampo/imunologia , Índice de Gravidade de Doença , Fatores de Tempo
12.
Neurology ; 70(19 Pt 2): 1753-62, 2008 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-18077799

RESUMO

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.


Assuntos
Complexo AIDS Demência/líquido cefalorraquidiano , Complexo AIDS Demência/diagnóstico , Oxirredutases Intramoleculares/líquido cefalorraquidiano , Lipocalinas/líquido cefalorraquidiano , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/líquido cefalorraquidiano , Estresse Oxidativo , Complexo AIDS Demência/fisiopatologia , Adulto , Biomarcadores/análise , Biomarcadores/líquido cefalorraquidiano , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Estudos de Coortes , Progressão da Doença , Regulação para Baixo/fisiologia , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitrosação , Valor Preditivo dos Testes , Abuso de Substâncias por Via Intravenosa/líquido cefalorraquidiano , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquidiano , Regulação para Cima/fisiologia
13.
Biochem Pharmacol ; 74(5): 758-67, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17658485

RESUMO

Previous research has shown that injection of high-dose of morphine into the spinal lumbar intrathecal (i.t.) space of rats elicits an excitatory behavioral syndrome indicative of severe vocalization and agitation. Substance P N-terminal fragments are known to inhibit nociceptive responses when injected i.t. into animals. In this study, we investigated the effect of i.t. substance P (1-7) on both the nociceptive response and the extracellular concentrations of glutamate and nitric oxide (NO) metabolites (nitrite/nitrate) evoked by high-dose i.t. morphine (500 nmol). The induced behavioral responses were attenuated dose-dependently by i.t. pretreatment with the substance P N-terminal fragment substance P (1-7) (100-400 pmol). The inhibitory effect of substance P (1-7) was reversed significantly by pretreatment with [d-Pro2, d-Phe7]substance P (1-7) (20 and 40 nmol), a d-isomer and antagonist of substance P (1-7). In vivo microdialysis analysis showed a significant elevation of extracellular glutamate and NO metabolites in the spinal cord after i.t. injection of high-dose morphine (500 nmol). Pretreatment with substance P (1-7) (400 pmol) produced a significant reduction on the elevated concentrations of glutamate and NO metabolites evoked by i.t. morphine. The reduced levels of glutamate and NO metabolites were significantly reversed by the substance P (1-7) antagonist (40 nmol). The present results suggest that i.t. substance P (1-7) may attenuate the excitatory behavior (vocalization and agitation) of high-dose i.t. morphine by inhibiting the presynaptic release of glutamate, and reducing NO production in the dorsal spinal cord.


Assuntos
Morfina/administração & dosagem , Morfina/farmacologia , N-Metilaspartato/metabolismo , Óxido Nítrico/metabolismo , Dor/tratamento farmacológico , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacologia , Substância P/administração & dosagem , Substância P/farmacologia , Analgésicos/administração & dosagem , Analgésicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Ácido Glutâmico/líquido cefalorraquidiano , Masculino , Nitratos/líquido cefalorraquidiano , Nitritos/líquido cefalorraquidiano , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Substância P/análogos & derivados , Fatores de Tempo
14.
J Neurosci Methods ; 164(1): 155-60, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17512604

RESUMO

Elevated cerebrospinal fluid (CSF) concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), are assumed to be related to delayed vasospasm after subarachnoid haemorrhage (SAH). However, data on CSF concentrations of L-arginine, ADMA and its structural isomer symmetric dimethylarginine (SDMA) are very sparse in humans. We here present a new hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS-MS) method for the precise determination of these substances in CSF. The method requires only minimal sample preparation and features isotope labeled internal standards. First data of patients with SAH showed that on the day of admission CSF concentration values of L-arginine and ADMA were not significantly different from controls, but increased markedly during the course of the hospital stay. The decrease of the L-arginine to ADMA ratio points to a progressive impairment of the NO production rate in the brain after SAH which is confirmed by a simultaneous decrease in nitrate and nitrite concentrations in CSF.


Assuntos
Arginina/análogos & derivados , Arginina/líquido cefalorraquidiano , Líquido Cefalorraquidiano/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neuroquímica/métodos , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/biossíntese , Nitritos/líquido cefalorraquidiano , Hemorragia Subaracnóidea/diagnóstico , Regulação para Cima/fisiologia
15.
J Neurol ; 254(5): 575-80, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17426914

RESUMO

BACKGROUND: The causes of Amyotrophic Lateral Sclerosis (ALS) are unknown. A bulk of evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis. METHODS =: We assessed, in cerebrospinal fluid (CSF) and in plasma of 49 ALS patients and 8 controls, the amount of oxidized proteins (AOPP, advanced oxidation protein products), the total antioxidant capacity (FRA, the ferric reducing ability), and, in CSF, two oxidation products, the 4-hydroxynonenal and the sum of nitrites plus nitrates. RESULTS: The FRA was decreased (p = 0.003) in CSF, and AOPP were increased in both CSF (p = 0.0039) and plasma (p = 0.001) of ALS patients. The content of AOPP was differently represented in CSF of ALS clinical subsets, resulting in increase in the common and pseudopolyneuropathic forms (p < 0.001) and nearly undetectable in the bulbar form, as in controls. The sum of nitrites plus nitrates and 4-hydroxynonenal were unchanged in ALS patients compared with controls. CONCLUSION: Our results, while confirming the occurrence of oxidative stress in ALS, indicate how its effects can be stratified and therefore implicated differently in the pathogenesis of different clinical forms of ALS.


Assuntos
Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Antioxidantes/análise , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Oxirredução , Idoso , Aldeídos/sangue , Aldeídos/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/sangue , Análise de Variância , Feminino , Humanos , Proteínas Ferro-Enxofre/análise , Masculino , Pessoa de Meia-Idade , Nitratos/sangue , Nitratos/líquido cefalorraquidiano , Nitritos/sangue , Nitritos/líquido cefalorraquidiano
16.
J Neural Transm (Vienna) ; 114(8): 1011-5, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17393066

RESUMO

The study aimed to assay the cerebrospinal fluid (CSF) levels of protein S100B, a biomarker of astrocyte activation in relation to kynurenic acid (KYNA) and nitric oxide (NO) metabolites, nitrate/nitrite (NOx) concentrations in acute relapse multiple sclerosis (MS) patients. Twenty relapsing-remitting MS (RR-MS) patients and 10 controls were enrolled. RR-MS patients were assessed on the expanded disability status scale (EDSS) and underwent lumbar puncture. The CSF KYNA, NOx and S100B levels were significantly higher in RR-MS group compared to controls (p = 0.01, 0.001, 0.04, respectively). There was a significant correlation between CSF S100B and KYNA (p = 0.01) but not NOx (p > 0.05) in RR-MS. CSF KYNA, NOx or S100B concentrations did not correlate with disease characteristics of MS patients. Our study suggests the activation of the kynurenine pathway leading to the increase of neuroprotective KYNA in the CSF of MS patients during acute relapse what contrasts with chronic phases of the disease.


Assuntos
Astrócitos/metabolismo , Encefalite/fisiopatologia , Gliose/fisiopatologia , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Doença Aguda , Adulto , Astrócitos/imunologia , Biomarcadores/análise , Biomarcadores/líquido cefalorraquidiano , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Encefalite/líquido cefalorraquidiano , Encefalite/imunologia , Feminino , Gliose/líquido cefalorraquidiano , Gliose/imunologia , Humanos , Ácido Cinurênico/análise , Ácido Cinurênico/líquido cefalorraquidiano , Masculino , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/imunologia , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/líquido cefalorraquidiano , Nitratos/análise , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/metabolismo , Nitritos/análise , Nitritos/líquido cefalorraquidiano , Valor Preditivo dos Testes , Recidiva , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/análise , Proteínas S100/líquido cefalorraquidiano , Sensibilidade e Especificidade
17.
Brain Behav Immun ; 21(5): 668-76, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17129705

RESUMO

Complex Regional Pain Syndrome is a severe chronic pain condition characterized by sensory, autonomic, motor and dystrophic signs and symptoms. The pain in CRPS is continuous, it worsens over time, and it is usually disproportionate to the severity and duration of the inciting event. This study compares cerebrospinal fluid (CSF) levels of pro- and anti-inflammatory cytokines, chemokines and several biochemical factors (glial fibrillary acidic protein (GFAP), the nitric oxide metabolites (nitrate plus nitrite), the excitatory amino acid neurotransmitter glutamate, calcium, total protein and glucose) in patients afflicted with CRPS to levels found in patients suffering with other non-painful or painful conditions. The aim of the study is to determine the degree of involvement of glial cells and immune system mediators in the pathophysiology of CRPS. There was no elevation or reduction of a CSF marker that was specific for CRPS patients. However, there were several patterns of markers that could be helpful in both elucidating the mechanisms involved in the disease process and supporting the diagnosis of CRPS. The most common pattern was found in 50% (11 out of 22) of the CRPS patients and consisted of; elevated IL-6, low levels of IL-4 or IL-10, increased GFAP or MCP1 and increases in at least two of the following markers NO metabolites, calcium or glutamate. The results from this and other similar studies may aid in elucidating the mechanisms involved in the pathophysiology of CRPS. A better understanding of these mechanisms may lead to novel treatments for this very severe, life-altering illness.


Assuntos
Cálcio/líquido cefalorraquidiano , Síndromes da Dor Regional Complexa/líquido cefalorraquidiano , Citocinas/líquido cefalorraquidiano , Proteína Glial Fibrilar Ácida/líquido cefalorraquidiano , Ácido Glutâmico/líquido cefalorraquidiano , Radiculopatia/líquido cefalorraquidiano , Idoso , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/metabolismo , Biomarcadores/líquido cefalorraquidiano , Quimiocinas/líquido cefalorraquidiano , Quimiocinas/imunologia , Síndromes da Dor Regional Complexa/imunologia , Síndromes da Dor Regional Complexa/metabolismo , Citocinas/imunologia , Feminino , Glucose/líquido cefalorraquidiano , Humanos , Hidrocefalia/líquido cefalorraquidiano , Hidrocefalia/imunologia , Hidrocefalia/metabolismo , Masculino , Pessoa de Meia-Idade , Neuroglia/metabolismo , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/metabolismo , Nitritos/líquido cefalorraquidiano , Doenças do Sistema Nervoso Periférico/líquido cefalorraquidiano , Doenças do Sistema Nervoso Periférico/imunologia , Doenças do Sistema Nervoso Periférico/metabolismo , Radiculopatia/imunologia , Radiculopatia/metabolismo , Espondilolistese/líquido cefalorraquidiano , Espondilolistese/imunologia , Espondilolistese/metabolismo
18.
J Chromatogr B Analyt Technol Biomed Life Sci ; 851(1-2): 250-6, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17110176

RESUMO

Citrulline and nitric oxide (NO) are synthesized by NO synthase (NOS) in a 1:1-stoichiometry. In this study, we determined by HPLC arginine and citrulline concentrations by fluorescence detection and nitrate levels by UV absorbance detection in the cerebrospinal fluid (CSF) from patients with acute hydrocephalus that underwent ventricular drainage. We found increased citrulline concentration (50.6+/-17.2 versus 20.9+/-2.0 microM) and decreased arginine/citrulline molar ratio (0.42+/-0.11 versus 1.12+/-0.16) in hydrocephalus patients, while arginine and nitrate concentrations and citrulline/nitrate molar ratio remained with little change. Citrulline has been determined as a marker of NOS activity in some studies, but it remains to be determined the extent at which this statement holds true, since other biochemical pathways also regulate the concentration of this amino acid. Our results suggest that citrulline is primarily synthesized from NOS in acute hydrocephalus. The evaluation of sample deproteinization by addition of methanol for the analysis of amino acids in CSF is also reported.


Assuntos
Arginina/líquido cefalorraquidiano , Citrulina/líquido cefalorraquidiano , Hidrocefalia/líquido cefalorraquidiano , Nitratos/líquido cefalorraquidiano , Adulto , Albuminas/isolamento & purificação , Aminoácidos/líquido cefalorraquidiano , Estudos de Casos e Controles , Humanos , Pessoa de Meia-Idade
19.
Int J Neurosci ; 116(12): 1391-402, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17145675

RESUMO

Evidence from clinical and experimental studies supports the hypothesis of free radical-mediated damage of dopaminergic neurons in the pathology of Parkin's disease (PD). The present study was undertaken to evaluate the role of nitric oxide and oxidative stress in PD. Estimation of the stable metabolites of nitric oxide (NO, nitrite, nitrate) and malondialdehyde (MDA), an acceptable marker of lipid peroxidation, can provide indirect evidence of involvement of free radicals. Nitrite and malondialdehyde (MDA) levels were estimated in the lumbar cerebrospinal fluid (CSF) of 20 controls and 21 patients with PD. Nitrite and MDA content was not significantly altered in the CSF of PD patients as compared to the controls. Nitrite and MDA levels in CSF of PD patients exhibited no correlation with age, duration of disease, and severity of illness (measured by the Unified Parkinson's Disease Rating Score). There was no correlation between the CSF nitrite and MDA level. Findings of the present study do not provide evidence for the involvement of nitric oxide and oxidative stress in PD.


Assuntos
Malondialdeído/líquido cefalorraquidiano , Nitritos/líquido cefalorraquidiano , Estresse Oxidativo , Doença de Parkinson/metabolismo , Feminino , Radicais Livres/metabolismo , Humanos , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Nitratos/líquido cefalorraquidiano , Óxido Nítrico/líquido cefalorraquidiano , Índice de Gravidade de Doença
20.
Free Radic Biol Med ; 40(8): 1466-73, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16631536

RESUMO

Subarachnoid hemorrhage (SAH) resulting from aneurysmal rupture is the major cause of nontraumatic SAH. We hypothesized that oxidative stress could be increased following aneurysmal SAH due to hemoglobin release and ischemia-reperfusion injury and that may further contribute to poor outcome. We collected plasma and cerebrospinal fluid (CSF) samples from 11 non-SAH controls and 15 aneurysmal SAH patients for up to 10 days after surgery and investigated status of oxidative stress in patients. Results showed that mean or peak levels of F(2)-isoprostanes (F(2)-IsoPs), a specific marker of lipid peroxidation, and total nitrate/nitrite, metabolites of nitric oxide and peroxynitrite, in CSF and plasma were significantly higher in SAH patients than in controls. First-day levels were also higher in CSF, but not in plasma, in SAH patients. Moreover, mean and peak levels of CSF F(2)-IsoPs were positively correlated with poor outcome or severity of clinical conditions in patients. Furthermore, levels of retinol, delta-tocopherol, beta+gamma-tocopherol, lutein, beta-carotene, and coenzyme Q(10) in plasma were significantly lower in SAH patients than in controls. Our results indicate that oxidative damage may play important roles in the severity and complications of aneurysmal SAH and suggest that means to suppress lipid peroxidation may be beneficial in improving the outcome of aneurysmal SAH.


Assuntos
Aneurisma/sangue , Aneurisma/patologia , F2-Isoprostanos/sangue , Hemorragia Subaracnóidea/sangue , Hemorragia Subaracnóidea/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aneurisma/líquido cefalorraquidiano , Antioxidantes/metabolismo , Biomarcadores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitratos/sangue , Nitratos/líquido cefalorraquidiano , Nitritos/sangue , Nitritos/líquido cefalorraquidiano , Estresse Oxidativo , Solubilidade , Hemorragia Subaracnóidea/líquido cefalorraquidiano
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