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1.
Ann Biol Clin (Paris) ; 69(4): 470-2, 2011.
Artigo em Francês | MEDLINE | ID: mdl-21896414

RESUMO

Screening of alloantibodies is required before each transfusion. As part of our blood bank quality assurance, we have developed a quality indicator to monitor these false positive antibody results. We have studied 25.162 samples: sera were first screened by automated column agglutination technology (CAT). Positive results were found in 1.365 of the 25.162 samples. False positive results, ie positive screening test followed by a negative identification, were found in 271 (20%) cases. In the 116 patients remaining (43%) no factor could be evidenced. Interestingly, the percentage of patients with false positive antibody screening was stable month after month. In our experience, this percentage is very stable, it may be used as an indicator of quality laboratory and its unusual variation allows to suspect alterations of the reagents (hemolysis, loss of specificity, sensitivity).


Assuntos
Incompatibilidade de Grupos Sanguíneos/prevenção & controle , Transfusão de Sangue/normas , Reações Falso-Positivas , Fatores Imunológicos/sangue , Isoanticorpos/sangue , Programas de Rastreamento , Indicadores de Qualidade em Assistência à Saúde , Algoritmos , Bancos de Sangue/normas , Incompatibilidade de Grupos Sanguíneos/etiologia , Feminino , França , Humanos , Masculino , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade
3.
Artigo em Inglês | MEDLINE | ID: mdl-15905141

RESUMO

gamma-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l-2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the gamma glutamylaminobutyrate formed is measured at m/z=233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.


Assuntos
Glutamato-Cisteína Ligase/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida/métodos , Dipeptídeos/análise , Fibroblastos/metabolismo , Glutationa/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas/métodos , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Ratos , Reprodutibilidade dos Testes , Alinhamento de Sequência
4.
Nephrol Ther ; 1(2): 121-5, 2005 May.
Artigo em Francês | MEDLINE | ID: mdl-16895675

RESUMO

Accurate evaluation of oxidative stress is needed for patients on chronic hemodialysis (HD), as cardiovascular risk level seems related to it. Oxidative stress is often evaluated by measuring an end product of lipoperoxidation named malondialdehyde (MDA). However, the most common technique for measuring MDA, the Thio Barbituric Acid Reactive Substances method (TBARS), is known to be sensitive but poorly specific. We measured true total and free plasma MDA in fifty-four unselected patients on long-term HD, before and after HD sessions, by a new, highly specific HPLC method. Total and free MDA were higher before than after dialysis. Essentially, free MDA was decreased by HD but its fractional decrease was lower than that of urea or creatinine. This confirms that, in fact, free MDA is more or less bound to low molecular weight compounds and/or suggests that MDA may be produced mainly during HD sessions. We propose this new tool to further explore the relationship between oxidative stress, HD and true MDA.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Malondialdeído/sangue , Diálise Renal , Idoso , Biomarcadores/sangue , Creatinina/sangue , Feminino , Humanos , Falência Renal Crônica/etiologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Sensibilidade e Especificidade , Substâncias Reativas com Ácido Tiobarbitúrico/análise
5.
Artigo em Inglês | MEDLINE | ID: mdl-14643515

RESUMO

We describe a new, fast (6 min) and reliable method to measure reduced or oxidized glutathione (GSH) or (GSSG) in whole blood. The method is based on a LC/MS measurement in positive electrospray ionization mode after a chromatographic separation on a specific column which does not need any counter-ion in the mobile phase, improving the sensitivity of detection. A 50 microl sample of whole blood is sufficient for analysis. We demonstrate that the lack of an alkylating agent during the sample preparation brings out an underestimation of GSH and an artefactual production of GSSG, corresponding to 2-3% of GSH. The simultaneous use of N-ethyl-maleimide and a strong deproteinising acid prevents these two drawbacks. This efficient and new method of preparation and analysis lets us show that, unexpectedly, GSH is stable in whole blood for some hours and that deproteinised samples can be stored without GSH loss for at least three weeks at -20 or -80 degrees C. The reference interval, measured on 22 volunteers, on blood samples collected either with heparin or with EDTA, is 1310 +/- 118 microM for GSH and 0.62 microM for GSSG. The within-run precision of this method, with gamma glutamyl-glutamic acid as an internal standard, evaluated in three successive series (n = 30), lies between 2.1 and 4.8% for a GSH level at 580 or 1150 microM. The one step sample preparation we propose seems well suited for GSH routine measurements in hospital laboratories and avoids any underestimation of GSH, a now well accepted biomarker of oxidative stress.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dissulfeto de Glutationa/sangue , Glutationa/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Calibragem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Reprodutibilidade dos Testes
6.
Ann Biol Clin (Paris) ; 71(2): 215-8, 2013.
Artigo em Francês | MEDLINE | ID: mdl-23587591

RESUMO

Pediatric hemolytic uremic syndrome (HUS) is a rare complication of infections usually caused by Escherichia coli; Streptococcus pneumoniae may be a causative agent in 5% of cases and is often more serious in terms of morbidity and mortality. We report a case of pediatric HUS following an infection by a serotype of S. pneumoniae not included in the vaccine administered to the child. Bacterial neuraminidase revealed a T-antigen and a Tk-antigen and red blood cells polyagglutinability in the laboratory test. Transfusion has been reoriented by an indication of secondary preparations: deplasmatisation of red blood cells and platelets and abstention of therapeutic plasma administration. HUS evolved favorably in a few days but the child retains consequences of meningitis cerebral anoxia.


Assuntos
Antígenos Virais de Tumores/análise , Transfusão de Sangue , Síndrome Hemolítico-Urêmica/microbiologia , Síndrome Hemolítico-Urêmica/terapia , Infecções Pneumocócicas/complicações , Streptococcus pneumoniae/isolamento & purificação , Eritrócitos/imunologia , Feminino , Humanos , Lactente , Meningite Pneumocócica/líquido cefalorraquidiano , Meningite Pneumocócica/complicações , Meningite Pneumocócica/etiologia , Meningite Pneumocócica/terapia , Diálise Peritoneal , Testes Sorológicos
7.
Br J Nutr ; 95(1): 18-26, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16441913

RESUMO

The chemopreventive effects of dietary n-3 PUFA in various pathologies has so far remained controversial, and we were interested in studying their potential influence on cell redox status. DHA (22 : 6n-3), a typical highly unsaturated n-3 PUFA, was used at 30 micromol/l in a model of human fibroblast cell culture. A dose-response effect, roughly linear, was checked for DHA between 0 and 60 micromol/l, and was accompanied by a large increase in intracellular GSH content. A time course study of this effect shows that, after a short fall, as soon as 4 h after the beginning of the experiment, the large increase in the GSH content was associated with elevated catalytic activities of gamma-glutamyl-cysteinyl ligase, glutathione reductase and glutathione S-transferase. This coordinated response is characteristic of an antioxidant response and was confirmed by the induction of expression of mRNA for gamma-glutamyl-cysteinyl ligase, glutathione reductase and haem-oxygenase. This large increase in the GSH content contributes to decreasing the reactive oxygen species level, as assessed by the decreased accumulation of dichlorofluorescein inside cells. To our knowledge, this is the first report on a specific and potent effect of DHA for decreasing the oxidative stress of human fibroblasts.


Assuntos
Antioxidantes/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Fibroblastos/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa Redutase/metabolismo , Células Cultivadas , Ácidos Docosa-Hexaenoicos/farmacologia , Relação Dose-Resposta a Droga , Ácidos Graxos/análise , Fibroblastos/efeitos dos fármacos , Glutationa Transferase/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Oxirredução , Estresse Oxidativo/fisiologia , RNA Mensageiro/análise , Espécies Reativas de Oxigênio/análise , Compostos de Sulfidrila/metabolismo , Fatores de Tempo , Regulação para Cima/fisiologia
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