Determination of total and oxidized glutathione in human whole blood with a sequential injection analysis system

• Tipo: Artigo em Revista Científica Internacional
• Data: 2008

Autores

Andre R T S Araujo (Autor) Outra; Lucia L M F S Saraiva (Autor) FFUP; Jose L F C Lima (Autor) FFUP

Revista

Título: Talanta

Vol. 74 Nº 5

Páginas: 1511-1519

ISSN: 0039-9140

Editora: Elsevier

Indexação

ISI Web of Knowledge - 26 Citações

ISI Web of Science

Scopus - 32 Citações

Classificação Científica

CORDIS: Ciências da Saúde

FOS: Ciências exactas e naturais > Química

Outras Informações

ID Authenticus: P-004-1H1

DOI: 10.1016/j.talanta.2007.09.028

Resumo (PT): This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between the tGSH and GSSG concentrations. The determination was based on the DTNB–GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (λ = 412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions. The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 μM, with detection limits of 0.031 and 0.014 μM, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.) < 5.0% (n = 10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure. <br> <br> Keywords: Whole blood; Total and oxidized glutathione; Reduced glutathione; Glutathione reductase; SIA; In-line dilution <br> <a target="_blank" href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6THP-4PTMXP0-1&_user=2460038&_coverDate=02%2F15%2F2008&_rdoc=58&_fmt=high&_orig=browse&_srch=doc-info(%23toc%235288%232008%23999259994%23678809%23FLA%23display%23Volume)&_cdi=5288&_sort=d&_docanchor=&_ct=88&_acct=C000057398&_version=1&_urlVersion=0&_userid=2460038&md5=6e5c01fffbad40301eb9a0548d83c940"> Texto integral </a> <br> <br>

Abstract (EN): This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between. the tGSH and GSSG concentrations. The determination was based on the DTNB-GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (lambda = 412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions. The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 mu M, with detection limits of 0.031 and 0.014 mu M, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.) <5.0% (n = 10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure. (C) 2007 Elsevier B.V. All rights reserved.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Notas: autor para correspondência