title: Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides.
abstract: Endogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at -80°C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3 ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories.
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 4, 53100 Siena, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Giustarini
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, I-53100 Siena, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Galvagni
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, I-53100 Siena, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Orlandini
authors.affiliation: Division of Nephrology, Department of Medicine, The University of Texas Health Science Center San Antonio, and Audie L. Murphy VA Hospital, San Antonio, TX 78229, USA.
authors.forename: Paolo
authors.initials: P
authors.lastname: Fanti
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 4, 53100 Siena, Italy. Electronic address: ranieri@unisi.it.
authors.forename: Ranieri
authors.initials: R
authors.lastname: Rossi
mesh_terms: D000328:Adult; D001773:Blood Cells / Q000737:chemistry / Q000378:metabolism; D001793:Blood Preservation / Q000379:methods*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004220:Disulfides / Q000097:blood* / Q000737:chemistry; D006801:Humans; D008875:Middle Aged; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000097:blood* / Q000737:chemistry
chemical_list: D004220:Disulfides; D013438:Sulfhydryl Compounds
keywords: Blood; Disulfides; Plasma; Platelets; Red blood cells; Thiols