title: Quantification of reduced glutathione by HPLC-UV in erythrocytes of hemodialysis patients.
abstract: Reduced glutathione (GSH) is a well-known multifunctional antioxidant. Its depletion is linked to a number of pathologies, such as renal insufficiency. Feasible methodologies in clinical chemistry are vital. Therefore a methodology for GSH quantification was optimized and validated by HPLC-UV. Important aspects such as acid deproteinization and GSH stability were established. The erythrocytes were hemolyzed, deproteinized, derivatized with 5,5-dithio-bis (2-nitrobenzoic) acid and analyzed using HPLC, on an RP18 gradient elution, lambda=330 nm. The method was applied to hemodialysis patients (n=75) compared with healthy subjects (n=40). The assay was linear from 0.5 to 3.0 mm (r2>0.99). The intra- and inter-run reproducibilities were obtained with CV%<10%. The accuracy (bias %) ranged from 1.32 to -6.38%, and the recovery was >94%. The derivatized sample was stable for 60 days at -20 degrees C. The GSH levels in hemodialysis patients showed a significant increase compared with healthy subjects (p<0.05) and an inverse correlation with age (r=-0.286; p=0.013) was found. This method used UV detection, reduction of the phosphate concentration in the mobile phase and effective protein removal with trichloroacetic acid. The method proved to be reproducible, precise, accurate and stable. Thus, it can be suggested for routine laboratory tests for the verification of physiopathological conditions.
authors.affiliation: Laboratory of Toxicology (LATOX), Department of Clinical and Toxicology Analysis, Center of Healthy Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil. sgarpom@smail.ufsm.br
authors.forename: Solange Cristina
authors.initials: SC
authors.lastname: Garcia
authors.forename: Karen
authors.initials: K
authors.lastname: Schott
authors.forename: Mariele
authors.initials: M
authors.lastname: Charão
authors.forename: Angela
authors.initials: A
authors.lastname: Moro
authors.forename: Rachel
authors.initials: R
authors.lastname: Bulcão
authors.forename: Denise
authors.initials: D
authors.lastname: Grotto
authors.forename: Juliana
authors.initials: J
authors.lastname: Valentini
authors.forename: Denise
authors.initials: D
authors.lastname: Bohrer
authors.forename: Simone
authors.initials: S
authors.lastname: Cardoso
authors.forename: Valdeci
authors.initials: V
authors.lastname: Pomblum
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000378:metabolism*; D005978:Glutathione / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D006435:Renal Dialysis; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
chemical_list: D005978:Glutathione