title: Improving the reliability of human serum albumin-thiol group determination.
abstract: The thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-thiol determination with Ellman's method depends on the purity of isolated HSA. Determination of total serum thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum thiols, was assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native polyacrylamide gel electrophoresis. Precipitation of HSA from serum via a two-step method with AS produced HSA with 91.9±3.6% purity and 69.7±4.4% yield, allowing for precise (relative standard deviation of 3.2%) and reliable (comparing with total serum thiols) measurement of HSA-SH content with DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)]. The content of the HSA-SH group in patients with type 2 diabetes was significantly (P<0.05) lower compared with that of the healthy cohort (0.483±0.067 vs. 0.561±0.054 mmol -SH/mmol HSA). Because the proposed method of HSA isolation is simple, time-efficient, and technically less demanding, and it also enables reliable determination of HSA-SH content, it is suitable for clinical practice.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Belgrade 11158, Serbia.
authors.forename: Vesna B
authors.initials: VB
authors.lastname: Jovanović
authors.forename: Ana Z
authors.initials: AZ
authors.lastname: Penezić-Romanjuk
authors.forename: Ivan D
authors.initials: ID
authors.lastname: Pavićević
authors.forename: Jelena M
authors.initials: JM
authors.lastname: Aćimović
authors.forename: Ljuba M
authors.initials: LM
authors.lastname: Mandić
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D011232:Chemical Precipitation; D003924:Diabetes Mellitus, Type 2 / Q000097:blood; D006801:Humans; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry* / Q000302:isolation & purification; D013438:Sulfhydryl Compounds / Q000032:analysis*
chemical_list: D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D000645:Ammonium Sulfate