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How to improve the reliability of HSA-thiol group determination?


Anal Biochem. 2013 Apr 9;


Authors: Jovanović VB, Penezić-Romanjuk AZ, Pavićević ID, Aćimović JM, Mandić LM


Abstract

The thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress, and could therefore 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 were assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native PAG 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 to total serum thiols) measurement of HSA-SH content with DTNB. The content of HSA-SH group in patients with type 2 diabetes was significantly (p<0.05) lower compared to that of the healthy cohort (0.483±0.067 versus 0.561±0.054 mmol SH/mmol HSA, respectively). As the proposed method of HSA isolation is simple, time and technically efficient, as well as it enables a reliable determination of HSA-SH content, it is therefore suitable for clinical practice.

PMID: 23583909 [PubMed - as supplied by publisher]