Improving the reliability of human serum albumin-thiol group determination
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AuthorsJovanović, Vesna B.
Penezić-Romanjuk, Ana Z.
Pavicevic, Ivan D.
Aćimović, Jelena M.
Mandić, Ljuba M.
Article (Published version)
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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 lt 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.
Keywords:Albumin thiol group determination / Albumin isolation / Two-step precipitation method / Diabetes / Oxidative and carbonyl stress
Source:Analytical Biochemistry, 2013, 439, 1, 17-22
- Academic Press Inc Elsevier Science, San Diego