Total protein in common duct bile measured by acetonitrile precipitation and a micro bicinchoninic acid (BCA) method.

Abstract

Bile protein assays are complicated due to interference by other bile substances. In the present study we describe a microtiter plate method for the purification and quantification of bile proteins. The method is based on addition of acetonitrile in three steps to reconstituted freeze-dried bile, followed by ethanol washing of the precipitated proteins. Finally, protein in the precipitate is quantitated by two-point colour development using micro BCA reagents. Overall recoveries of protein in reconstituted bile spiked with exogenous protein (Seronorm) ranged from 91.0% (coefficient of variation; CV = 7.0%) to 97.1% (CV = 2.4%) by recoveries of 125I-Fibrinogen and 125I-Albumin. Bile pigments were largely removed during precipitation and washing, as verified by high pressure liquid chromatography (HPLC). Preferably the samples should be freeze-dried initially, as this lowered the blank readings. Two-point colour development with the BCA reagents were identical for standards assayed directly and standards added to protein depleted bile, and processed through all steps. Hence, no interference by either residual bile constituents nor the reagents upon the BCA protein assay could be detected. Standard curves ranged from 0.05 to 5.0 gl-1 (r > 0.98). Within day reproducibility (n = 15) was 7.8% (CV) and day to day (n = 10) was 12.1% (CV). Mean protein concentration in common duct bile from 30 patients was 1.20 gl-1 (range 0.34-3.87 gl-1). The method appears suitable for assay of bile protein, requires only limited sample volumes and allows processing of many samples within a short time.