A novel method to determine total and free serum bilirubin. |
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Authors: | S Nagaoka M L Cowger |
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Institution: | 1. Department of Pediatrics Albany Medical College, Albany, New York 12208 USA;2. Laboratory of Bioenergetics, State University of New York at Albany, Albany, New York 12222 USA |
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Abstract: | The determination of total (unconjugated) and free serum bilirubin concentrations using a novel and sensitive method based on static fluorescence quenching of daneyl bovine serum albumin was developed. The method allowed the use of a sample of 5 μl or less to determine total bilirubin over a range of 10–200 μg/ml with good recovery (94.9 ± 2.2%). For the determination of total bilirubin, a denaturation medium containing 8 m urea, 10 mm dithloerythreitol, and 0.1 m Tris was employed to eliminate interference by human serum albumin itself. The method was also tested with patients' sera containing negligible conjugated bilirubin in order to compare it to a commonly used “diazo” method. The correlation between the two methods gave a practically linear relation (γ = 0.99). The effects of a number of potentially interfering substances were tested and the results showed the test was specific for bilirubin. Concentrations of free bilirubin were determined without adding a denaturation agent. The experimental values were in agreement with those calculated theoretically using the isotherm of a single binding site and an association constant of human serum albumin to bilirubin of 1.5 × 108m?1. |
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