Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: results of a novel ultrafiltration method.
Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [(14)C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [(14)C]bilirubin was strongly dependent on assay conditions, falling from (5.09 +/- 0.24) x 10(7) liters/mol at lower albumin concentrations (15 microm) to (0.54 +/- 0.05) x 10(7) liters/mol at higher albumin concentrations (300 microm). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 +/- 0.26) x 10(7) liters/mol to (0.65 +/- 0.03) x 10(7) liters/mol). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.
Weisiger
R A
RA
Department of Medicine and the Liver Center, University of California, San Francisco, California 94143-0538, USA. dickw@itsa.ucsf.edu
Ostrow
J D
JD
Koehler
R K
RK
Webster
C C
CC
Mukerjee
P
P
Pascolo
L
L
Tiribelli
C
C
D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism*; D002021:Buffers; D002713:Chlorine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008962:Models, Theoretical; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014462:Ultrafiltration / Q000379:methods*
D002021:Buffers; D012709:Serum Albumin; D012965:Sodium Chloride; D002713:Chlorine; D011189:Potassium Chloride; D001663:Bilirubin