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) ؋ 10 7 liters/mol at lower albumin concentrations (15 M) to (0.54 ؎ 0.05) ؋ 10 7 liters/mol at higher albumin concentrations (300 M). 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) ؋ 10 7 liters/mol to (0.65 ؎ 0.03) ؋ 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.

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

No abstract

The i.r. spectra of bilirubin isomers that differ in number and position of vinyl groups were examined to verify the assignment of the 988 cm-1 peak of bilirubin (991 cm-1 peak in calcium bilirubinate) to its pendant vinyl groups. There were only small changes in this peak with changes in position of vinyl groups (exo-2- and -18-vinyl versus endo-3- and -17-vinyl), but progressive loss of peaks in this region was observed when vinyl groups were reduced to ethyl groups (dihydrobilirubin and mesobilirubin). Methylvinylmaleimide, a monopyrrole derived from the outer (A and D) rings of bilirubin, has a pendant vinyl group and exhibits a prominent peak at 986 cm-1, but haematinic acid methyl ester derived from the inner (B and C) rings has no vinyl group and shows no peak near 988 cm-1. These observations verify the assignment of the 988 cm-1 peak of bilirubin to its pendant vinyl groups. This supports our previous proposal that a decrease in the peak at 985-995 cm-1 in the i.r. spectra of pigment gallstones, as compared with unconjugated bilirubin or calcium bilirubinate, indicates a consumption of vinyl groups in the process of formation of the polymer in the pigment stones.

Congenitally jaundiced (jj) Gunn rats had a greater hepatic microsomal content of a cytochrome P-450 isoenzyme, P-450c, than did the non-jaundiced (Jj) rats. No differences in content of P-450b, P-450d and pregnenolone-16 alpha-carbonitrile-induced (PCN) P-450 were found between jj and Jj rats. This is the first demonstration of a constitutive increase in a specific cytochrome P-450 isoenzyme in association with a genetic defect.