ALBUMIN-BINDING OF UNCONJUGATED [H-3] BILIRUBIN AND ITS UPTAKE BY RAT-LIVER BASOLATERAL PLASMA-MEMBRANE VESICLES

Using highly purified unconjugated [H-3]bilirubin (UCB), we measured U CB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence an d presence of an inside-positive membrane potential. Free UCB concentr ations ([B-f]) 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 H SA concentrations from 12 to 380 mu M, K'(f) (litre/mol) was inversely related to [HSA], irrespective of the [B-t]/[HSA] ratio. K'(f) was 2. 066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KCl was isoosmotically substituted for sucrose, the K'(f) value was significantly lower {2.0 77 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an os motic-sensitive space. Below saturation ([B-f] less than or equal to 6 5 nM), both electroneutral and electrogenic components followed satura tion kinetics with respect to [B-f], with K-m values of 28 +/- 7 and 5 7 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The V-max w as greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB . mg(-1) of protein . 15s(-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 up take of organic anions observed with increasing [HSA]. (b) UCB is take n up at the basolateral membrane of the hepatocyte by two systems with K-m values within the range of physiological free UCB levels in plasm a. The electrogenic component shows a lower affinity and a higher capa city than the electroneutral component. (c) It is important to calcula te the actual [B-f] using a K'(f) value determined under the same expe rimental conditions (medium and [HSA]) used for the uptake studies.