Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

Abstract

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.

Affinity of Human Serum Albumin for Bilirubin Varies with Albumin Concentration and Buffer Composition

“…Because Ͼ99.8% of bilirubin was bound at all albumin concentrations studied, bound and total bilirubin concentrations are considered identical for modeling purposes. (5). This discrepancy probably reflects higher levels of unbound impurities in our earlier study because of exchange of tritium with the solvent.…”

“…Preparation of Stock [ 14 C]Bilirubin-Radiolabeled bilirubin conjugates were purified from bile after intravenous infusion of ␦- [5][6][7][8][9][10][11][12][13][14] C]aminolevulinic acid (PerkinElmer Life Sciences, Boston, MA) to bile-fistula rats using a protocol approved by the Animal Committee of Northwestern University Medical School and the Chicago VA Lakeside Medical Center. Purified 14 C-unconjugated bilirubin was then obtained by a modification of the method of Ostrow et al (21).…”

“…This approach assumes that K F is a constant that is independent of the albumin concentration. This assumption has never been adequately tested and has recently been questioned (5,6). Moreover, many properties of albumin are known to depend on its concentration (7)(8)(9)(10).…”

The case of chicken attachment region binding protein reinforces the importance of pre-screening of several libraries for cDNA cloning

“…The [$H]bilirubin was made by the Wilzbach procedure, which involves exposure of unlabelled bilirubin to $H gas at high pressure for 3 weeks. This procedure was first used to prepare [$H]bilirubin by Grodsky et al [2], whose work was not cited in the paper and who found that it caused significant decomposition of bilirubin. The decomposition and other reactions that occur during the Wilzbach treatment of bilirubin are unknown, but are likely to involve free-radical processes, disproportionation to non-IXα isomers, and addition to double bonds [3][4][5][6].…”

“…For example, a high expression rate is not a guarantee that a corresponding high number of positive clones will be obtained, and cases have been reported of successful screening of a library prepared from a weakly expressing tissue [1]. Furthermore, particular mRNAs are difficult to reverse-transcribe, possibly owing to a high CG content or base-paired structures, leading to an under-representation of the desired cDNA clone in the library, CG-rich sequences generally behave problematically in polymerase reactions, even at elevated temperatures (72 mC), and several attempts have been made to solve these problems [2,3].…”