Intracellular distribution of tritiated bilirubin during hepatic uptake and excretion

Abstract

Physiologic amounts of bilirubin-H3 were injected into normal rats and the distribution of isotope was determined in serum, subcellular fractions of the liver, and bile at intervals during the phases of uptake and excretion. Recovery in the three spaces after 2, 5, 15, and 30 min was 75–85%, indicating little bilirubin entered the extrahepatic tissues. At 5 min, 50% of the dose (excluding trapped serum bilirubin-H3) was in the liver, a bilirubin concentration greater than in serum; biliary excretion of bilirubin was less than 3%. By 30–60 min, most of the label was excreted in the bile as intact, diazotizable and crystallizable bilirubin. Bilirubin-H3 appeared principally in the hepatic cell sap at a rate and concentration too high for an albumin-linked transfer. No differences in the distribution of isotope in subcellular fractions were detectable during the phases of uptake or excretion or when the quantity injected was increased 20–40 times. Thus, intracellular distribution was primarily a constant function of intracellular bilirubin concentration. When compared to in vitro experiments, significant amounts of bilirubin accumulated within the microsomes and the acid hydrolase-rich fraction (lysosomes) but not in the mitochondrial or nuclear fractions.

Hepatic intracellular distribution of tritium-labeled unconjugated and conjugated bilirubin in normal and Gunn rats.

“…Starch gel electrophoresis of the supernatant fraction of normal liver containing UCB-3H revealed that the pigment was not bound to albumin but to a soluble protein or liproprotein that did not enter the gel at pH 7 to 8 (18). This observation is consistent with the conclusions of Brown and co-workers that transfer of UCB from plasma albumin into the liver cell is too rapid for an albumin-linked transfer (3).…”

“…This relatively invariable pattern revealed about 60%o of radioactivity in the supernatant fraction, approximately 25% in the microsomal fraction, and the rest distributed equally among mitochondria, lysosomes, and nuclei. These findings in normal rats confirm the results of Brown and associates (3). Starch gel electrophoresis of the supernatant fraction of normal liver containing UCB-3H revealed that the pigment was not bound to albumin but to a soluble protein or liproprotein that did not enter the gel at pH 7 to 8 (18).…”

Identification, Purification, and Partial Characterization of an Organic Anion Binding Protein from Rat Liver Cell Plasma Membrane

“…The hepatic uptake of bilirubin and BSP is rapid. After the intravenous injection of tracer amounts of [3H]bilirubin or [35S]BSP in rats, -40% of injected radioactivity is present within the liver 1.5 min later (2,(4)(5)(6). In the isolated perfused rat liver, 30 '251-albumin is injected simultaneously, all 1251 is recovered in the hepatic vein effluent confirming that albumin does not accompany bilirubin or BSP into the liver cell (7,8).…”

Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions

“…Although bilirubin is bound to albumin in plasma (5), only small amounts of albumin are present in liver cells (6), and the rate of transfer of bilirubin into liver is greater than the corresponding rate of transfer of albumin (1). These observations suggest that bilirubin in the cytoplasm of liver cells is primarily bound to protein(s) other than albumin (1,2,7,8).…”