THE ROLE OF THE CANALICULAR MULTISPECIFIC ORGANIC ANION TRANSPORTER IN THE DISPOSAL OF ENDOBIOTICS AND XENOBIOTICS

Bile is an important excretory route for the elimination of amphiphilic organic anions, and hepatocytes are the primary secretory units of bile formation. The hepatocytic basolateral and canalicular membranes are equipped with various carrier proteins. Transport across the canalicular membrane represents a major concentrative step. Various ATP-dependent transporters have been identified, such as a multispecific organic anion transporter (canalicular multispecific organic ion transporter, cMOAT), a bile acid transporter and several P-glycoproteins. TR(-) rats, which lack cMOAT activity, have been valuable in defining the substrate specificity of cMOAT. A wide range of glucuronide-, glutathione- and sulfate-conjugates are transported by this system

IMMUNOAFFINITY PURIFICATION AND RECONSTITUTION OF THE HUMAN BILIRUBIN PHENOL UDP-GLUCURONOSYLTRANSFERASE FAMILY

When membrane proteins are solubilized and subjected to purification procedures, the loss of lipids surrounding the protein often results in irreversible inactivation. We describe a procedure for the immunoaffinity purification of the membrane protein UDP-glucuronosyltransferase from human liver. This procedure reduces exposure of the protein to detergent, thereby reducing lipid loss. Triton X-100 was used to solubilize human Liver microsomes. The solubilized proteins were applied to a sephacryl 300 (S-300) gel filtration column equilibrated with detergent-free buffer. UDP-glucuronosyltransferase activity eluted in turbid fractions in the void volume. During passage through the column Triton X-100 can partition in the buffer, while lipids are reconstituted into vesicular structures. Proteins with a high affinity for phospholipids remain associated with the lipid and elute in the void volume. The active fractions from the S-300 column were resolubilized with Triton X-100 and applied to a column with immobilized antibody. Washing this column with buffer containing phosphatidylcholine liposomes and no detergent removed unbound proteins and minimized loss of lipid from bound UDP-glucuronosyltransferase. Raising the pH of the washing buffer to 11.5 in the presence of liposomes resulted in elution and simultaneous reconstitution of active UDP-glucuronosyltransferase. Antibodies against membrane proteins are often available but immunoaffinitypurification of active enzyme is difficult. The approach described here could be useful for the isolation of other membrane proteins. (C) 1995 Academic Press, Inc

The ins and outs of reverse cholesterol transport

It is generally assumed that HDL is the obligate transport vehicle for 'reverse cholesterol transport'. the pathway for removal of excess cholesterol from peripheral tissues via the liver into bile and subsequent excretion via the feces. During the last few years, intensive research has generated exciting new data on the separate processes involved in reverse cholesterol transport. Many 'new' proteins, particularly members of the ABC transporter and nuclear receptor subfamilies, that mediate or influence cholesterol fluxes have been identified and characterized. An important role of the intestine in regulation of cholesterol homeostasis is emerging. In this paper, new insights into mechanisms of reverse cholesterol are reviewed

Influence of dietary calcium phosphate on the disposition of bilirubin in rats with unconjugated hyperbilirubinemia

The aim of this study was to test a possible form of therapy that could be used in the management of unconjugated hyperbilirubinemia. We hypothesized that unconjugated bilirubin (UCB) can permeate the intestinal wall and can thus be secreted with the feces. We have previously observed that UCB binds to amorphous calcium phosphate in vitro, Orally ingested amorphous calcium phosphate may act as a trapping agent for bilirubin in the intestine, thereby preventing back-diffusion across the intestinal wall, In this study, we tested whether feeding calcium phosphate leads to enhanced excretion of unconjugated bilirubin in Gunn rats. When a purified control diet was substituted by a high calcium phosphate diet, a decrease in bilirubin levels of 30% to 50% in male Gunn rats and of 23% in female rats was observed, The fecal output of bilirubin was more than doubled in Gunn rats in the first 3 days after the normal diet had been replaced by the high calcium-phosphate diet, The biological half-life of H-3-labeled bilirubin in blood was 89.8 +/- 17.2 hours in rats fed the purified control diet and 50.9 +/- 1.4 hours in rats fed the high calcium phosphate diet (P = .004). After 30 weeks, plasma bilirubin levels were still significantly lower in Gunn rats fed a high calcium phosphate diet, No differences were found in plasma concentrations of calcium, magnesium, phosphate, urea, and creatinine in both Gunn rats and Wistar rats on control or high calcium phosphate diets, This therapy might be useful in the management of Crigler-Najjar patients, for example, as an adjunct to phototherapy