EFFECT OF INFUSION OF TRIS-GALACTOSYL-CHOLESTEROL ON PLASMA-CHOLESTEROL, CLEARANCE OF LIPOPROTEIN CHOLESTERYL ESTERS, AND BILIARY-SECRETION IN THE RAT

As shown by us previously (van Berkel et al. 1985. J. Biol. Chem. 260: 2694-2699 and van Berkel et al. 1985. J. Biol. Chem. 260: 12203-12207) the clearance of both low density lipoproteins (LDL) and high density lipoproteins (HDL) from the blood can be greatly enhanced by pretreatment of these lipoproteins with a tris-galactosylated cholesterol derivative, which makes these particles recognizable by hepatic galactosyl-receptors. Here we report that intravenous infusion of the (water-soluble) tris-galactosyl-cholesterol in rats caused a dose-dependent decrease of the plasma cholesterol level. This fall was sustained long after termination of the infusion. It was not observed upon infusion of tris-glucosyl-cholesterol. The fall in plasma cholesterol was accompanied by an increase in hepatic cholesterol. Upon injection of rat HDL and LDL labeled in their cholesteryl ester moieties, plasma clearance of label in both lipoproteins was enhanced in rats infused with tris-galactosylcholesterol, the stimulation being more pronounced when the label was in HDL. The appearance of label in bile was also enhanced in the rats receiving the compound, again more markedly when the label was given as HDL. Ninety four percent or more of the radioactivity excreted in the bile was in the form of bile salts, with conjugated cholate being the major species in both control and treated rats; 6% or less of the radioactivity in the bile was as free cholesterol. Infusion of tris-galactosyl-cholesterol constitutes a new and defined method of lowering plasma lipoprotein levels by enhancing their uptake in the liver. Chemicals/CAS: Cholesterol Esters; Cholesterol, 57-88-5; Lipoproteins, HDL; Lipoproteins, LDL; N-(tris((beta-galactopyranosyloxy)methyl)methyl)-N(alpha)-(4-(5-cholest en-3 beta-yloxy)succinyl)glycinamide, 91202-79-

COUPLING OF THE ANTIVIRAL DRUG ARA-AMP TO LACTOSAMINATED ALBUMIN LEADS TO SPECIFIC UPTAKE IN RAT AND HUMAN HEPATOCYTES

We covalently coupled 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) to the carrier molecule lactosaminated human serum albumin using a water-soluble carbodiimide with a two-step conjugation method (pH 4.5 and pH 7.5) instead of the commonly used single-step conjugation at pH 7.5. This resulted in a predominantly monomeric conjugate (lac27-HSA-ara-AMP9). The conjugate was stable in buffer (pH 7.4) and blood plasma. After in vivo injection, the carrier and the monomeric conjugate were subjected to selective endocytosis in rat hepatocytes, as shown on immunohistochemical study and cell-separation techniques using I-125-labeled material. In competition experiments with other ligands for the asialoglycoprotein receptor N-acetylgalactosamine and asialofetuin, we showed that both lactosaminated human serum albumin and lac27-HSA-ara-AMP, are subject to endocytosis by this receptor system. Although the coupling of ara-AMP significantly increased the net negative charge of the conjugate compared with the native carrier, liver uptake was not affected by coadministration of an excess of succinylated human serum albumin (suc-HSA), a negatively charged ligand for the scavenger receptor. Incubation studies with purified rat liver lysosomes showed that in this acidic and proteolytic environment, mainly ara-AMP and, to a much lesser extent, ara-A itself were released from the carrier. After injection into the rat in vivo and in isolated perfused rat liver, no free ara-AMP or 9-B-D-arabinofuranosyladenine (ara-A) could be detected in plasma and perfusate, respectively, indicating proper retention of the virally active components in hepatocytes. Uptake experiments with freshly isolated rat and human hepatocytes indicated that human hepatocytes can also carry out endocytosis of lactosaminated human serum albumin and the ara-AMP conjugate by means of the galactose-recognizing receptor