Biliary excretion of FITC metabolites after administration of FITC labeled asialo orosomucoid as a measure of lysosomal proteolysis

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An isolated perfused rat liver system was used to study the hepatic uptake and degradation of asialo orosomucoid (asialo α 1-acid glycoprotein). To this aim we coupled the fluorochrome FITC to the asialoglycoprotein. The covalent attachment of FITC to the glycoprotein did not affect its perfusate dissappearance. The dissappearance rate was characterized by a t 1 2 ⋍ 6.1 min , the clearance being 11.2 ml/min. The internalized ligand was probably extensively degraded in the lysosomes as demonstrated by the appearance of low molecular weight fluorescent compounds in the bile, having a higher fluorescence yield than the native conjugate. Lysosomal degradation of ASOR-FITC was shown to be the rate limiting step in FITC excretion into the bile. Treatment of a perfused liver with varying doses of the protease inhibitor leupeptin did not influence the perfusate disappearance rate of the protein. However, leupeptin inhibited the biliary output of FITC metabolites in a dose dependent fashion, half maximal inhibition occuring at 210 nM (in the perfusion medium), corresponding with a dose of 0.05 mg leupeptin per 10 g liver. It is concluded that the rate of lysosomal degradation of proteins in vivo can be determined by measuring the biliary excretion of fluorescent material originating from fluorescent probes covalently coupled to the particular protein.