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

Sluijs, Peter van der; Oosting, Roelof; Weitering, J.G.; Hardonk, M. J.; Meijer, Dirk K. F.

doi:10.1016/0006-2952(85)90676-8

Cited by 17 publications

(6 citation statements)

References 33 publications

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“…Together with the "cluster effect" (31), this may explain why lz51-Lz5HSA is cleared less efficiently than the naturally occurring asialoglycoprotein ASOR (19) in the same perfusion set up. Fluorescence measurements of fluorescein-protein conjugates are difficult to interpret at low concentrations due to spuriously high background fluorescence, especially if theses conjugates are internalized and intracellularly de-graded with subsequent release of highly fluorescent degradation products (19,32). However, our initial observations [Bootsma H. P. et al, J .…”

Section: Discussionmentioning

confidence: 99%

“…Proteins were reacted with fluorescein isothiocyanate to produce a protein-fluorescein derivative exactly as described before (19). The molar fluorescein/protein ratio as assessed by the method of Jobaggy (20) was to 2/3 for the albumin derivatives.…”

Section: Chemicalsmentioning

confidence: 99%

“…Perfusate samples of the experiments with fluorescein-tagged proteins were assayed fluorometrically as described before (19). Protein was measured by the method of Lowry et al (24) using BSA as standard.…”

Section: Ex Vivo Perfused Rat Livermentioning

confidence: 99%

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Drug targeting to the liver with lactosylated albumins: Does the glycoprotein target the drug or is the drug targeting the glycoprotein?

et al. 1986

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The isolated perfused rat liver preparation was employed to study hepatic disposition of the model drug-carrier conjugate fluorescein-lactosylated albumin (F-LnHSA) with special reference to the influence of the organic anion fluorescein on liver cell specificity of the endocytosed neoglycoprotein. Hepatic clearance of fluoresceinated neoglycoproteins was significantly faster than clearance of radioiodinated neoglycoproteins. Perfusate clearance of F-L7HSA and F-L25HSA could not completely be inhibited by a dose of 10 mg asialoorosomucoid that saturates the hepatocyte receptor-mediated endocytic process. From these data, we inferred an additional hepatic uptake mechanism, competing with the Ashwell-receptor-mediated internalization of galactose-terminated glycoproteins. Clearance experiments with fluoresceinated 125I-human serum albumin in the presence of the polyanionic probe dextran sulfate revealed a nearly complete (approximately 90%) inhibition of hepatic uptake, while also a pronounced effect was obtained with colloidal carbon. These data point to nonparenchymal cell uptake of fluoresceinated protein via interaction with scavenger receptors. In wash-out studies, it was shown that about 25% of ligand sequestrated by sinusoidal liver cells escaped degradation and recycled to the perfusion medium. Our results show that care should be taken in the use of neoglycoproteins as drug carriers to hepatocytes, since a load of only 2 to 3 moles fluorescein per mole neoglycoprotein considerably affects intrahepatic distribution. The relative contribution of nonparenchymal cell uptake by coupling of acidic drugs to the neoglycoproteins is very probably inversely related to the number of exposing galactose groups per molecule neoglycoprotein. This phenomenon of "inversed targeting" could therapeutically both be useful or detrimental, dependent on the spectrum of cell types that should be reached by the drug.

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Section: Discussionmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

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Drug targeting to the liver with lactosylated albumins: Does the glycoprotein target the drug or is the drug targeting the glycoprotein?

et al. 1986

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“…Degradation of ASGP results in the generation of lowmolecular-weight compounds that are released into the circulation as well as into bile [11]. In addition, a minor fraction of internalized ASGP escapes the lysosomal route, enters the transcytotic pathway and is secreted intact into bile [12,13].…”

Section: Introductionmentioning

confidence: 99%

Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: Analysis by free-flow electrophoresis

Ellinger¹,

Klapper²,

Courtoy³

et al. 2002

Electrophoresis

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Endocytosis at reduced temperature has been used to define and characterize endosome subpopulations. Thus, the temperature sensitivity of endosome subpopulations involved in transport to lysosomes and transcytosis in rat hepatocytes was analyzed applying endosome labeling in the isolated perfused rat liver with route-specific ligands in combination with temperature shift protocols. Free-flow electrophoresis (FFE) that separates membranes and organelles based on their surface charge was then applied to isolate functional endosomes. Using asialoorosomucoid (ASOR) and polymeric immunoglobulin A (pIgA) as specific ligands of the lysosomal and transcytotic route, respectively, two distinct endosome subpopulations along either pathway were separated by FFE. Upon a short (1-3 min) internalization at 37 degrees C, 125I-ASOR and fluorescein isothiocyanate (FITC)-pIgA were colocalized in common early endosomes. Following a 5-10 min chase of the ligands at 37 degrees C endosomes labeled with 125I-ASOR were separated from endosomes labeled with FITC-pIgA, indicative of two distinct late compartments along the lysosomal and transcytotic route. Internalization at 16 degrees C resulted in accumulation of both ligands in common early endosomes and, consequently, in inhibition of transport to lysosomes and transcytosis. When 125I-ASOR or 125I-pIgA were first chased into late compartments at 37 degrees C and the temperature was subsequently lowered to 16 degrees C, biliary secretion of 125I-ASOR-derived counts was arrested, while biliary output of 125I-pIgA continued. In summary, ASOR en route to lysosomes can be blocked in early as well as in late endosomes at 16 degrees C, while biliary secretion of pIgA cannot be prevented by temperature reduction once the ligand had been transferred from early to late compartments.

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“…The conjugation of FITC was performed as described by van der Sluijs et al (1985) with minor modi® cations. The succinylation of FITC-LZM was performed according to Habeeb (1967).…”

Section: Preparation and Characterization Of Conjugatesmentioning

confidence: 99%

Renal Drug Delivery With Low-Molecular-Weight Proteins: The Effect of Charge Modifications on the Body Distribution of Drug-Lysozyme Conjugates

et al. 1999

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

Cited by 17 publications

References 33 publications

Drug targeting to the liver with lactosylated albumins: Does the glycoprotein target the drug or is the drug targeting the glycoprotein?

Drug targeting to the liver with lactosylated albumins: Does the glycoprotein target the drug or is the drug targeting the glycoprotein?

Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: Analysis by free-flow electrophoresis

Renal Drug Delivery With Low-Molecular-Weight Proteins: The Effect of Charge Modifications on the Body Distribution of Drug-Lysozyme Conjugates

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