Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

U Schramm,A Dietrich,S Schneider,Hp Buscher,W Gerok,G Kurz

doi:10.1016/s0022-2275(20)41631-1

Abstract

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.

Highlights

Introduction of theNBD-amino group in the 7-position, whether axial or equatorial, leads to a dependency of the extent of conjugation on the applied concentration of the unconjugated fluorescent derivatives
The presence of albumin causes a small hypsochromic shift of the longest wavelength absorption maxima, as shown for 7P-NBD-NCT, where the maximum is shifted from 497 nm in aqueous solution to 486 nm in presence of 5% albumin (Fig. 1)
These results clearly indicate that the fluorescent bile salt derivatives interact with all the polypeptides that were identified in subfractions enriched with sinusoidal membranes as bile salt binding proteins

Summary

Introduction

Introduction of theNBD-amino group in the 7-position, whether axial or equatorial, leads to a dependency of the extent of conjugation on the applied concentration of the unconjugated fluorescent derivatives. All taurine-conjugated fluorescent bile salt derivatives were secreted into bile unmetabolized, and, with the exception of 3a-NBD-NCT, in all cases more than 95% of the applied compound could be detected in bile within 30 min (Fig. 4,insert).

Results

Conclusion