Elsevier

Journal of Hepatology

Volume 31, Issue 4, October 1999, Pages 678-684
Journal of Hepatology

Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR rats

https://doi.org/10.1016/S0168-8278(99)80348-1Get rights and content

Abstract

Background/Aims: Fluorescent bile acids have proved useful for characterizing bile salt transport mechanisms. The aim of this study was to further validate the use of lysyl-fluorescein conjugated bile acid analogues as surrogate bile acids.

Methods: We analyzed biliary excretion kinetics of cholyl lysyl fluorescein (CLF), lithocholyl lysyl fluorescein (LLF) and sulfo-lithocholyl lysyl fluorescein (sLLF), both in the isolated rat hepatocyte couplet model and in TR rats with a selective canalicular transport defect of non-bile acid organic anions.

Results: CLF and LLF, which like their natural nonsulfated bile acid congeners are expected to behandled by the canalicular bile salt export pump, were transferred into the bile canaliculus much faster than sLLF, a putative substrate for the canalicular multispecific organic anion transporter in both the in vivo and the in vitro models employed. The contention that different transport systems are involved in sulfated and non-sulfated lysyl fluorescein conjugated bile acids biliary excretion was supported further by studies using TR rats, in which the cumulative biliary excretion of sLLF was reduced to 6% as compared with that of normal Wistar rats, in good agreement with values for its naturally-occurring radiolabeled parent compound sulfoglycolithocholate. In contrast, CLF and LLF were reduced to 66% and 52%, similar values to these for their congeners, [14C] glycocholate and [14C] lithocholate.

Conclusion: The close similarity in behavior of lysyl fluorescein conjugated bile acids to that of their naturally-occurring parent compounds in these different models gives support for both sulfated and nonsulfated lysyl fluorescein conjugated bile acids as substitute molecules for studies of bile acid transport.

Section snippets

Materials and Methods

CLF and LLF were synthesized as described previously, with minor modifications 22., 23.. Briefly, the synthetic method used excess N-ε-CBZ-1-lysine methyl ester hydrochloride and either cholic acid or lithocholic acid via N-ethoxycarbonyl-2 ethoxy-1, 2-dihydroquinolone (EEDQ) to give cholyl-lysine or lithocholyl-lysine. CLF or LLF was then prepared using equimolar amounts of choly-lysine or lithocholyl-lysine and fluorescein isothiocyanate (FITC), in bicarbonate buffer, pH 9.5. Purities of

Results

Fig. 2 shows cVA of CLF, LLF and sLLF in isolated hepatocyte couplets. The kinetic behaviors of non-sulfated (CLF and LLF) and sulfated (sLLF) bile acids were strikingly different. Whereas a plateau in cVA of non-sulfated bile acids was reached at 20–40 min of bile acid exposure, the sulfated bile acid studied did not appear to approach maximal canalicular accumulationuntil 90 min.

Representative examples of couplets treated with CLF, LLF and sLLF are shown in Fig. 3. Whereas sLLF exhibited a

Discussion

There have been numerous attempts to conjugate bile acids to fluorescent probes without affecting their physiological properties as bile acids 29., 30., 31.. We have synthesized LFCBAA and have shown that CLF and LLF have similar physical properties to their natural bile salt congeners 22., 23.. Moreover, CLF has been shown to have similar properties to cholylglycine when used as a probe in various transport studies 16., 17., 18., 19.. We have shown that CLF is closely similar to cholyglycine

Acknowledgements

Dr P. Milkiewicz was supported by an EASL Fellowship and the Midlands Gastroenterological Society. Dr M.G. Roma was supported by a Royal Society/CONICET Fellowship.

Portions of this work have been presented previously in abstract form at the 32nd EASL Meeting in London, UK, April, 1997.

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Dr. C.O. Mills and Dr P. Milkiewicz contributed equally to this work.

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