SYNTHESIS, PHYSICAL AND BIOLOGICAL PROPERTIES OF LITHOCHOLYL-LYSYL-FLUORESCEIN - A FLUORESCENT MONOHYDROXY BILE-SALT ANALOG WITH CHOLESTATIC PROPERTIES

We have synthesised and characterised a fluorescent monohydroxy bile s alt analogue, lithocholyl-lysyl-fluorescein and compared its physical and biological properties with those of lithocholate, glycolithocholat e, sulpholithocholate, lithocholic acid glucuronide and taurocholate. The synthetic method used excess N-epsilon-CBZ-L-lysine methyl ester h ydrochloride and lithocholic acid via N-ethoxycarbonyl-2-ethoxy-1,2-di hydroquinolone (EEDQ) to give lithocholyl-lysine. Lithocholyllysyl-flu orescein (LLF) was then prepared using equimolar amounts of lithocholy l-lysine and fluorescein isothiocyanate (FITC) in bicarbonate buffer. LLF retained an apple green fluorescence, similar to that of fluoresce in. Unlike lithocholate, the critical micellar concentrations (CMCs) o f LLF, glycolithocholate (GLC), lithocholic acid glucuronide (LG) and sulpholithocholic acid (SLC) were similar. HPLC retention times (t(R)s ) of LLF and GLC were similar with a ratio of LLF/GLC of 1.05. In cont rast, the t(R) of SLC (6.52 min) but not of LG (21.2 min) was more com parable to that of taurocholate (5.73 min). In rats under pentobarbita l anaesthesia, the plasma half-life (t(1/2 alpha)) (min) was 4.5 +/- 1 .3 (n = 6) for LLF, 2.9 +/- 0.4 (n = 5) for [C-14]sulpholithocholate ( C-14-SLC) and 4.3 +/- 0.3 (min) for [C-14]lithocholic acid glucuronide (C-14-LG). Plasma clearances of C-14-SLC, LLF and C-14-LG were 15.5 /- 2.2 (n = 6), 18.1 +/- 4.2 (n = 6) and 17.8 +/- 0.5 ml/min/kg (n = 6 ) (P = 0.15), respectively. Biliary excretion in bile-fistula rats gav e cumulative 20 min biliary output as a percentage of injected dose as follows: LLF, 71.6 +/- 0.8% (n = 10); C-14-SLC, 75.5 +/- 2.8% (n = 6) and C-14-LG, 61.7 +/- 0.5% (n = 6) (P = NS). Peak biliary excretion r ates, given as % dose/2 min, were 10.2 +/- 0.3 for LLF, 13.5 +/- 0.6 f or C-14-SLC and 12.8 +/- 0.4 for C-14-LG. In another group of bile-fis tula rats, a 3.0 mu mol/500 mu l saline i.v. bolus of LLF caused a 15. 4 +/- 1.9% decrease in bile flow and, similarly, sodium lithocholate i n a solution of albumin caused a 17.9 +/- 1.8% (P = NS) diminution in bile flow. Despite the similar cholestatic properties of LLF and litho cholate, LLF was more soluble than lithocholate, with a relative reten tion time on HPLC similar to that of GLC. LLF is a divalent 'unipolar' anionic fluorescent monohydroxy bile salt analogue with physical, bio logical and cholestatic properties that are similar to those of lithoc holate, glycolithocholate and their derivatives and thus offers a pote ntially useful probe for studying mechanisms of monohydroxy bile salt- induced cholestasis at the hepatocellular level. (C) 1997 Elsevier Sci ence B.V.