Synthesis, physical and biological properties of lithocholyl-lysyl-fluorescein: a fluorescent monohydroxy bile salt analogue with cholestatic properties.
We have synthesised and characterised a fluorescent monohydroxy bile salt analogue, lithocholyl-lysyl-fluorescein and compared its physical and biological properties with those of lithocholate, glycolithocholate, sulpholithocholate, lithocholic acid glucuronide and taurocholate. The synthetic method used excess N-epsilon-CBZ-L-lysine methyl ester hydrochloride and lithocholic acid via N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinolone (EEDQ) to give lithocholyl-lysine. Lithocholyl-lysyl-fluorescein (LLF) was then prepared using equimolar amounts of lithocholyl-lysine and fluorescein isothiocyanate (FITC) in bicarbonate buffer. LLF retained an apple green fluorescence, similar to that of fluorescein. Unlike lithocholate, the critical micellar concentrations (CMCs) of LLF, glycolithocholate (GLC), lithocholic acid glucuronide (LG) and sulpholithocholic acid (SLC) were similar. HPLC retention times (tRs) of LLF and GLC were similar with a ratio of LLF/GLC of 1.05. In contrast, the tR of SLC (6.52 min) but not of LG (21.2 min) was more comparable to that of taurocholate (5.73 min). In rats under pentobarbital anaesthesia, the plasma half-life (t(1/2alpha)) (min) was 4.5 +/- 1.3 (n = 6) for LLF, 2.9 +/- 0.4 (n = 5) for [14C]sulpholithocholate (14C-SLC) and 4.3 +/- 0.3 (min) for [14C]lithocholic acid glucuronide (14C-LG). Plasma clearances of 14C-SLC, LLF and 14C-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 gave cumulative 20 min biliary output as a percentage of injected dose as follows: LLF, 71.6 +/- 0.8% (n = 10); 14C-SLC, 75.5 +/- 2.8% (n = 6) and 14C-LG, 61.7 +/- 0.5% (n = 6) (P = NS). Peak biliary excretion rates, given as % dose/2 min, were 10.2 +/- 0.3 for LLF, 13.5 +/- 0.6 for 14C-SLC and 12.8 +/- 0.4 for 14C-LG. In another group of bile-fistula rats, a 3.0 micromol/500 microl saline i.v. bolus of LLF caused a 15.4 +/- 1.9% decrease in bile flow and, similarly, sodium lithocholate in a solution of albumin caused a 17.9 +/- 1.8% (P = NS) diminution in bile flow. Despite the similar cholestatic properties of LLF and lithocholate, LLF was more soluble than lithocholate, with a relative retention time on HPLC similar to that of GLC. LLF is a divalent 'unipolar' anionic fluorescent monohydroxy bile salt analogue with physical, biological and cholestatic properties that are similar to those of lithocholate, glycolithocholate and their derivatives and thus offers a potentially useful probe for studying mechanisms of monohydroxy bile salt-induced cholestasis at the hepatocellular level.
Mills
C O
CO
Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, UK.
Milkiewicz
P
P
Molloy
D P
DP
Baxter
D J
DJ
Elias
E
E
D000818:Animals; D001646:Bile / Q000187:drug effects / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism*; D002779:Cholestasis; D005452:Fluoresceins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D005965:Glucuronates / Q000378:metabolism; D007202:Indicators and Reagents; D008095:Lithocholic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008239:Lysine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D008297:Male; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility
D001647:Bile Acids and Salts; D005452:Fluoresceins; D005965:Glucuronates; D007202:Indicators and Reagents; C109091:lithocholyl-lysyl-fluorescein; D008095:Lithocholic Acid; D008239:Lysine