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Inhibitors of side chain degradation in bile salt biosynthesis
ABNORMAL CYT P450 - DEPENDENT FUNCTION IN HEALTHY HBsAg CARRIERS. 125
A.P. Geubel, S. Pauwels, J.P. Buchet,E. Dumont, C. Dive. St-Luc University Hospital(U.C.L.), Bruxelles, Belgium.
Chronic HBV infection has been associated with both liver cell ultrastructural changes and increased susceptibility to hepatocellular carcinoma. Therefore, our AIM was to determine whether the HB Ag carrier state is by itself, associated with an unapparent s abnormal liver biotransformation system. METHODS : Nineteen HBeAg negative carriers without any clinical or biochemical evidence of liver dysfunction were investigated for aminopyrine demethylation (ABT). Eight were also tested for 6 8 -hydroxycortisol excretion (HPLC, 6 8 -OHF/FF ratio) and inorganic arsenic monomethylation capacity (MMA, % of total As) which has been shown to be only minimaly influenced by maximal phenobarbital induction (Buchet J.P. et al, Arch. Toxicol. 55: 151, 1984). RESULTS : ABT values were significantly higher in patients (0.74 + 0.21, mean ~ SD) than in controls (0.61 + 0.13, p < 0.05). Among the 8 cases with ABT, MMA and 6 8 -OHF determinations, results--were as follows :
Controls (mean _+ SD) HB Ag pos. (mSean + SD)
ABT(spec. activ.,%) MMA (% of total As) 6 ~-OHF/FF (mcg/24hr) 0.61 + 0.13 12.7 + 2.5 4.38 + 1.2 (nL/-7) (n----13) (n--9) 0.75 + 0.13 8.6 + 2.2 7.2 + 2.9 (n~-8) (n--8) (n--8) p < 0.05 p < 0.005 p < 0.02
CONCLUSIONS : Healthy HBsAg carriers exhibit unapparent hepatocellular dysfunctions which are indicative of an increased Cyt P450 dependent biotransformation system.
126
INHIBITORS OF SIDE CHAIN DEGRADATION IN BILE SALT BIOSYNTHESIS U. Giese and G. Kurz, Institut f~r Organische Chemic und Biochemie, Freiburg, FRG
In order to study the metabolic compartmentation of bile salt biosynthesis by EM-autoradiography with the aid of photoaffinity labelling besides photolabile derivatives of intermediary metabolites selective inhibitors of metabolic sequences are required. (i) As a reactive intermediate, easily taken up by isolated hepatocytes, 24,25-epoxy-5~-cholestane-3a,7a,12a,26-tetrol was synthesized and shown to inhibit oxidation of the side chain of 58-cholestane-3a,7a,12a-triol. The inhibitor does not impair taurocholate formation starting with 5B-cholestane-3a,7a,12a,26-tetrol and 3a,7a,12=-5B-cholestan-26-oate. Presence of inhibitor resulted in an altered polypeptide labelling pattern after photoaffinity labelling of isolated hepatocytes with 7,7-azo-5~-[3B-3H]cholestane-3a,12a-diol (sp. act. 2 Ci/mmol), but not with 7,7-azo-3a,12a-dihydroxy-5B-[3~-3H]cholestan-26-oate (sp. act. 20 Ci/mmol). (2) 2-Bromopalmitate, as an inhibitor, turned out to stop normal side chain degradation of all intermediates, leaving unaltered only the conjugation reactiones. Whereas the labelling pattern of polypeptides in photoaffinity labelling studies with isolated hepatocytes by 7,7-azo-5B-[3~-3H]cholestane-3a,12=-diol and by 7,7-azo-5~-[3B-3H]cholestane-3a,12a,26-triol (sp. act. 2,5 Ci/mmol) was unchanged in presence of 2-bromopalmitate the one by 7,7-azo3a,12a-dihydroxy-5~-[38-3H]cholestan-26-oate was affected. 24,25-Epoxy-5B-cholestane-3a,7a,12=,26-tetrol blocks selectively metabolism of 58-cholestane3a,7a,12a-triol and of its photolabile analogue down to the corresponding cholestan-26-oates, whereas 2-bromopalmitate blocks further side chain degradation of 3a,7a,12a-trihydroxy-5Beholestan-26-oate and of 7,7-azo-3a,12a-dihydroxy-5B-cholestan-26-oate. This offers an excellent possibility of studying enzymatic reactiones and intracellular transport in bile salt biosynthesis and allows indication of compartmentation with the aid of photoaffinity labelling.
$241
A.P. Geubel, S. Pauwels, J.P. Buchet,E. Dumont, C. Dive. St-Luc University Hospital(U.C.L.), Bruxelles, Belgium.
Chronic HBV infection has been associated with both liver cell ultrastructural changes and increased susceptibility to hepatocellular carcinoma. Therefore, our AIM was to determine whether the HB Ag carrier state is by itself, associated with an unapparent s abnormal liver biotransformation system. METHODS : Nineteen HBeAg negative carriers without any clinical or biochemical evidence of liver dysfunction were investigated for aminopyrine demethylation (ABT). Eight were also tested for 6 8 -hydroxycortisol excretion (HPLC, 6 8 -OHF/FF ratio) and inorganic arsenic monomethylation capacity (MMA, % of total As) which has been shown to be only minimaly influenced by maximal phenobarbital induction (Buchet J.P. et al, Arch. Toxicol. 55: 151, 1984). RESULTS : ABT values were significantly higher in patients (0.74 + 0.21, mean ~ SD) than in controls (0.61 + 0.13, p < 0.05). Among the 8 cases with ABT, MMA and 6 8 -OHF determinations, results--were as follows :
Controls (mean _+ SD) HB Ag pos. (mSean + SD)
ABT(spec. activ.,%) MMA (% of total As) 6 ~-OHF/FF (mcg/24hr) 0.61 + 0.13 12.7 + 2.5 4.38 + 1.2 (nL/-7) (n----13) (n--9) 0.75 + 0.13 8.6 + 2.2 7.2 + 2.9 (n~-8) (n--8) (n--8) p < 0.05 p < 0.005 p < 0.02
CONCLUSIONS : Healthy HBsAg carriers exhibit unapparent hepatocellular dysfunctions which are indicative of an increased Cyt P450 dependent biotransformation system.
126
INHIBITORS OF SIDE CHAIN DEGRADATION IN BILE SALT BIOSYNTHESIS U. Giese and G. Kurz, Institut f~r Organische Chemic und Biochemie, Freiburg, FRG
In order to study the metabolic compartmentation of bile salt biosynthesis by EM-autoradiography with the aid of photoaffinity labelling besides photolabile derivatives of intermediary metabolites selective inhibitors of metabolic sequences are required. (i) As a reactive intermediate, easily taken up by isolated hepatocytes, 24,25-epoxy-5~-cholestane-3a,7a,12a,26-tetrol was synthesized and shown to inhibit oxidation of the side chain of 58-cholestane-3a,7a,12a-triol. The inhibitor does not impair taurocholate formation starting with 5B-cholestane-3a,7a,12a,26-tetrol and 3a,7a,12=-5B-cholestan-26-oate. Presence of inhibitor resulted in an altered polypeptide labelling pattern after photoaffinity labelling of isolated hepatocytes with 7,7-azo-5~-[3B-3H]cholestane-3a,12a-diol (sp. act. 2 Ci/mmol), but not with 7,7-azo-3a,12a-dihydroxy-5B-[3~-3H]cholestan-26-oate (sp. act. 20 Ci/mmol). (2) 2-Bromopalmitate, as an inhibitor, turned out to stop normal side chain degradation of all intermediates, leaving unaltered only the conjugation reactiones. Whereas the labelling pattern of polypeptides in photoaffinity labelling studies with isolated hepatocytes by 7,7-azo-5B-[3~-3H]cholestane-3a,12=-diol and by 7,7-azo-5~-[3B-3H]cholestane-3a,12a,26-triol (sp. act. 2,5 Ci/mmol) was unchanged in presence of 2-bromopalmitate the one by 7,7-azo3a,12a-dihydroxy-5~-[38-3H]cholestan-26-oate was affected. 24,25-Epoxy-5B-cholestane-3a,7a,12=,26-tetrol blocks selectively metabolism of 58-cholestane3a,7a,12a-triol and of its photolabile analogue down to the corresponding cholestan-26-oates, whereas 2-bromopalmitate blocks further side chain degradation of 3a,7a,12a-trihydroxy-5Beholestan-26-oate and of 7,7-azo-3a,12a-dihydroxy-5B-cholestan-26-oate. This offers an excellent possibility of studying enzymatic reactiones and intracellular transport in bile salt biosynthesis and allows indication of compartmentation with the aid of photoaffinity labelling.
$241