Volume 120, Issue 7, Pages 1801-1809 (June 2001) Expression of sterol 12α-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo  William M. Pandak, Patricia Bohdan, Clifton Franklund, Darrell H. Mallonee, Gösta Eggertsen, Ingemar Björkhem, Gregorio Gil, Z.Reno Vlahcevic, Phillip B. Hylemon  Gastroenterology  Volume 120, Issue 7, Pages 1801-1809 (June 2001) DOI: 10.1053/gast.2001.24833 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of (A) sterol 12α-hydroxylase (CYP8b1) and (B) cholesterol 7α-hydroxylase (CYP7a1) in primary rat hepatocytes. Hepatocytes were infected with recombinant adenoviruses (1 pfu/cell) encoding either CYP8b1, CYP7a1, or a control virus (Ad-CMV-control) and incubated for 48 hours as described in Materials and Methods. Total RNA was isolated, and mRNA levels for CYP8b1 and CYP7a1 were determined by RPA using cyclophilin as the loading control. Gastroenterology 2001 120, 1801-1809DOI: (10.1053/gast.2001.24833) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Thin-layer chromatography of [14C]bile acids formed by primary rat hepatocytes infected with adenoviruses encoding sterol 12α-hydroxylase (CYP8b1) and/or cholesterol 7α-hydroxylase (CYP7a1). Primary hepatocytes were infected (1 pfu/cell) with a recombinant adenovirus encoding CYP8b1(Ad-CMV-CYP8b1), CYP7a1 (Ad-CMV-CYP7a1), control virus (Ad-CMV-control), or a combination and incubated for 48 hours in medium containing [14C]cholesterol. Conjugated bile acids were extracted into methanol:water, base hydrolyzed, and separated on thin-layer chromatography in a solvent system of ethyl-acetate:cyclohexane:acetic acid (7.7:2.3:1 vol/vol/vol). [14C]Bile acids were visualized by radioautography. Gastroenterology 2001 120, 1801-1809DOI: (10.1053/gast.2001.24833) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of sterol 12α-hydroxylase (CYP8b1) and cholesterol 7β-hydroxylase (CYP7a1) overexpression on bile acid composition in primary rat hepatocytes. Primary hepatocytes were infected (1 pfu/cell) with an adenovirus encoding CYP8b1 or CYP7a1 or control virus and incubated for 48 hours in the presence of [14C]cholesterol. Radiolabeled conjugated bile acids were extracted from culture medium into methanol:water, base hydrolyzed, and chromatographed on thin-layer chromatography (Figure 2), and the bile acids were located by radioautography. Individual bile acids were scraped into liquid scintillation vials and radioactivity quantitated by liquid scintillation counting. MCA, α/β-muricholic acids. Gastroenterology 2001 120, 1801-1809DOI: (10.1053/gast.2001.24833) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Expression of sterol 12α-hydroxylase (CYP8b1) in intact rats. Male Sprague–Dawley rats were infected with 1.88 × 1011 viral particles encoding CYP8b1 or control virus via tail vein injection. Animals were killed 5 days after injection. Livers were harvested for enzyme activity and mRNA analysis. (A, B) RPAs for CYP8b1 and CYP7a1 mRNA, respectively. Gastroenterology 2001 120, 1801-1809DOI: (10.1053/gast.2001.24833) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of sterol 12α-hydroxylase (CYP8b1) overexpression on the biliary bile acid composition profiles in rats. Male Sprague–Dawley rats were infected with 1.88 × 1011 viral particles encoding CYP8b1 or control virus via tail vein injection. After 5 days, animals were placed under methoxyfluorane anesthesia, the bile duct was cannulated, and bile was collected, extracted into methanol:water, and analyzed for bile acid composition using reverse-phase high-performance liquid chromatography as described in Materials and Methods. Gastroenterology 2001 120, 1801-1809DOI: (10.1053/gast.2001.24833) Copyright © 2001 American Gastroenterological Association Terms and Conditions