The ��cortisol�� area under the curve generated following cortisone acetate administration was used as an index of hepatic 11��-HSD1 activity as previously described [17]. Urine samples were analyzed by gas chromatography/mass spectrometry, as reported previously [18], [19], measuring free and conjugated cortisol metabolites. The THF+5��THF/THE, the cortols/cortolones, and the 11OH-androsterone+11OH-etiocholanolone/11-oxoetiocholanolone till ratios represent acknowledged markers of global 11��-HSD1 activity, with a high ratio indicating increased 11��-HSD1 reductase activity, with the proviso that the urinary free F/E (UFF/UFE) ratio, reflecting 11��-HSD2 activity is normal. The 5��THF/THF was used as a marker of 5��-reductase activity with a high ratio in the setting of increased absolute levels of urinary 5��-THF indicating increased activity.
Real Time PCR 11��-HSD1, Glucocorticoid receptor �� (GR��), and 5��-reductase 2 (SRD5A2) hepatic mRNA levels were measured by real-time PCR using an ABI 7500 system (Perkin-Elmer, Biosystems, Warrington, UK). PCR was performed in 25 ��l reactions on 96-well plates. Reactions contained TaqMan universal PCR master mix (Applied Biosystems, Warrington, Cheshire, UK), 900 nmol primers, 100�C200 nmol TaqMan probe and 25�C50 ng cDNA. All reactions were multiplexed with primers specific for 18S rRNA (provided as a preoptimized mix; Perkin-Elmer, Beaconsfield, Bucks, UK) as an internal reference. All target gene probes were labelled with the fluorescent label FAM, and the 18S probe with the fluorescent label VIC.
Reactions were as follows: 50��C for 2 min, 95��C for 10 min, and then 40 cycles of 95��C for 15 s and 60��C for 1 min. Data were analysed according to the manufacturer’s guidelines and were obtained as Ct values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine dCt values (dCt=Ct of the target gene minus Ct of the internal reference, 18S). Probes and primers for all genes were provided by ��assay on demand�� (Applied Biosystems). Arbitrary units were used with the transformation [AU=1000*2?dCt] to express results obtained. Immunohistochemistry and Immunofluorescence Five micron thick acetone fixed frozen liver sections with severe NASH and normal donor livers were cut onto coated glass slides. The slides were treated with methanol-hydrogen peroxide 0.
1% to block endogenous peroxidase activity for 20 minutes. After washing in phosphate buffered saline (PBS) sections were incubated in 20% normal donkey serum for 30 minutes and then with polyclonal antibody to 11��-HSD1 [20] at Drug_discovery a dilution of 1 in 100 in 10% donkey serum for 45 minutes. Secondary antibody, donkey antisheep IgG peroxidase conjugate (1200), was added to sections for 30 min. Slides were developed using 3,3��-diaminobenzidine and were counterstained with Mayer’s hematoxylin.