2.7. Liver Microsome Assay

Microsome experiments with [¹⁸F]12 in the presence of NADPH were performed according to the procedure recently described by us with slight modifications [43,44]. Incubations had a final volume of 250 µL. The radiotracer dissolved in EtOH (4 µL; 5 MBq/µL) was diluted with PBS (496 µL, 0.8% EtOH). This radiotracer solution (100 µL, 0.32% EtOH and 16 MBq/mL or 1.6 µM final) was mixed with PBS (112.5 µL) and human liver microsomes (12.5 µL of 20 mg/mL stock; 1 mg/mL final; Gibco™ Cat. No. HMMCPL, Lot. No. PL050E-B) in a 1.5 mL Eppendorf tube and the mixture was warmed for 5 min at 37 °C. Subsequently, NADPH (25 µL of freshly prepared 20 mM solution in PBS, 2 mM final) was added and the mixture was incubated at 37 °C. As a control incubation, NADPH was omitted and replaced by PBS. After distinct time points (10, 20, 30 and 60 min), an aliquot (40 µL) was withdrawn and added to ice-cooled CH₃CN (160 µL). The mixture was vortexed for 30 s, stored on ice for 4 min and centrifuged (5 min at 15,000 rpm). The resulting supernatant was used for radio-HPLC analysis using the Shimadzu system described in Section 2.2 with the following conditions. A C₁₈ column Kinetex^® from Phenomenex (5 µm, 100 Å, LC Column 250 × 4.6 mm) served as stationary phase. The eluent consisted of (A) 0.1% trifluoroacetic acid in H₂O and (B) MeCN; flow rate 1 mL/min; elution profile A/B: t_{0 min} 70/30–t_{10.0 min} 70/30–t_{11.0 min} 5/95–t_{16.0 min} 5/95–t_{17.0 min} 70/30–t_{22.0 min} 70/30.

Testosterone (40 µM final) was used as a positive control for the activity of the HLM and was separately incubated under the conditions described above (0% EtOH final). The metabolization was analyzed at distinct time points (10, 20, 30, 40 and 60 min) with the HPLC system specified above (254 nm, elution profile A/B: t_{0 min} 55/45–t_{10.0 min} 55/45–t_{11.0 min} 5/95–t_{16.0 min} 5/95–t_{17.0 min} 55/45–t_{25.0 min} 55/45) and was completed after >30 min.