1480 wizard gamma counter

The general procedure for radiometabolite analysis was described previously^{39 (link), 41 (link)} with minor modification in this work. Briefly, Following the intravenous injection of tracers 48–50, CD-1 mice were sacrificed at 30 min (n = 2). Either whole brain (for 48) or blood (for 49 and 50) samples were quickly removed and the blood samples were centrifuged at 15,000 × g for 2 min at 4 °C to separate the plasma. The supernatant (0.5 mL) was then collected in a test tube containing CH₃CN (0.5 mL) and the resulting mixture was vortexed for 15s and centrifuged at 15,000 × g for 2 min for deproteinization. The rat brain was homogenized in an ice-cooled CH₃CN/H₂O (1 mL, 1/1, v/v) solution. The homogenate was centrifuged at 150,000 rpm for 2 min at 4 °C and the supernatant was collected. The recovery of radioactivity into the supernatant for all three tracers 48–50 was > 90% based on the total radioactivity in the brain or blood homogenate. An aliquot of the supernatant (100 μL) obtained from the plasma or brain homogenate was injected into the HPLC system together with unlabelled 8, 17 or 37, and analyzed using a Phenomenex C18 column (10.0 mm ID × 250 mm). The radioactivity collected was measured by a 1480 Wizard gamma counter (PerkinElmer, USA). The percentage of 48, 49 or 50 to total radioactivity was calculated as (counts for 48, 49 or 50/total counts) × 100.

Circulating progesterone levels were assessed in plasma samples obtained at the time of genital sampling using a solid-phase ¹²⁵I radioimmunoassay kit, Coat-A-Count^® Progesterone (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). EDTA-treated plasma samples were processed according to the manufacturer’s basic protocol and counted for one minute on a 1480 Wizard Gamma Counter (Perkin Elmer, Waltham, MA). Final concentrations were determined by extrapolation of counts per minute to a calibration curve. The limit of detection in this assay is 0.02 ng/mL.

The general procedure for ex vivo biodistribution studies was described previously^{39 (link), 41 (link)} with minor modification in this work. Briefly, a solution of 48–50 (50 μCi / 150–200 μL) was injected into CD-1 mice via tail vein. These mice (each time point n = 4) were sacrificed at 5, 15, 30 and 60 min post tracer injection. Major organs, including whole brain, heart, liver, lung, spleen, kidneys, small intestine (including contents), muscle, testes, and blood samples were quickly harvested and weighted. The radioactivity present in these tissues was measured using 1480 Wizard gamma counter (PerkinElmer, USA), and all radioactivity measurements were automatically decay corrected based on the half-life of carbon-11. The results are expressed as the percentage of injected dose per gram of wet tissue (% ID/g).