80 mkg/day

Immediately prior to the assay, a reaction mixture containing 45 mL of HNG buffer, 450 μL of ovalbumin, 75 μL of rat amniotic fluid, and 45 μL of 1 × 10−6 M 2,4,6,7-[3H]-estrone was prepared. Increasing concentrations of radioinert Isohexestrol (10 μL) were incubated with 2 mL of the reaction mixture in 13 × 100 mm borosilicate glass tubes at room temperature for 30 min. Tubes were subsequently transferred to ice and refrigerated (4 °C) for 1 h. While constantly being stirred, 200 μL of the cold HNG-charcoal suspension was added to each assay tube after the final incubation period. Tubes were vortexed and immediately centrifuged at 1,400g for 15 min at 4 °C. Following centrifugation, a 1 mL aliquot of the supernatant was removed from each tube and transferred to a scintillation vial containing 10 mL of Ultima Gold liquid scintillation cocktail, and the radioactivity was counted on a Packard Tri-Carb 1600 TR Liquid Scintillation Analyzer