Placental tissue was removed from a ewe at 127 days' gestation and, without separating foetal from maternal contributions, incubated as a mince (4 g) with equimolar amounts of [4-14C]androstenedione (2 μCi, 56 mCi/mmol) and [1,2-3H]epitestosterone (15 μCi, 420 mCi/mmol) at 39·5 °C. The reactions were stopped after 2 h by adding acetone and freezing. The non-radioactive carrier steroids, androstenedione, testosterone, epitestosterone, oestrone, oestradiol-17β, oestradiol-17α, testosterone sulphate and epitestosterone sulphate were added in 500 μg amounts.
After mixing, steroids and their conjugates were extracted from the incubation medium with 2 × 100 ml acetone followed by 2 × 100 ml ether: ethanol (3:1, v/v). Nonpolar lipid material was removed by partitioning the pooled, dried extract between 70% (v/v) aqueous methanol and light petroleum (b.p. 40–60 °C). The methanol was evaporated and unconjugated steroids extracted from the aqueous residue with 3 × 30 ml ether before saturating with ammonium
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