Synthesis of deuterium labeled cholesterol and steroids and their use for metabolic studies

A simple method is described for the preparation of [6,7,7(-2)H3] sterols and steroids. The synthesis starts with a Δ(5)-sterol or steroid and involves preparation of the 6-oxo-3α,5α-cyclosteroid, base exchange in the presence of deuterium oxide to introduce two deuteriums at the C-7 position and sodium borodeuteride reduction of the 6-oxo group to introduce the third deuterium atom at C-6. Rearrangement of the [6,7,7(-2)H3]6α-hydroxy-3α,5α-cyclosteroid then gives the desired [6,7,7(-2)H3]-Δ(5) sterol or steroid. [6,7,7(-2)H3]Cholesterol, [6,7,7(-2)H3]pregnenolone and [6,7,7(-2)H3]3β-hydroxyandrost-5-en-17-one were synthesized in this fashion and [6,7,7(-2)H3]progesterone was prepared from the [6,7,7(-2)H3]pregnenolone. Three examples of the use of these deuchromatography-mass spectrometry. The chrysophyte alga,Ochromonas malhamensis, was shown to be capable of introducing an extra methyl or ethyl group at C-24 of the side chain of [6,7,7(-2)H3]cholesterol to yield brassicasterol and poriferasterol, respectively. The ovary of the echinoderm,Asterias rubens, was demonstrated to metabolize [6,7,7(-2)H3]progesterone to yield mainly the 5α-isomers of pregnane-3,20-dione and 3β-hydroxypregnan-20-one. However, the 5β-isomers of these compounds were also detected as minor products for the first time as progesterone metabolites in this animal. Isolated oocytes of the frog,Xenopus laevis, produced a number of metabolites of [6,7,7(-2)H3]progesterone. In this report, two of them were shown to be 17α-hydroxy-pregn-4-en-3,20-dione and 20α-hydroxypregn-4-en-3-one.