Location of hydroxy groups introduced to steroid molecules by adrenal and testicular enzymes. I

Abstract

Incorporation of molecular oxygen into steroids by hydroxylases from adrenal and testicular glands was demonstrated by using 18O 2 as the tracer for molecular oxygen, and analyzing the hydroxylated products by a special mass-spectrometric technique. Methods for determining the definite position and form of oxygen atoms thus incorporated were proposed. After confirmation of 18O atom incorporation into the products, the presence of the 18O atom was examined in the fragments which were found on mass spectrometry. 17α-Hydroxyprogesterone, which was produced from progesterone under 18O 2 by microsomal enzymes of testis, was found to retain the 18O atom in the form of a 17α-hydroxy group, since the dehydrated fragment of 17α-hydroxyprogesterone was devoid of 18O. Testosterone produced from progesterone under the same conditions was found to retain the 18O atom in the 17β-hydroxy group, since the fragment which lost a CH 2CO group in the A ring of testosterone acetate retained the 18O atom. The pregnenolone acetate which was produced from 20α, 22 R-dihydroxycholesterol under 18O 2 by adrenal enzymes, contained no 18O in the form of the 20-ketone of pregnenolone, since the fragment which was produced by removal of the 3β-acetoxy group as acetic acid was devoid of 18O. The hydroxylated steroid was also chemically degraded, and the degradation product was further analyzed mass-spectrometrically to locate the 18O atom in the molecule. Deoxycorticosterone and 11-deoxycortisol were produced from progesterone and 17α-hydroxyprogesterone, respectively, by adrenal homogenate under 18O 2 and were found to have incorporated 18O. These corticoids were then chemically degraded to remove parts of the side-chains, and the degradation products were found to be devoid of 18O. This suggests that the 18O atoms were incorporated in the form of a 21-hydroxy group.