Studies on the Metabolism of Ethynylestradiol in vitro and in vivo: The Significance of 2-Hydroxylation and the Formation of Polar Products

Abstract

1. The metabolic fate of ethynylestradiol in vitro and in vivo has been followed by examining the displacement of tritium from [6,7-³H]ethynylestradiol and [2,4,6,7-³H]ethynylestradiol by other substituents. Incubations with rat liver microsomes and NADPH demonstrated that 2-hydroxylation is the major pathway of ethynylestradiol metabolism. In contrast, hydroxylations of ethynylestradiol at C-6 and C-7 are only of minor importance.

2. The easy inducibility of this microsomal 2-hydroxylation by pretreatment with phenobarbital, the requirement for NADPH and inhibition by CO and SKF 525A indicate that a haeme protein is involved.

3. The microsomal elimination of ³H from C-2 and C-4 of ethynylestradiol was markedly increased by glutathione, which is known to bind at C-1 and C-4 of estrogens forming ‘polar’ products. This formation of polar products was dependent on NADPH and was inhibited by CO and SKF 525A, supporting the concept that 2-hydroxylation of an estrogen is prerequisite for binding of glutathione.

4. In rats, ³H was eliminated from C-2 and C-4 of ethynylestradiol to an extent of 75%, but only 51% in the case of estradiol. This demonstrates that conjugation of ethynylestradiol to glutathione or other nucleophilic groups occurs in vivo and that the 2-hydroxylation of ethynylestradiol is greater than that of estradiol. This is due to the impossibility of hydroxylation of ethynylestradiol at C-16 because of its 17α-alkyl substituent, and to the competitive nature of 16α- and 2-hydroxylation of estrogens.