The isolation of 6-alpha-hydroxyoestrone from the urine of pregnant women.

1. In view of previous experiments in vitro and in vivo, in which 6-hydroxylation of phenolic steroids by various tissues was demonstrated, an attempt was made to isolate 6-hydroxy oestrogens from the urine of pregnant women. 2. During this work it was found that free 6alpha- as well as 6beta-hydroxylated oestrogens were extremely unstable under acidic conditions (pH <3); it was therefore necessary to establish experimental conditions under which no rearrangement of the hydroxyl group would take place. 3. By avoiding acid steps during the experimental procedures, a ketonic-phenolic fraction was obtained from enzymically hydrolysed late-pregnancy urine that was subjected to paper chromatography in various systems. 4. By using similar methods on a large scale and partition chromatography on Celite columns, a crystalline material was obtained from 1001. of late-pregnancy urine that was identified as 6alpha-hydroxyoestrone by various chemical reactions and by infrared spectroscopy.