The Isolation from Rabbit Urine of a Conjugate of 17α-Estradiol with N-Acetylglucosamine

The Formation of Estradiol-3-glucuronoside-17α-N-acetylglucosaminide by Rabbit Liver Homogenate*

“…The major peak with K of 3.5 (peak B, Figure 1) was redistributed through 99 transfers in ethyl acetatecyclohexane-ethanol-water, 7:3:3:7. No radioactivity was found at K = 0.39, which is the value given by estradiol-17a-A-acetylglucosaminide in this system (Layne et al, 1964).…”

“…After the addition of 2500 units of Ketodase, the tubes were incubated at 37°for 16 hr. The contents of each tube was extracted three times with 10 ml of chloroform to remove free steroid and then three times with 10 ml of ethyl acetate to remove estradiol-17-Aacetylglucosaminide (Layne et al, 1964). The ethyl acetate extracts were evaporated to dryness and chromatographed on thin-layer silica gel G in chloroformethanol (3:7) in parallel with authentic estradiol-17a-A^-acetylglucosaminide (Layne et al, 1964).…”

“…The contents of each tube was extracted three times with 10 ml of chloroform to remove free steroid and then three times with 10 ml of ethyl acetate to remove estradiol-17-Aacetylglucosaminide (Layne et al, 1964). The ethyl acetate extracts were evaporated to dryness and chromatographed on thin-layer silica gel G in chloroformethanol (3:7) in parallel with authentic estradiol-17a-A^-acetylglucosaminide (Layne et al, 1964). The chromatogram was sprayed with Folin-Ciocalteu reagent to locate the standard material and was then assayed for radioactivity by eluting small areas into vials for liquid scintillation counting (Arai et al, 1962).…”

“…Smith and Breuer (1963) reported that estrone-3glucuronoside is formed by the incubation of estrone and UDP-glucosiduronic acid with washed rabbit liver microsomes. Recent work (Layne et al, 1964;Layne, 1965) has shown that much of the 17a-estradiol excreted in rabbit urine is in the form of the 3-glucuronoside-17a-Ar-acetylglucosaminide. This indicates that the rabbit possesses an enzyme system capable of transferring iV-acetylglucosamine to the estrogen molecule.…”

“…Experimental Procedure and Results Materials. Solvents were purified and stored as previously described (Layne et al, 1964). 17a-Estradiol-16-14C was obtained in previous experiments from the urine of rabbits injected with estrone-16-14C (Layne, 1965).…”