| Description |
Four in vitro incubation experiments have been carried out with cortisone and an attempt was made to isolate and identify any metabolic products formed. All incubations were carried out in a phosphate buffer medium at 35° for a period of four hours. Incubation I was carried out using slices of rat liver and a total of four hundred micrograms of cortisone. The incubation mixture was fractionated and a qualitative test for the presence of 17-ketosteroids in each fraction was made. A few questionable positive tests were found, but these were negative as a whole. The attempt to determine quantitatively the amount of alpha, beta-unsaturated ketone present in the fractions was severely hampered by the interference of background absorption. Incubation II was carried out with rat liver slices and ten milligrams of cortisone. The incubation mixture was extracted with hexane and ether, and the residues obtained were separated into ketonic and non-ketonic fractions. The ketonic fraction obtained from the ether extract was acetylated and chromatographed, and a substance was isolated that was believed to be the acetate of the starting material. The small amounts of this substance and the difficulty of obtaining it in pure form prevented positive identification. Incubation III was carried out by using slices of rabbit liver and one hundred milligrams of cortisone acetate. The incubation mixture was extracted with acetone, and this extract was concentrated and extracted successively with petroleum ether and ethylene dichloride. The residues obtained were separated into ketonic and non-ketonic fractions. The latter were saponified in ethanolic potassium hydroxide. Quantitative measurement of the ultraviolet absorption spectra indicated an overall recovery of eighty per cent. The ketonic fractions and the non-ketonic fraction obtained from the ethylene dichloride extract were chromatographed, and free cortisone was isolated from all three of these fractions. It was identified by the melting point and mixed melting point of both the free compound and the acetate. A small amount of an unidentified material melting 173-175°C was isolated from a ketonic fraction obtained from the residue of the petroleum ether extract. Incubation IV was carried out using rabbit liver slices and 196.2mg of free cortisone. Rabbit serum was used as a suspending medium for the steroid. The incubation mixture was worked up in the same was as was done for Incubation III. A control sample of 10.1mg of cortisone that was not incubated was carried simultaneously through the same procedure. The overall recovery for the control sample, a measured by periodates oxidation and ultraviolet absorption, was ninety and eighty-five per cent, respectively. The overall recovery for the incubation was thirty-two and forty-eight percent, respectively, as measured by the same methods. The ketonic and non-ketonic fractions were chromatographed, and no metabolites or starting material were isolated from the non-ketonic fractions. Free cortisone was isolated from the ketonic fraction obtained from the petroleum ether extract and characterized as before by the melting point and the mixed melting point of both the free compound and the acetate. A small amount of the unknown substance, m.p. 173-175°C, was isolated from the ketonic fraction obtained from the residue from the ethylene dichloride extract. Various fractions obtained from Incubation III and IV were analyzed fro 17-ketosteroids by the Zimmerman reaction. Only traces were found, and these were questionable because the color produced in the reaction was atypical. The unknown substance melted at 173-175°C on the Fisher-Johns melting point apparatus and 175-179°C on the Kofler Block. This substance reduced alkaline ammoniacal silver solution, gave a negative fluorescence test with concentrated sulfuric acid, and a negative Zimmerman reaction, and showed an ultraviolet absorption maximum at 238 mu. The molar extinction coefficient, based on an assumed molecular weight of 360, was 10,600. One hundred fifty-four micrograms of the substance, when oxidized with periodic acid, produced enough formaldehyde to be equivalent to 154 micrograms of cortisone. The acetate of the substance melted 226-230°C with much preliminary softening. A mixed melting point with known cortisone acetate apparently showed a small depression. Infrared analysis of the acetate indicated the presence of cortisone acetate. It is thought that a molecular complex of two molecules of cortisone and one molecular of compound with a molecular weight of approximately 360 by do alpha, beta-unsaturated ketone group would explain the observations, although this is not the only possible explanation. It is not certain whether or not the unknown member of the complex is a steroid. The lack of sufficient material prevents further efforts at the present time. The relatively low recovery from Incubation IV indicates that a substantial amount of the starting material was converted to unrecognized products. |
