Edward A. Doisy

Hydrolysis of conjugated steroids by bacterial glucuronidase.

Summary Bacterial glucuronidase which hydrolyzes the glucuronides of estriol, pregnanediol, menthol and phenolphthalein liberates a large proportion of estrogen, ketosteroid and corticosteroid of human urine.

Role of vitamin A in induction of vitamin K deficiency in the rat.

Summary Evidence is presented to indicate that the level of dietary Vit. A is closely related to development of symptoms of Vit. K deficiency in the rat. Under appropriate experimental conditions, 0.5 and 5 i.u. of Vit. A per gram of diet gave reduced prothrombin concentrations which were not observed on corresponding rations deficient in Vit. A. This extends the previously known hemorrhagenic toxicity of Vit. A to physiological levels and emphasizes the need for careful consideration of the level of Vit. A in studies of Vit. K deficiency in the rat. The effect of Vit. A was more severe in male than in female rats. In further studies with male rats, Vit. A acid was markedly more hemorrhagenic than Vit. A acetate.

Hydrolysis of Conjugates of Neutral Ketosteroids

Summary The hydrolysis of steroid conjugates has been studied by 5 methods. Continuous ether extraction of urine made 7.2 N with respect to HCl gave values appreciably higher than those obtained by boiling with 15 vol. % HC1 and these values approximated the sum of ketosteroids released by β-glucuronidase and by continuous extraction with ether of urines brought to pH 0.7 with HCl

Cornification of Vaginal Epithelium of Ovariectomized Rat Produced by Smearing

The histological examination of the vaginae of a number of spayed rats showing an unexpected response to the examination of the vagina by the smear method revealed that smearing alone, without the injection of an estrogenic hormone, may produce, under certain conditions, full cornification of the vaginal epithelium. The results of some of our experiments are summarized in Table I. The spayed rats which were neither smeared nor injected presented a thin, smooth vaginal epithelium usually 2 cell layers in thickness with leucocytes migrating into the lumen. When the animals were smeared once, twice, and 3 times daily the typical section from the vagina showed a progressive thickening of the epithelium up to 12 or more layers with the usual desquamation of the surface cells, thus presenting a picture very similar to that obtained after the administration of estrogenic material. 1 , 2 The vaginal smear picture 3 likewise underwent a progressive change when the animals were examined once, twice, and 3 times daily. The usual — smear observed when the animals were examined once daily changed to a ± or occasionally a ± ± on and after the third day if the animals were examined twice daily. When the vaginal smears were made 3 times daily about 25% of the animals showed full + smears on the third or fourth day of treatment.∗ In order to observe whether smearing 3 times daily would change the typical picture observed after the administration of theelin, 3 animals injected daily with one rat unit of theelin and smeared 3 times each day were compared with 3 other animals injected with the same amount of theelin but smeared once daily.

The Crystalline Ovarian Follicular Hormone

Due to the low concentration of estrus-producing material in ovaries, we abandoned their use several years ago in favor of the much cheaper and more concentrated source, namely, the urine of pregnant women and mares. However, with the isolation of so many different estrogenic compounds, it seemed desirable to determine the nature of the active substance in the ovary. Starting last year seriously to work on this problem we soon obtained the hormone in a crystalline condition but owing to the very low concentration in hog ovaries, have not secured enough to complete our work. Preliminary assays of the crystalline follicular hormone give the following data: (1) ovariectomized mice by the Marrian-Parkes procedure, 200,000 units and by the Butenandt procedure, 70,000 units per mg.; (2) ovariectomized rats by our usual procedure, 16,000 units per mg.; (3) immature rats by the Curtis-Doisy procedure, 5,000 units per mg. In our laboratory these results are from 4 to 8 times the values that we obtain for theelin, but with the immature rat the potency is equal to that of theelol. The assays for dihydro-theelin and for the follicular hormone by the respective methods give similar values. Although we have not yet accumulated sufficient material for complete analysis, our results indicate the identity of the hormone with dihydro-theelin. The m-bromobenzoate of the hormone was prepared, and after 3 crystallizations had a melting point of 154°–155°. After 4 crystallizations the m-bromobenzoate prepared from a sample of pure dihydro-theelin melted at 155°–156°, and the dihydro-theelin obtained from it by saponification with dilute alcoholic alkali melted at 171°–172° after one crystallization. By saponification of the m-bromobenzoate of the hormone in the same manner the crystalline hormone was recovered and found to melt at 170°–171°. All melting points are uncorrected but were taken with a Bureau of Standards long stem thermometer.

Effect of Indigestible Oils on Vitamin K Deficiency in the Rat

Studies of indigestible oils such as mineral oil and squalene were undertaken to determine their effect on vitamin K deficiency in the rat. Severe hypo- prothrombinemia occurred in male rats fed a vitamin K-deficient diet containing as little as 0.5% of the oil and the effect was reversed by feeding vitamin K. Female rats were partially resistant to the treatment. Dietary squalene also interrupted the assimilation of vitamin K in chicks where 0.5% of dietary oil inhibited the utiliza tion of 0.1 fig of phylloquinone/g of diet. Similar results were obtained in chicks fed retinyl acetate and retinole acid.

Assay of vitamin K concentrates.

Summary A curative method of assay of Vitamin K based on Trevans principles of bioassay has been found to give satisfactory results.

Bile acids XLIV. Quantitation of bile acids from the bile fistula rat given [4-14C] cholesterol

The bile acids derived from [4-14-C]cholesterol administered intracardially to rats with cannulated bile ducts were identified and quantitated. Over a period of 28 days about 90% of the administered 14-C was found in bile of which 73% was retained in the biliary acid fraction. [7beta-3-H]cholic acid, alpha-muri[3beta-3-H]cholic acid, beta-muri[3beta-3-H]cholic acid and litho[3beta-3-H]cholic acid were prepared with specific activities of about 30 muCi/mg by reduction of appropriate ketonic precursors with NaB3H4 and were added to the biliary acid fraction. After separation and purification of the bile acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids accounted for 70, 16, 7.5 and 6.1%, respectively, of the 14-C in the biliary acid fraction. The specific activities of these isolated 14-C-labeled acids were almost identical. Lithocholic acid accounted for a maximum of 0.2% and ursodeoxycholic acid and 7-oxolithocholic acid could account for no more than 2% of the biliary 14-C. Gas-liquid chromatography on 3% OV-17 of the trimethylsilyl ether derivatives of the methyl esters of the common bile acids of rat bile results in their complete separation and provides a convenient means of estimating the relative proportions of these acids in rat bile. By this method, the relative amounts of the four major acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids were 63, 20, 8 and 6%, respectively.

Bile acids. LXX: Preparative separation of kryptogenin from companion sapogenins by high performance liquid chromatography

Abstract Commercial samples of kryptogenin or its acetate can be purified by preparative high performance liquid chromatography on a PrepPAK-500/Silica cartridge. The free alcohol is separated from accompanying sapogenin with a mixture of chloroform:methanol (50:1), whereas the acetates are separated well with a mixture of methylene chloride:hexane (2:3). The companion sapogenins, diosgenin and yamogenin, 25R- and 25S- isomers, were separated by analytical HPLC with hexane-isopropanol (100:1) or as the acetates with hexane:isopropanol (250:1). Characterization of kryptogenin and yamogenin was completed with 1H-NMR, IR and MS spectrometry.

Inositol and the toxicity of four isomers of benzenehexachloride for the rat.

Summary The toxicities of the α, β, γ, and δ isomers of hexachlorocyclohexane were not affected by inositol in the rat. The convulsive and narcotic effects of the γ and β isomers are discussed in detail. The authors are indebted to Drs. Vicente Moragues and John P. Wyatt of the Department of Pathology of St. Louis University for performing the microscopic examinations.