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Orientating investigations of neutral steroids in urine of cattle and horses



Orientating investigations of neutral steroids in urine of cattle and horses



Skand Vet Tidskr 38(5): 278-298



To detn. the content of 17-ketosteroids in urine and blood of cattle and horses, a micromethod requiring only 4 hrs. for complete analysis has been devised. To 20 ml. urine add 1.5 ml. of concentrated HCl, boiling for 1 hr. in a water-bath with reflux condenser, then add 40 ml. of benzene followed by renewed boiling for 1 hr., cooling, decanting and purification by filtering, freeing the benzene from water, and drying in vacuo. One extraction is sufficient. Dissolve the remainder in ether, fractionating in an acid, phenolic and a neutral fraction. Shake the ether fraction 3 times with 20 ml. of concentrated Na bicarbonate soln., remove the phenolic fraction by shaking the ether soln. 3 times with 10% NaOH soln. Male hormones remain in the ether soln. To eliminate pigment from this, filter it through charcoal (Norit). Free the purified ether soln. from water by means of Na2SO4, evaporate and mix with Pincus' or Zimmermann's reagent. Color intensity is detd. in Pulfrich's photometer (5 ml. cuvette). In blood samples the procedure is as follows mix 50 ml. blood with dried Na phosphate, place in a 500-ml. flask, extract in the cold (+ 1[degree] C) with 300 ml. of benzol, shake regularly during a 16-hr. period. Decant the benzol, filter and distill, dissolve the residue in ether, and thereafter carry out the procedure as descr. for urine. Examinations were carried out on urine specimens of pregnant and non-pregnant animals, of newborn calves and foals, of bulls, stallions and geldings, of cattle in different stages of pregnancy, at different stages in the sexual cycle, as well as of animals with disturbances in sexual function. Blood specimens of healthy cattle and horses were examined. By means of the Pincus reagent it is possible to divide neutral steroids into a blue and a violet colored group. Steroids belonging to the blue group were extracted from bovine and equine blood, and in urine extracts of humans, pregnant mares and newborn foals. With Pincus' reagent these steroids give a blue color with absorption max. S. 57-61. Steroids reacting with violet color appear in urine extracts of cattle and horses. They have an absorption max. at S. 53 with Pincus' reagent. With Girard's separation the substances producing color with SbCl3 in extracts of urine from animals, could be separated from the non-ketonic fraction, whereas the blue color-producing substances from human urine were in the ketonic fraction, showing that the blue color-producing substances in human and animal urine are not identical. The expts. suggest that the neutral steroids in the blood are chemically changed when secreted into urine so that they cannot be set free on hydrolysis with HC1. The variations in arat. of steroids in animal urine seem to depend on the specific gravity of the urine, the time of day when collected, and the state of health of the animals. The highest values were found in heifers with adrenal virilism and in bulls, the lowest in cows with cystic ovarian degeneration. Zimmermann's reagent showed no difference in the color gradation in blood and urine steroids, nor between urine extract of human and urine extracts of cattle and horse.

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