A Quantitative Procedure for Determining Normal Excretion of Prolan

Abstract

Several methods have been developed for the preparation of active extracts of the gonad-stimulating substance of pregnancy urine. Although some of these procedures give good recoveries from pregnancy urine no one has reported a satisfactory procedure for the quantitative estimation of the small quantities of prolan that occur in normal urines. The excretion of prolan in conditions unassociated with pregnancy has been determined exclusively with untreated urine or by means of Zondek's alcohol precipitation method which gives only a 5-fold concentration. Since alcohol causes an appreciable loss of activity (Wiesner and Marshall, Katzman and Doisy,) this procedure is not suitable for use in normal excretion of the anterior pituitary-like substance. In attempting to purify pregnancy urine extracts by means of various protein precipitants we observed that precipitation with tungstic acid invariably gave remarkably complete recovery. Wiesner and Marshall and Zondek, Scheibler and Krabbe found that phosphotungstic acid quantitatively precipitates the active material of pregnancy urine but they have not reported studies on the recovery of the small amounts of prolan which occur in normal urine. We have not used phosphotungstic acid but it is evident from Table I that our tungstic acid method gives a surprisingly good recovery of minute quantities. The procedure is as follows: A 24-hour urine sample to which has been added 10 cc. of 10% sodium tungstate and 10 cc. of 0.5% casein is made faintly acid to congo red with dilute H2SO4. The precipitate is collected by centrifugation, washed with acetone, freed from acetone by reduced pressure, and dissolved by adding dilute NaOH until the mixture is faintly alkaline to phenolphthalein. BaCl2 and Ba(OH)2 are then added in the proportion necessary to prevent the solution from becoming acid, until precipitation is complete.