Alcohol Precipitation Method Research Articles

Aluminum hydroxide as an adsorbing agent for urinary gonadotropins.

THE concentration of urinary gonadotropins in normal individuals is so low that concentrated extracts must be prepared as free from toxic material as possible with a minimum loss of hormone activity. This laboratory has used the alcohol-precipitation method of Zondek (1) as later modified by Klinefelter et al. (2) for determining the urinary gonadotropic substances in male patients with spinal cord injuries. Because of the low gonadotropin levels encountered, the dialysis method was used and it was possible to test for an excretion of less than 6.6 mouse uterine units (m.u.u.) in twenty-four hours with a minimum of toxic reactions in the test animals. Patients on this service were maintained on a well hydrated regimen; consequently, daily urine volumes would be as much as 8 liters. Because of the large urine volumes obtained, adsorption concentration techniques were examined. Katzman et al. (3) demonstrated that chorionic gonadotropins could be adsorbed on a permutit column and later both Dekanski (4) and...

THE EXTRACTION AND ASSAY OF GONADOTROPIC HORMONES FROM HUMAN MALE URINE

IN THE ASSAY of gonadotropic substance from normal human urine, two major difficulties have been encountered: (a) that of accomplishing complete extraction of hormone from the urine and (b) that of eliminating toxic substances from the extracts. Although several precipitating agents give good yields of hormone, it is well known that extracts of normal urines are frequently so toxic that completion of an assay may be impossible due to the death of all the animals employed. During the course of our investigation of the gonadotropic content of the urine of men, Heller1,2 published a similar study of the gonado tropic content of the urine of normal women. In his method, the hormone was precipitated with alcohol and the toxicity decreased by dialysis. We also use a simple modification of the Aschheim-Zondek alcohol precipitation method, but we avoidthe necessity of dialysis by taking advantage of fractional alcoholic precipitation.

MINERAL SALTS AS TOXIC FACTORS IN URINARY PROLAN CONCENTRATES1

DURING A recent investigation of the urinary prolan excretion of a nonpregnant human female (i), we experienced great difficulty in that when using the alcohol precipitation method and the original AschheimZondek technic, the urinary concentrate proved toxic to many of the animals. Believing that the source of toxicity might be due to the mineral salts it contained, 3 specimens of the solutions of concentrates which had proven toxic were analysed for various cations.2 The results were as follows; the metals being reported as the chlorides: In order to determine the relation of these concentrations to the M.L.D., 2,i′day′old female rats were injected subcutaneously with solutions of pure salts. I believed it unwise to use the data in the literature on the toxicity of these salts for adult rats. Solutions were made up in the convenient strengths of 10% NaCl and 5% KC1, CaCl2, and MgCl2, and the dose was always diluted to 1 cc.

AN EVALUATION OF THE FRANK METHOD FOR THE DETERMINATION OF PROLAN (GONADOTROPIC PRINCIPLE) IN THE URINE OF NONPREGNANT WOMEN1

For some time we have been seeking a quantitative method for determining prolan in the urine of nonpregnant women when it is present in amounts of less than 66 r.u. per liter. The Aschheim-Zondek alcohol precipitation method (1), which employs 60 cc. of morning urine, does very well when amounts of prolan in the urine are in excess of 66 r.u. We found that many women suffering from vasomotor disturbances of the menopause did not have sufficient prolan in 60 cc. of urine to give a positive test; yet we felt that their urinary prolan must be in excess of normal. It is not so important to have a method for the detection of excess prolan in the urine of women who are able to describe typical vasomotor waves, but in cases of those suffering from some bizarre syndrome or psychosis, or those whose

THE QUANTITATIVE DETERMINATION OF THE GONADOTROPIC MATERIAL OF URINE OF WOMEN AFTER CASTRATION AND THE MENOPAUSE AND OF NORMAL MEN

During the past few years we have been intermittently engaged in the development of methods for the quantitative estimation of small amounts of gonadotropic substances. We have reported that small quantities of the gonadotropic substances of urine of pregnant women and of unfractionated anterior lobe extracts which were added to boiled urine of normal men were satisfactorily recovered by adsorption on benzoic acid or preciptation with tungstic acid 1, 2). In that work the assay was based on the induction of vaginal opening and cornification in immature rats. Since the active material from these sources was successfully recovered by these methods, we assumed that the gonadstimulating substances of various urines could likewise be determined. However, other investigators who were using the alcohol precipitation method and a different method of assay found that the benzoic acid method gave low results when applied to urine obtained from women after castration or the menopause (H. M. Evans, O. W. Smith, L. Le...

A Quantitative Procedure for Determining Normal Excretion of Prolan

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.

THE GONADOTROPIC HORMONES (ρ‐FACTORS). —I. THE PREPARATION AND PROPERTIES OF EXTRACTS OF ANTERIOR LOBE, PLACENTA, AND PREGNANCY URINE

(1) Two methods for the preparation of potent extracts containing the ρ‐factors from pregnancy urine have been elaborated, one of which involves the use of phosphotungstic acid as a precipitant, whilst the other is an alcohol precipitation method with various refinements introduced.(2) Methods are also given for the preparation of active extracts from the anterior lobe of the pituitary and human placenta.(3) The effect of prolonged injections of small quantities or fewer injections of large quantities of ρ‐factors is to inhibit the œstrous cycle in the mature mouse, whilst small quantities actually induce premature œstrus in immature mice. A theory is enunciated to account for these phenomena.(4) The active factors are not precipitated to any appreciable extent by dialysed iron, nor can they be ultrafiltered through an “ultrafein” membrane filter which is just impermeable to Congo red. Other chemical properties are in accord with the observations of previous workers.