I
‘I
Androsterone and Testosterone P. E. HILMER AND W. C. HESS Georgetown University School of Medicine, Washington, D. C. The 2,4-dinitrophenylhydrazones of androsterone and testosterone have been prepared according to the method of Veitch and Milone. The absorption spectra of these compounds after treatment with 0.1 N alcoholic potassium hydroxide have been determined. Androsterone hydrazone exhibited maximum absorption at 430 millimicrons, and the maximum for testosterone hydrazone was at 460 millimicrons. A chromatographic method for the separation of the two androgens from the estrogens and a sensitive spectrophotometric method fnr quantitative estimation of androsterone and testosterone are described.
approxiniately 150 ml. of 207, acetone in petroleum ethei. Thr two bands separated, and the androgens were eluted in the first portion of the acetone-petroleum ether washing. The solvent was evaporated, and the residue was dissolved in 1.25 ml. of chloroform and made to a volume of 25 ml. with 9570 alcohol. Thib solution was used for the spectrophotometric determination. A 2-ml. aliquot was treated with 8 ml. of 0.1 N alcoholic potassium hydroxide, and its absorption was read a t 430 and 460 millimicrons. Then 2 ml. of standard solutions of androsterone and testosterone hydrazones, containing 7.75 micrograms of hydrazone per ml. (equivalent to 4.8 micrograms of hormone), were similarly treated with alcoholic potassium hydroxide and read at the two wave lengths. The concentrations of the individual androgens were calculatrd by means of the formulas of Knudson u t
HE use of 2,4-dinitrophenylhydrazinederivatives by Veitch and Milone (4) in this laboratory for the quantitative estimation of the estrogens suggested the possibility of a similar method for the determination of the male hormones. Following their procedure, the dinitrophenylhydrazones of the androgens were prepared by Johnston ( I ) , but no attempt was made to separate them from the estrogens. The present paper gives details of a method for the separation of estrone and progesterone from androsterone and testosterone, and a method for the quantitative estimation of these androgens EXPERILMENTAL
01
Preparation of 2,4-Dinitrophenylhydrazones of Androgens. Thirty milligrams of the androgen were dissolved in 50 ml. of redistilled, aldehyde-free ethyl alcohol and refluxed for 2 hours with 10 ml. of a saturated alcoholic solution of 2,4-dinitrophenylhydrazine. After 2 hours, 1 ml. of concentrated hydrochloric acid was added, and refluxing was continued for 2 minutes more. Distilled water was added to the point, of cloudiness, and the solution was allowed to cool to room temperature, then placed in the icebox until precipitation was complete. The crude hydrazone was filtered off and washed with 957, ethyl alcohol and water. Further purification of the compound was effected by the inethod of Johnston (1). The hydrazone was dissolved in a minimum amount of benzene and adsorbed on a column (100 X 15 mm.) of Fisher alumina. Treatment of the column with approsimately 100 ml. of a 127, solution of chloroform in benzene resulted in the formation of two hands, one of which (unreacted hydrazine) was eluted by this solvent. The other hand (hydrazone) was eluted with approximately 1.50 ml. of chloroform. The elution of the two se arate hands can he easily followed visually. Biter removal of tge solvent b y distillation in vacuo, the hydrazone was recrystallized from alcohol and water, and dried in a vacuum desiccator. The hydrazones of the androgens melted in the same range as those reported by Johnston ( I ) . Spectroscopic Analysis. Solutions of androsterone and testosterone hydrazones in chloroform, 2.5 ml. containing 120 and 104 micrograms of hydrazone or 74 and 64 micrograms of hormone per ml., respectively, were treated n-ith 5 ml. of 0.1 S alcoholic potassium hydroxide, diluted to a voluine of 50 ml. with 95Y6 alcohol, and submitted to spectroscopic analysis in the Beckman spectrophotometer. Readings were made at intervals of 10 millimicrons. The hydrazone of androsterone showed maximum absorption a t 430 millimicrons; the maximum for testosterone hydrazone was a t 460 millimicrons (Figure 1). Separation of Androgens from Estrogens. .1 benzene solutiori containing not more than 20 micrograms of each of the hydrazones of androsterone, testosterone, estrone, and progesterone was adsorbed on a 100-mm. column of AZerck alumina (according to Brockmann) in the apparatus described by Yeitch and Milone (4). The column was washed with 10 ml. of benzene, 50 nil. of 17, acetone in petroleum ether, and finally chloroform until the washings were colorless. This procedure eluted all the hydrazones except estrone. After evaporation of the solvent from t,he eluate, the residue was dissolved in benzene and adsorbed on a 300-mm. column of F l o r i d This was washed with 5 ml. of henzene, then ~ l u t e r lwith
(2). (’, =
Lvhrrt: C1 C2
=
k;’UM- kiU” kAkB - kBk4 2 1 2 1
ouncelitration uf androsteroiie
= concentration of testosterone
k< = slope of androsterone hydrazone a t 430 inilliinicrone
k i = slope of testosterone hydrazone a t 430 millimicrons = slope of androsterone hydrazone a t 460 millimicrons k: = slope of testosterone hydrazone a t 460 millimicrons D” = reading of density of mixture a t 430 millimicrons DB = reading of density of mixture a t 460 millimicrons
kf
0.6
0.5
+
cIn
0.4
2
a g
On3
0 0.2
0.1
350
400
450
500
550
600
WAVE LENGTH
Figure 1.
Absorption Curves for Androsterone arid ’restosterone Solid line. Androsterone hydrawne Broken line. Tratn8trrone hydramnr
822
V O L U M E 21, NO. 7, J U L Y 1 9 4 9 Table 1.
I
2
.+ 4.
5.
823
Recovery of Androsterone and Testosterone from Solutions R
Hydrazoneso hndrosteronr Testosterone Androsteronr Testosterone Androsterone Testosterone Androsteronr Testosterone Androsterone Testosterone
Found, y 15.9 ( 9 . 8 ) b 17.3 (10.6) 17.1 (10.5) 1 4 . 8 (9.1) 17.1 (10.5) 14.8 (9.1) 16.1 (9.9) 15.8 ( 9 . 7 ) 16.1 (9.9) 15.8 (9.7)
Recovery 102 110 109 95
IO9 95 I03 101 103
Five milliliters of blood were deproteinized by the method of Somogyi ( 3 ) . The protein-free filtrate was extracted for 2 hours in a continuous extractor with carbon tetrachloride. After removal of the solvent by distillation, the residue was dissolved in alcohol, and the hydrazone was prepared as described in this paper. The hydrazones were submitted to the chromatographic procedure, and the androgens were estimated in the Coleman spectrophotometer. Initial investigations indicated that 100 ml. of blood contain approximately 2 mg. of testosterone and somewhat more androsterone. This work is .till in progress, and these valiies are n n l r tentative.
101 _-
Average androsteronr .4 verage testosterone '1
This procedure has been used io, the determination of the androgens in blood in several preliminary studies.
16.4 (10.1) 105 18.5 (9.6) I no 15.5 y (Y.6 y J of each ubrd 111 all tabts. Values In parenthesee are androgen erciiiralents of hydrazones.
The slopes were calculated by dividing the optical densities of the standards a t the two wave lengths by the number of micrograms of hydrazone in the volume of standard tested. 111 a series of five determinations on solutions containing 15.5 micrograms of the hydrazone of each androgen, the average concentration of androsterone hydrazone was found to be 16.4 micrograms, and the average concentration of testosterone hydrazone was 15.5 micrograms. In terms of free hormone, the average concentration of androsterone was 10.1 micrograms, and of testost,erone 9.6 micrograms. These results are summarized in Table I.
A C K I \ O W L E D C ; M E ~I
The authors are indebted to Arthur St. Xndr6 of Ciba Pharmaceutical Products, Inc., who supplied the androsterone and testosteronr used in this investigation LITEK4'1'L~KEC11 LII
(1) Johristoii, C. U., Science, 106, 91 (1947).
(2) Knudson, H. W., *Veloche. T. W.. and Juda3. ('.. Ivn E;ur; CHEM.,AVALED., 12, 715 (1940). (3) Somogyi, IM.. J . Bid. C'hem., 86, 655 (1930). (4) Yeitch. F. P., Jr., and Milone, H. S..Ibid., 158, 61 (1945) RECEIVEDSeptember 25, 1948. Presented before the Division of Biolodcttl Chemistry a t the 114th Meeting of the AMERICANCHEMICAL SOCIETY, Washington, D. C. Investigation qiippnrted hp a g r a n t from the TTnited .States Piihlic Healt,h Serrirr.
MICROBIOLOGICAL 'ASSAY OF PANTHENOL ELMER DE RITTER
AND
SAUL H. RUBIN, Hoffrncinn-La Roche, Inc., 'Vutley, N. ./.
The method of Walter for the microbiological assaj of panthenol (the biologically active hydroxy analog of pantothenic acid) has been modified to permit assays in the presence of pantoyl lactone as well as pantoic acid. The lactone is removed quantitatively from water solution by continuous extraction (1 to 2 hours) with ethyl ether. Panthenol in the aqueous residue is then hydrolyzed to pantoic acid for micro-
P
ASTHESOL, ol,y-dihydro~\~-,~-(3-hydroxypropyl)-P,B-d1methylbutyramide, the hydroxy analog of pantothenic acid, was first shown by Pfaltz (7) to have the full vitamin activity of the latter in animals. Later work by Burlet ( I ) and in this laboratory (IO) demonstrated that the physiological availability of panthenol to humans exceeds that of pantothenic acid under stated conditions. This circumstance suggested superior stability. R premise that has been borne out in studies in vitro (9), in which panthenol displayed hetter stability a t p H 3 to 5, and ?specially a t pH 4, to which considerable practical intrrrst attaches. Becauae panthenol is oxidized to pantothenic ami in the mammal, it has been possible to elaborate a rapid (2-day) bioassay in rats ( 3 ) wherein the urinary excretion of pantothenic acid is measured after a test dose of panthenol and related to a standard curve of excretion. In the present report, suitable microbiological assay procedures are described. h direct microbiological assay of panthenol has been described by Walter ( 1?'), utilizing .4cetobacter suboxydans as the test organism, but the presence of even a small percentage of pantothenic acid or pantoic acid (a,-y-dihydroxy-p,p-dirnethylbutvric acid) invalidates surh assays because of the far
biological assa? with Acetobacter suboxydurrn. Assay before hydrolysis provides a correction for preformed pantoic acid. By modifying the medium used by Sarett and Cheldelin and using EvelJn colorimeter tubes with continuous shaking, the 60to 70-hour growth period has been reduced to 20 to 24 hours. In stationary Erlenmeyer flasks suitable growth curves are obtained after 40 hours.
greater activity of pantothenic and pantoic acids a b giowth promoters as compared to panthenol. Subsequently i\-alter (18)reported a method using the same organisms for determining panthenol as pantoic acid after complete alkaline saponification. This procedure, a modification of the turbidimetric assay of Sarett and Cheldelin ( I @ , employs a fully synthetic medium and is based upon the oxidation of sorbitol to sorbose and determination of the reduction value as a measure of bacterial growth. Preformed pantoic acid, if present, is determined by assay before hydrolysis. This method, however, is not applicable if significant amounts of pantoic acid have undergone lactonization, for pantoyl lactone is also measured as pantoic acid after alkaline hydrolysis but is not corrected for by the assay before hydrolysis because of its slight activitr per se as compared to pantoic acid. I n a study of the stability of pantothenic acid, Frost (6) presented data obtained by optical rotation measurements regarding the relation between pH and the lactonization of pantoir acid. At pH above 6.0, no lactonization was observed. At pH hrlow 6.0, the rate of lactonixation increases as the pH decreases, and a t equilibrium, below p H 4.7, lactonization is essentially complete. Because panthenol is considerably more stable i n vitro than Pantothenic acid hetn-een p H 3 and 5 (9).
