Microchemical Identification of Radioactive and Wonradioactive Steroids DAVID
L. BERLINER
and HILTON A. SALHANICK’
Department of Obstetrics and Gynecology, University o f Utah College
Methods are needed for identifying 1 y or less of steroids, for this is the level at which they function biologically. By isotopic dilution and subsequent chromatography of the unknown substance and a derivative, as little as 0.01 y of radioactive steroids can be identified. About 20 y of a nonradioactive substance can be characterized. The method is simple, sensitive, and accurate within the limits of instrumentation, and identifies total molecular structure.
T
HE increasing recognition of the importance of very small amounts of substances in biological analysis has emphasized the need for methods that can measure and identify quantities in the range of tenths and hundredths of micrograms] for this is the order of concentration of these potent substances in the organism.
C H CIx-FORMAMIDE
i””3
BENZENE-FORMAMIDE
“OrRS RF
01
ACETYLATION
CORTISONE AND CORTISONE-$l4
CORTISONE ACETATE AND CORTISONE-C;~ ACETATE
Figure 1. Identification of cortisone
Probably, the most sensitive methods of detection of a compound available a t present are based upon the determination of radioactivity. It can be made as sensitive as the radioactivity of the compound to be analyzed; certainly, less than 0.01 y of many substances can be detected with ease. In the analysis of a number of steroids extracted from biological sources, it was found that the method of isotopic dilution followed by crystallization t o constant activity (5) was fraught with technical difficulties reflecting variations in temperature, coprecipitation phenomena, plating factors, self-absorption corrections, and unknown variables. For carbon-14-labeled steroids this method requires a minimum activity of at least 500 counts per minute (CPM), so that subsequent aliquots can be counted with reasonable reliability. 1 Present address, Department of Obstetricsand Gynecology, Washington University School of Medicine, St. Louis, Mo.
o f Medicine, Salt Lake City, Utah
With the advent of paper partition chromatography of steroids, minute amounts of substances can be separated and partially characterized on the basis of the ratio of movement of a solvent to the distance moved by a compound on filter paper (R,). Although the R , is not so definitive as the partition coefficient, it is as dependable a physical property as a melting point for the characterization of a substance. By combining the sensitivity of radioactivity measurement, the physical characteristic of the R f , and the concept of isotopic dilution, it has been possible to devise a simple procedure for the identification of less than 0.01 y of a radioactive steroid and about 20 y of a nonradioactive substance.
MATERIALS AND METHODS
All steroids used in this study were as pure as could be obtained by chromatography and crystallization. Solvents were purified by standard procedures (3) and redistilled prior to use. “Unknown substances” were obtained by isolation from biological sources. The chromatographic methods used in this study were described in detail by Zaffaroni (7). Quantitation of the steroids was done by ultraviolet spectroscopy in a Model DU Beckman spectrophotometer using silica cells of 1-cm. light path containing either 3 or 0.5 ml. of the solution to be analyzed. Radioactivity was measured in a three-chamber internal flow counter in the Geiger range. Samples were either eluted from the paper and plated on aluminum disks or counted directly from 1-cm. squares of the paper chromatogram. It has been the authors’ experience that the self-absorption of this paper can be corrected with reasonable consistency by multiplying the observed count rate of a 1-cm. square of the chromatogram by 4.2. Because many of the substances had as few as 20 counts per minute above background, in some cases the counting error might have been as high as 10%. Usually, however, the samples were counted for a sufficient length of time to make the error less than 5%. The spots on the chromatogram were located by viewing the paper in a modified Haines ultraviolet scanner (4). Substances which absorb ultraviolet light in the region of 240 mp stand out clearly in this instrument and as little as 5 y can be detected on a strip 1 em. in width. With the exception of tetrahydrocortisone, all the substances analyzed in the study had an a-p unsaturated ketone moiety and were therefore readily detectable. Tetrahydrocortisone was detected by triphenyltetrazolium reagent (TPTZ), which gives a pink color in the presence of the primary a-keto1 side chain. Formation of Derivatives. Two methods of derivative formation were performed for this study. For acetylation, the material was disolved in 0.5 ml. of pyridine-acetic anhydride (1 to 3) and the solution allowed to remain a t room temperature overnight. Oxidation was performed by dissolving the steroid in 0.5 ml. of glacial acetic acid, adding 0.5 mg. of chromic acid, and allowing the solution to stand overnight a t room temperature. The reaction was stopped by adding water and the products were extracted with ethyl acetate. Procedure. The procedure for this analysis is gratifyingly simple. To about 50 counts per minute of the unknown radioactive substance is added about 20 y of the “authentic” substance which has been postulated. The specific activity is determined. The mixture is then chromatographed and the area of ultraviolet absorption and radioactivity measured. If the substances are identical, they occupy the same region and the specific activity remains constant. For the sake of convenience, it is often not necessary to measure 1608
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
1609 separated the radioactivity frpm the tetrahydrocortisone diacetate. Thus, the unknown substance was cortisol. To c o d r m this identification, the cortisol acetate mixture was oxidized to cortisone acetate and rechromatographed. I n this case, the specific activity wm 19.7 counts per minute per y. The difference in specific activities of these three determinations is within the error of counting. Identification of 6&Hydroxpprogesterone. A product of the incubation of carbon-14ring-labeled progesterone with placental tissue is 6-&hydroxyprogesterone (1). An aliquot of the isolated radioactive substance was added t o crystalline &a-hydroxyprogesterone and recrystallized three times from cyclohexane, hexanecyclohexane (1 to l), and heptane-methanol (9 to 1). The specific activities were 394, 397 and 401 counts per minute per mg., verifying the structure by recrystallization to constant activity. The recrystallization procedure was compared with the method reported here. Another aliquot was diluted with 50 y of authentic &@-hydroxyprogesterone and chromatographed in the benzene-formamide system. The authentic material as determined by ultraviolet absorption and the isolated substance as determined by radioactivity occupied the same spot. A fraction of the material was acetylated with acetic anhydride in pyridine and the procedure repeated in the heptane-formamide system. Again, both located in the same area ( R j 0.20). Finally, a portion was oxidized nith chromic oxide and the resulting spot identified as 6-ketoprogesterone ( R f 0.71 in hexane-benzene-formamide). This experiment shows that, if one couples the formation of derivatives with the scanning technique and counts only the paper vithout elution, structure can be identified conclusively. Attempted Identification of 17a-Hydroxyprogesterone (Figure 3). From the incubation of tissues with carbon-14-ring-labeled progesterone, an unknown substance was isolated. It was p o s tulated that this substance was 17a-hydroxyprogesterone because of the similarity in polarity. Authentic 17 a-hydroxyprcgesterone (100 y ) was mixed with 3724 counts per minute of the unknown compound, (specific activity 37.2 counts per minute per 7). After purification by chromatography, the specific activity was 32.7 counts per minute per y . Acetylation was attempted (a “negative” proof because the 17-hydroxyl group does not acetylate by this procedure) and the mixture was again chromatographed. The same Rf was observed, but the specific activity was reduced to 29.4 counts per minute per y . While a drop from 37 to 32 or from 32 to 29 may be within experimental error, the large difference between 37 counts per minute and 29 counts per minute per y was sufficient to arouse suspicion. The substance and the 17a-hydroxyprogesterone carrier were oxidized and the products chromatographed in the hexane-formamide system. h sharp discrepancy was observed (Figure 3 F t h e radioactivity remained a t the origin and 4-androstene-3,17-dione,the oxidation product of the 17a-hydroxyprogesterone,traveled 10 cm. down the paper. Clearly, the unknown substance was not 1701hydroxyprogesterone.
the specific activity of the material. If they have identical R1’s, derivatives such as acetates or oximes can be made directly and rechromatographed. In this case, the two areas should again correspond. In a t least one of these tests, however, the specific activity should be determined. Another simplification should be mentioned. If an absorption area is divided in segments and the specific activity of each segment determined, one can readily demonstrate the presence of a substance which has an R , so close to that of the unknown as to be inseparable by other procedures. Even though the quantity of a substance in different portions of the same spot may differ because of tailing, unequal distribution of the substance a t the starting line, etc., the specific activities remain constant. EXPERIMENTAL WORK
This technique is now used routinely in this laboratory Several examples of the identification of steroids are given in Figures 1 to 3. Identification of Cortisone (Figure 1). After the injection of radioactive cortisone into a patient, it was expedient to learn the degradation products. One compound suspected to be the unchanged injected substance was isolated. To an aliquot of this ‘‘unknown compound” (150 counts per minute) was added 200 of authentic cortisone. The mixture was chromatographed in the chloroform-formamide system ( 3 ) and had an Rj of 0.52. The specific activity of the eluted substance was 0.66 counts per minute per y. The mixture was acetylated and rechromatographed in the benzene-formamide system. The radioactivity was concentrated in the area which absorbed ultraviolet light. The specific activity of the acetate derivative was 0.66 count per minute per y. Identification of Cortisol (Figure 2). From this same patient, a substance was isolated which had the same R/ as tetrahydrocortisone and cortisol; these two compounds are inseparable by this chromatographic procedure, particularly if minute amounts are present. An aliquot of the unknown (400 counts per minute) was added to 20 y of tetrahydrocortisone and 20 y of cortisol. This mixture of three substances was chromatographed in the chloroform-formamide system for 8 hours. A single spot was observed by ultraviolet absorption, triphenyltetrazolium reaction, and radioactivity determination, further confirming the difficulty in separating these substances. The specific activity on the basis of ultraviolet absorption was 18.4 counts per minute per 7. The mixture was acetylated and rechromatographed in the benzene-formamide system. h4ost of the radioactivity remained near the origin a t the same place as the ultraviolet absorbing area was visualized. The triphenyltetrazolium reaction located the tetrahydrocortisone diacetate over 10 cm. down the paper and with it was a small amount of radioactivity. The specific activities were 17.5 counts per minute per y for the cortisol acetate area and 1.2 counts per minute per y for the tetrahydrocortisone diacetate spot. Rechromatography of the latter spot completely
CHC13-FORMAMIDE TIME - 8 HOURS
DISCUSSION
Criteria for Identification of Micro Quantities of Steroids I n the analysis of invisible quantities of steroids, unfortunately
B E N Z E N E - FORMAMIDE
BENZENE-FORMAMIDE
TIME-SHOURS
TIME-9 HOURS
S4.17.5
CPM/,ug
5 A = I 2 CPM/pg OXIDATION
ACETYLATION
25CPM
25CPM
50
MIXTURE OF: TETRAHYDROCORTISONE (I),
Figure 2.
50
CORTISOL (11).
AND CORTISOL-CI”
Identification of cortisol
ANALYTICAL CHEMISTRY
1610 HEXANE:BENZENEFORMAMIDE
HEXANE: BENZENEFORMAMIDE
TIME 3 HOURS
HEXANE-FORMAMIDE
TIME 3 HOURS
ACETYLATION
0.52
RF 0.52
P S.A.829.4 CPM/&g
......
100
+
I ........
500
300
CPM I70H-PROGESTERONE UNKNOWN -Ch4
~L
I70H-PROGESTERONE UNKNOWN-Ct
+
TIME 3 HOURS
100
300
500
CPM
*ANDROSTENDIONE
UNKNOWN-CY
+
Figure 3. -4ttempted identification of 17a-hydroxyprogesterone
structure must be demonstrated by indirect methods. The similarity of the physical characteristics of many substances renders a conclusive proof extremely difficult. However, often the great biological differences that can accompany a “minor” chemical change-e.g., a ketone or a hydroxyl group a t an important locus such as carbon 3-make precise identification of utmost importance. Because of the close similarity of many steroidal structures, there are many pitfalls in the identification of a substance. One of the misleading forms of data is the R f . Theoretically, it may be said to reflect a partition coefficient, but the many technical details of obtaining the R , introduce such error that it probably is no more reliable than a melting point. The problem is dramatically illustrated by the attempted identification of a 17a-hydroxpprogesterone described above. Only after oxidation could the unknonm compound be separated from the postulated steroid, and, had it not been radioactive, it could never have been detected by chemical or visual procedures. Another pitfall is the use of chromophoric reactions. Three categories may be used to describe those employed most routinely. First is the use of a single absorption maximum or minimum with or without absorption coefficients. In the authors’ experience, a single maximum has no value other than to confirm a functional grouping such as an a+ unsaturation. A second method is based upon making a chromophoric derivative. This may be useful in identifying a functional grouping such as a ketone, but hardly establishes the entire molecular configuration. Finally, certain reactions such as the chromogen of a steroid in concentrated sulfuric acid ( 6 ) , fuming sulfuric acid, or phosphoric acid have yielded valuable information of the total structure. This type of identification is of definitive value under conditions where more than one absorption maximum is present; the general shape of the curve, including shoulders and minima, is taken into consideration; and a control curve is determined simultaneously. A comment should be made on the behavior of radioactive steroids in paper chromatography. A careful examination of Figures 1 to 3 emphasizes that a certain amount of radioactivity remains a t the origin and also flows to the front. This is to be anticipated and rechromatography would verify the authenticity of the compound. Furthermore, in the presence of contaminating substances, a small amount of radioactivity is often, but not always, carried with the substances. This applies to every method of paper partition chromatography that has been tested in this laboratory (%,6), and is particularly true of the chromatography of nonpolar substances. For this reason, the use of a t least one derivative is essential.
For most purposes, two are preferred. In the use of radioactive substances, the determination of specific activity a t each chromatography and formation of a derivative are of great value. When the method described above is used, the determination of many of the specific activities can probably be safely eliminated, provided one derivative (preferably two) is made. Advantages of Identification by Isotopic Dilution. The benefits of this isotopic dilution method are its simplicity, its identification of total structure, and its sensitivity. If carried through a suitable analysis, almost any radioactive steroid can be determined in quantities of less than 0.01 y , if a steroid of greater specific activity than 1 microcurie per mg. is used. (Steroids with specific activity as high as 5 to 10 pc. per mg. are now obtainable from commercial sources.) The procedure can be reversed by mixing a nonradioactive unknown substance with a known amount of the postulated radioactive compound. As little as 20 y of unknown material can be detected in this way, but its usefulness is limited by the availability of labeled test substances. ACKNOWLEDGMENT
The authors are indebted to E. G. Holmstrom for his support and interest in this investigation and to T. F. Dougherty for generously permitting the use of laboratory facilities, without which this work could not have been done. The carbon-14-labeled cortisol was obtained from the Radioactive Steroids Allocation Committee, Endocrinology Study Section, Public Health Service. LITERATURE CITED (1) Berliner, D. L., Salhanick, H . A , , J . C h . Endocrinol. and Mefabolism 16, 903, (1956). (2) B u s h , I. E., Biochem. J . 5 0 , 3 i O (1951). (3) Fieser, L. F., “Experiments in Organic Chemistry,” p a r t 11, p. 358, “Solvents,” D. C. H e a t h , Boston, 1941. (4) Haines, W. J., “Recent Progress in Hormone Research,” vol. VII, p. 255, -4cademic Press, S e w York, 1952. (5) Hevesy, G., “Radioactive Indicators.” Interscience, Kew York, 1948. (6) Zaffaroni, A., J . A m . Chem. SOC.7 2 , 3828 (1950). (7) Zaffaroni, A . , “Recent Progress in Hormone Research,” vol. VIII, p. 51, Academic Press, New York, 1953. RECEIVED for review March 13, 1956. Accepted June 16, 1956. Investigation supported by a research grant, G-3843(C), from the Sational Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service.
