The assay of androgens and related substances involves the considera-tion, first, of the bioassay methods which are dependent upon the biologi-cal activity of this class of compounds, and second, of the chemibiologi-cal methods which essentially involve the color produced by C-17 ketones under special conditions. Although the biological and chemical methods both measure some of the same compounds, certain obvious differences exist. Urinary steroids such as androsterone (I) and dehydroisoandro-sterone (III) both possess androgenic activity of varying degrees and give roughly, by the chemical methods usually employed, about the same intensity of color. On the other hand, a steroid such as etio-cholanol-3(a)-one-17 (XVIII) gives a positive test by the chemical methods but is inactive biologically.
A. BIOASSAY OF ANDROGENS
In studies of extracts containing androgenic material, the biological method is obviously indispensable. Biological methods are needed for
XII. BIOCHEMISTRY OF ANDROGENS 489 the characterization of a new androgen which must include its physiologi-cal action (qualitative) and its relative activity (quantitative).
In studies on urinary concentrates, it has been shown that a reasonable parallelism exists between the quantity of androgenic material (biological assay) and the amount of 17-ketosteroids (chemical assay) present.
Oesting (168) has demonstrated that relatively good correlation exists between the androgenic and 17-ketosteroid titer of normal children's urine. The work of Callow (31) indicates a close relationship between the two methods in a variety of urines. Holtorff and Koch (116) have studied this relationship, and, although their correlation appears to be poorer than that found by the earlier mentioned workers, it appears to be adequate. In human urines, for comparative studies, either the biological methods or chemical methods may be employed. For special studies, the choice of either the biological method, the chemical method, or both must be dependent upon the specific objectives.
1. Capon's Comb Assay by Intramuscular Injection
The capon's comb has served as a test object for the evaluation of androgenic activity since the time of Berthold's classic experiments.
It has served not only as a qualitative measure of androgenic activity, but has been used for quantitative studies. As a quantitative tool, many variables had to be discovered and controlled. Sueh factors as age at which cocks are caponized, breed of capon, weight of capon, influence of light, age of bird, and initial comb size have been subjected to critical analysis. Various methods of measuring comb size have been employed such as direct measurement of the comb with a millimeter rule, photograph of the comb with subsequent measurement of the areas, etc.
Detailed studies on the capon method have been reported particu-larly by Gallagher and Koch (91), Greenwood, Blythe, and Callow (96), and McCullagh and Cuyler (146). The method of Gallagher and Koch is representative of the capon method. This method consisted in the use of brown leghorn capons. Before administration of the test material, the length and height of the comb was obtained by direct measurement with a millimeter rule. The capons were injected intramuscularly once daily for five days and the combs again measured one day after the last injection. Each daily dose was contained in 1 ml. of oil. The increase in the length (L) plus the increase in height (H) was taken as the response.
The response (L plus H) was plotted against dosage and the charac-teristic curve determined. It was found that a response of 3 to 7 mm. in L plus H was the desirable range for assays. The capon unit was defined as the amount of material which, injected per day for five days, yields an average of 5-mm. increase in L plus H. This unit is approximately
equal to one international unit. The standard preparation employed was a highly purified bull testis preparation in which the activity \vas due principally to testosterone. These workers reported a mean error of 22.6% when the unknown was run in parallel with a standard and groups of 16 to 25 capons were used for both the unknown and standard.
Greenwood et al. (96) have also studied the dose-response relationship using androsterone and the brown leghorn capon. The five-day period was employed and the measurement of the comb done in a manner similar to that utilized by Gallagher and Koch. A log dose-response curve was constructed between the limits of 0.5 and 8 mg. of androsterone and was found to give a linear relationship within these limits. The authors found a slope of 12.6 when the comb response (L plus H) was expressed in millimeters and the dose expressed as logarithm of milli-grams. The authors claimed an accuracy of ± 1 8 % for the determina-tion of an unknown, using five capons, and claimed that if the number of capons employed was increased to ten, the error was decreased to ± 12%.
Although early studies indicated that the initial size of the comb was unimportant (7,89), more detailed studies seemed to indicate that the initial size of the comb must be taken into account for precise assays (91). The weight of the animals makes a slight difference in response.
No significant difference in response could be attributed to animals varying in age from four months to six years. Responses to subcuta-neous and intramuscular injections were similar but the amount and nature of the solvent employed was an important factor. Variations in intensity of light were reflected in changes in response to a standard dose.
A question of the strain of capons suitable for androgenic studies has been investigated. It has been found that, in addition to the white and brown leghorn, the English game bantam may be employed. How-ever, the heavier breeds such as the Rhode Island Red and the Plymouth Rock are not sensitive enough for the test (171).
2. Capon Assay by Direct Application to the Comb
A more sensitive method for the utilization of the capon's comb has been the direct inunction of the androgen on the comb. Studies of these methods (50,52,88,146) have indicated that this method is approxi-mately 100 to 200 times as sensitive as subcutaneous and intramuscular injection methods. The author is not aware of any statistical studies on the method using capon's comb b}' inunction, and the accuracy is difficult to evaluate.
3. Chick's Comb Method of Assay
The early observations of Ruzicka (192), Burrows, Byerly, and Evans (14), Danby (44,45), Dorfman and Greulich (59), and Frank
X I I . B I O C H E M I S T R Y OF A N D R O G E N S 491 et al. (82,83) indicated the advisability of using the chick comb as the test object for androgen assays. Ruzicka painted the chick's comb with a 0.5% solution of androsterone in oil each day for a period of several weeks and obtained large increases in comb area. He did not, however, study this reaction quantitatively. Frank and Klempner (83) applied the androgens in oil solutions directly to the base of the comb of white leghorn chicks. Applications were begun on the sixth day after hatching and were repeated on ten successive days. The animals were sacrificed and the comb weights were determined on the day following the last application. These workers were able to evoke a definite response with as little as 20 μg. of androsterone. Burrows and co-workers injected both androsterone and testosterone either into the base of the chick's comb or into the breast muscles and found that both these androgens stimulated comb growth. In all the studies mentioned, the end point consists of the weight of the comb, which perhaps represents an advan-tage over the less exact methods of measurement of size of the capon's comb. However, the capon's comb method has the advantage that each animal serves as its own control.
The method of Hollander et al. (115) and Frank et al. (84) is the most precise of the chick methods suggested and has been demonstrated to be an adequate method for the determination of androsterone and urinary androgen. This test was designed to utilize the two-to-three-day-old white leghorn chick. The total dose of material in 0.35 ml. of oil was administered in seven divided doses at 24-hour intervals. The material was administered by applying the test solution from a hypodermic needle moving lightly over the surface of the comb. Twenty-four hours after the last administration the animals were killed with chloroform and the combs removed and weighed. Mixed male and female chicks were employed. The calculations take into consideration the initial and final body weights as well as the sex of the animal and weight of the comb.
The following formulation was developed to calculate the andro-sterone equivalent in terms of milligrams:
A =
1.06(Sw) - 0.0043(2w2) - 0.397(Σ#0 - 0.267(EBt) + U.75Nm + 18MNf
Nm + Nf
where A = androsterone equivalent in mg., Σιν = sum of comb weights in mg., Σιυ2 = sum of squared comb weights, ΣΒχ = sum of initial body weights in g., 2Bt = sum of terminal body weights in g., Nm = number of males, and Nf = number of females.
Using this formulation, Klempner (120) has shown that in 24 deter-minations of androsterone using sixteen animals in a determination in
the dosage range of 20-40 Mg., the mean error was 13%, and in 39 deter-minations over the range of 10-50 Mg., the mean error was 24.6%. In another study, the results of Klempner were essentially confirmed (58).
In the latter study, with the range 20-40 μg. of androsterone, a mean error of 12% was found, and in the range 10-40 Mg., a mean error of 24%
was found.
The details of this method have been extended to the assay of testo-sterone propionate by Dorf man (58). Here the calculations were based on a simultaneous standard run according to the design formulated by Bliss (6). With the use of 32 chicks on the standard and 32 chicks on the unknown, errors in potency ratios of less than ± 3 8 % were realized.
Increase in light tends to increase the sensitivity of the comb to androgens, at least in the range of complete darkness to normal light (202). The body weights of animals in normal light were higher than those kept in darkness. The effect was still preserved, however, if the results were expressed as ratios of comb to body weight.
The sensitivity of the chick's comb to androgens varies with the breed employed. This is probably true for endogenous androgens as well as exogenous material since the comb ratios (comb weight per unit body weight) vary with the various breeds. Thus, when the comb ratios of the White Leghorn, Rhode Island Red, and Barred Rock untreated male chicks are compared, it is found that the White Leghorn is the largest followed in order by the Rhode Island Red and Barred Rock. The relative magnitudes of the ratios may be expressed as 8:6:4, respectively.
When relatively small doses of androgens were administered to male chicks of the three breeds, it was found that when the chick comb ratios of White Leghorns increased 300%, comb ratios of Rhode Island Reds increased 100% and ratios of Barred Rocks increased 70%, (58).
4. Mammalian Assay Methods
Various mammalian tests, usually on rodents, have been employed for the assay of androgens, such as the weight or histological change of the seminal vesicles or the prostate, the electrical ejaculation test (3,156), the ductus deferens test (210), the Cowper's gland test (104,105), and a pharmacological (pernoston and yohimbine) ejaculation test (127).
Among the various mammalian tests employed, the most important from the standpoint of sensitivity and accuracy has been the weight of the seminal vesicles, prostate, or both. The studies of Korenchevsky and Dennison (123), Deanesley and Parkes (49), Miescher, Wettstein, and Tschopp (149), Callow and Deanesley (32), Bulbring and Burns (10), and Greene and Burrill (93,95) are important in the development of
XII. BIOCHEMISTRY OF ANDROGENS 493 these methods. Recently Hays and Mathieson (100) and Mathieson and Hays (145) have reinvestigated the use of the seminal vesicles of the castrated rat for the assay of androgens. These workers specifically used testosterone propionate and the experimental design of Bliss (6).
The assay is so designed that a comparison could be made between standard and unknown solutions of testosterone propionate at two dose levels. By this method, an accuracy of ± 2 0 % could be achieved if each of the four groups of animals contained eight animals or a total of 32 animals on the standard plus the unknown.
In addition to the usual variables which influence biological assay methods—such as weight, strain, and age of animals, volume and nature of solvent (49), and diet of animals—the presence of contaminating estrogens may be considered in the seminal vesicle and prostate tests, since these substances have been shown to have an enhancing action.
A second factor is the question of such activators as palmitic acid, which apparently exert an enhancing action on the absorption of androgenic substances.
5. The International Androgen Standard
As a result of the League of Nations Committee meeting held in 1935, an international standard for androgens was established. The committee adopted 0.1 mg. of androsterone as equivalent to one international unit.
b. POLAROGRAPHIC DETERMINATION OF ANDROGENS AND RELATED COMPOUNDS
Studies on the applicability of the Polarographie method for the determination of androgens and related steroids have been reported by Wolfe, Hershberg, and Fieser (219). From the work of these investiga-tors, it appears that the 17-ketosteroids present in neutral urinary extracts can be determined accurately and rapidly by reacting these steroids with Girard's reagent Τ (trimethylacethydrazide ammonium chloride) and Polarographie analysis of a suitable aqueous solution of the reaction mixture. Under the conditions of analysis, 3-ketosteroids are indifferent, and the 20-ketosteroids give a distinctly different result than the 17-ketosteroids. The A4-3-ketosteroid may be easily distinguished from the 17-ketosteroids.
In a preliminary study of the relationship between 17-ketosteroid concentrations in urinary extracts, good agreement was found between the values obtained by the Polarographie and Zimmerman methods, although the range was from 1.7 mg. to 141 mg. of 17-ketosteroids per liter of urine.
C . CHEMICAL DETERMINATION OF ANDROGENS AND RELATED COMPOUNDS
For specific purposes such as the determination of urinary androgens and their related compounds, many of which are metabolites of body androgens, the chemical methods of detection have been applied as an alternative to biological assay. Zimmerman (224,225) demonstrated that pure ketonic steroids such as androsterone, testosterone, and estrone could be quantitatively determined by the use of the reaction of these substances with m-dinitrobenzene in alkaline solution to produce a characteristic color. This work was followed by that of Wu and Chou (223), who modified the test and studied concentrations of color-produc-ing material in urine, and expressed the results in terms of androsterone.
Following these initial efforts, an extensive literature has appeared deal-ing with modifications of the method as well as extensive applications to the study of urinary concentrations in normal and abnormal individuals.
Although numerous methods for the determination of 17-ketosteroids have been suggested, analysis of some of the factors operating in two of these methods (the details of which show differences) may suffice for our purposes. These two representative methods are those of Callow et al.
(28) and of Holtorff and Koch (116).
The method of Callow et at. (28) consists essentially in dissolving the material to be tested in absolute alcohol, adding a 2 % solution of m-dini-trobenzene in absolute alcohol, and finally a 2.5 Ν solution of potassium hydroxide in absolute alcohol. The solutions are mixed and incubated for one hour at 25 ± 0.1°C. and protected from strong light. A calibra-tion curve is constructed with known amounts of a crystalline standard such as androsterone. The "blank" consists of the solvent, absolute alcohol, plus the m-dinitrobenzene and potassium hydroxide solutions.
The spectroscopic studies of the reaction product between andro-sterone and m-dinitrobenzene showed a maximum at 5010 A, while the reagents alone gave a low general absorption with a maximum at 4650 A.
Callow suggested that any selective filter having maximum transmission somewhere between 5000 and 5400 A. would be suitable. It was noted that a broad absorption band with a maximum in the green was charac-teristic of carbonyl substitution at C-17. By this technique, distant substituents had little influence on the spectral characteristics of the color. Thus, dehydroisoandrosterone and estrone gave calibration curves similar to that of androsterone. Saturated 3-ketones show a very low general absorption after a one-hour development preceded by a rapid color development at five minutes. In the case of A4-stenones a longer time is required for the color development; the maximum is not
XII. BIOCHEMISTRY OF ANDROGENS 495 obtained at one hour. This group of compounds also shows a maximum in the yellow in addition to that found in the green. The 20-keto group has been shown to give only a low general absorption.
The studies on urinary extracts showed that both normal male and female urinary extracts had an absorption spectrum quite similar to that found for androsterone. However, in certain abnormal urines the read-ing in the green was partly due to substances other than 17-ketosteroids.
In such cases, it is found that relatively high absorptions were found in the region of the violet.
In the original work of Callow a good correlation was found between the chemical tests and the androgenic assay. In spite of the relatively high error of estimate in the biological assay, a correlation coefficient of 0.745 was found.
The question of nonspecific chromogen determined on the total neutral fraction has been studied by a number of workers. Essentially two methods have been employed, the first being to perform the deter-mination on ketonic fraction, the second, the use of a correction factor.
Talbot, Butler, and MacLachlan (206) have shown that higher accuracy can be attained with the Callow method when ketonic fractions are employed. Frazier et al. (85) have used a correction equation to compensate for thé overestimates inherent in measurements on the total neutral fraction. The interfering chromogens appear to absorb maxi-mally in the region of the violet at 4100 Α., as contrasted with the maximal absorption of the 17-ketosteroids at 5200 A. The validity of using a correction equation for the Callow procedure has been shown by the fact that net values so obtained agree well with the value derived from assays on the ketonic fractions (76,208).
Applying the formulations of Gibson and Evans (92) and making readings in the green and violet, the following correction equation may be used for the 17-ketosteroid determination by the Callow procedure:
Corrected reading in green = ^ _ ^ — For chromogens Ki = Ev/E0. For 17-ketosteroids Ks = EV/EG.
The Holtorff-Koch technique differs from the Callow method in a number of details. This method consists in the use of an aqueous 5 Ν potassium hydroxide solution and 95% ethanol solutions of the test material and a 2 % solution of ra-dinitrobenzene in 95% ethanol. The time of incubation was originally set at 45 minutes, but subsequent studies have indicated that the maximum color development is obtained at about 105 minutes (164). Unlike the Callow^ method, this method shows a difference in color produced by various 17-ketosteroids. This method shows a departure from linearity as the amount of total urinary
extract employed is increased. This is minimized if the measurements are made in the dilute range and completely removed if assays are done on the ketonic fraction even in an extended range. Since the curve departs from linearity, correction equations cannot be applied over an extended range of urinary concentrations (76).
Pincus (179) has described a colorimetric method for the determina-tion of urinary 17-ketosteroids which excludes a number of chromogens that react with m-dinitrobenzene. It involves reaction of neutral ketonic steroids with concentrated antimony chloride (SbCl3) in acid solution. Androsterone and its isomers produce an intense blue color, whereas the 20-ketosteroids and the 3-ketosteroids give yellowish or
Pincus (179) has described a colorimetric method for the determina-tion of urinary 17-ketosteroids which excludes a number of chromogens that react with m-dinitrobenzene. It involves reaction of neutral ketonic steroids with concentrated antimony chloride (SbCl3) in acid solution. Androsterone and its isomers produce an intense blue color, whereas the 20-ketosteroids and the 3-ketosteroids give yellowish or