ANALYTICAL
75,441-446
BIOCHEMISTRY
(1976)
Assay of A”-3-Oxosteroids and Their Conjugates by the lsonicotinic Acid Hydrazide Method MICHIO Kyoritsu
College
MATSUI
of Pharmacy,
AND Shibakoen.
MIE
TAKAHASHI Minuto-ku,
TOXJY~ 105. Japan
Received April 26, 1976: accepted May 19, 1976 Enzyme-catalyzed hydrogenation of A”-3-oxosteroids and their conjugates can be assayed without hydrolytic and chromatographic procedures. The assay medium is mixed with methanol, followed by centrifugation. The resultant supematant is evaporated to dryness and subsequently condensed with the isonicotinic acid hydrazide reagent. The hydrazone of Al-3-oxosteroid thus formed is determined at 372 nm.
Previous studies with rat liver microsomal and soluble fractions demonstrated the presence of enzyme systems capable of reducing the A4 double bond of testosterone 17-glucosiduronate, testosterone 17-N-acetylglucosaminide, and testosterone 17-sulfate to the corresponding 5~ and S/3-reduced steroid conjugates (l-3). Following incubation of the radioactive conjugates with liver enzymes, the metabolites were separated and purified by a combination of column chromatography, hydrolysis of the conjugates, and identification of the liberated aglycones. For continuation of the enzymic studies, a simple and sensitive method for the analysis of Ad-3-oxosteroids and their conjugates is needed. The spectrophotometric methods for the assay of the A?-hydrogenase activity are based on the measurement ofthe absorption either of the A”-3-0~0 grouping (4) or of the hydrazone formed by condensation of the Ad-3-oxosteroid with isonicotinic acid hydrazide (INH) (5), after the steroid is extracted with organic solvents such as methylene chloride. However, these methods are not applicable to steroid conjugates, because the conjugates are not extracted with such solvents. This paper describes a specific, sensitive, and simple method for the quantitative estimation of A”-3-oxosteroids and their conjugates using the INH method. MATERIALS
AND METHODS
Steroids and reagents. 2a- and 2P-Hydroxytestosterones were prepared by the procedure described by Sondheimer et al. (6). Progesterone, corticosterone, and isonicotinic acid hydrazide were purchased from Tokyo Kasei Kogyo Co. Hydrocortisone and testosterone were obtained from Sigma Chemical Co. Testosterone 3-isonicotinic acid hydrazone (mp, 441 Copyright All rights
Q 1976 by Academic Press. Inc. of reproductmn in any form reserved.
442
MATSUI
0.2
AND TAKAHASHI
-
0.1-
0
’ 530
350
370
39” wAvEw.NGTI~
FIG. 1. Absorption standard analysis.
410
430
450
1 470
(nm)
spectrum of testosterone 3-isonicotinic
acid hydrazone formed in the
214-219°C; lit. (7), 216-218”(Z) was prepared by condensation of testosterone (100 mg) with INH (100 mg) in acetic acid-methanol solution (lo%, 2 ml). Other reference steroids and reagents used in this study were described in previous papers (1,2,8). Acetic acid-methanol reagent: 80 ml of acetic acid is adjusted to 100 ml with methanol. Isonicotinic acid hydrazide (INH) reagent: 200 mg of INH is dissolved in 100 ml of acetic acid-methanol reagent. Tissue preparation. Male rats of the Wistar strain, weighing 150-300 g were killed by decapitation. Liver microsomal and soluble fractions were prepared in ice-cold 0.25 M sucrose solution as previously described (l), and the protein concentrations were about 1.4 and 15 mg/ml, respectively, as determined by the procedure of Lowry et al. (9). Procedures. Standard curves were prepared in the following manner: To 2.0 ml of the assay medium, which consisted of 3.0 ml of 0.25 M sucrose solution and 3.5 ml of 0.2~ Tris-maleate buffer (pH 6.5), was added methanol solution of the steroid or steroid conjugate (15-260 nmol) and made up to 10 ml with methanol to afford the buffer-methanol solution. Then, 3.0 ml of this solution was evaporated to dryness in vacua. The residue was dissolved in 3.0 ml of the INH reagent and kept at 37°C for 2 hr. The absorbance at 372 nm was determined in a Hitachi 124 spectrophotometer using tubes with l.O-cm light path. The reference consisted of the test reagent with the solvent blank.
ASSAY OF A’-3-OXOSTEROIDS
AND THElR CONJUGATES
FIG. 2. Standard curves for testosterone (0). testosterone 17-glucosiduronate testosterone 17-sulfate (x ).
443
(0). and
Recoveries of the steroids from the incubation medium were carried out as follows: 3.0 ml of the microsomal or soluble fraction was added to 3.5 ml of 0.2 M Tris-maleate buffer (pH 6.5), containing an NADPH-generating system as described in the previous paper (1). To this enzyme medium, which was kept at 4°C in an ice bath, was added a methanol solution of the steroid or a 50% methanol solution of the steroid conjugate (0.2-3.0pmol. 100 pl), followed by thorough mixing. Two milliliters of this incubation medium was removed and immediately made up to 10 ml with methanol. The resultant mixture was allowed to stand at room temperature at least for 1 hr and centrifuged at 3000 rpm for 10 min. Three milliliters of the supernatant was evaporated to dryness in vacua. The residue was treated with the INH reagent, and the absorbance was determined as described above. The corrected absorbance was obtained by subtracting the absorbance of the supernatant blank from that of the supernatant described above, and the recovery was calculated from the standard curve. RESULTS
Condensation of testosterone with the INH-HCl reagent (5.7) was first attempted in the buffer-methanol solution. However, no more than 70% of the testosterone reacted with INH. In order to improve the yield. the condensation was carried out in an anhydrous medium. Thus,
444
MATSLJI AND TAKAHASHI TABLE
1
MOLAR EXTINCTION COEFFICIENTS (E) OF STEROIDS AND THEIR CONJUGATES
Compound Testosterone Testosterone 17-sulfate Testosterone 17-glucosiduronate Androstenedione Progesterone Corticosterone Hydrocortisone Za-Hydroxytestosterone Z/3-Hydroxytestosterone 6/3-Hydroxytestosterone 4-Androstene-3.6.17-trione 17/3-Hydroxy-So-androstan-3-one 17P-Hydroxy-Sp-androstan-3-one 3a-Hydroxy-Sa-androstan-17-one 3ol-Hydroxy-5P-androstan-17-one 3/3-Hydroxy-S-androsten-17-one 4-Androstene-3P. 17~diol Slu-Androstane3a. 17@diol 5@-Androstane-3a, 17P-diol
A”
B*
14,800 12.500 11.300 16,300 15.000 14,700 14,800 6,200 7.500 7,100 7.900 800 640 0 0 0 0 0 0
15,300 13,400 11,000 14,400 15,000 15,300 15,300 6.850 7,500 9.400 8,170 870 880 0 0 0 0 0 0
h max(4” 372 372 372 372 372 372 372 364 362 366 358
(7,250) (9,550) (7,400) (12,400)
n The buffer-methanol solution of steroid was evaporated to dryness and condensed with the INH reagent (standard procedure). * The methanol solution of steroid was evaporated to dryness and condensed with the INH reagent.
the buffer-methanol solution was evaporated to dryness and treated with the INH reagent. As a condensation solvent, acetic acid-methanol solution had advantages over the widely employed HCI-methanol (or ethanol) solution with respect to sensitivity and stability of the colored product. The greatest absorbance was obtained when testosterone was treated at 20 to 37°C for 2 hr with the acetic acid-methanol reagent containing 0.2 to 0.3% INH. The color intensity was stable for 24 hr. The standard procedure was established based on these observations. The absorption spectrum of testosterone 3-isonicotinic acid hydrazone formed in the standard analysis revealed an absorption maximum at 372 nm (Fig. 1). The maximal molar extinction coefficient of testosterone after color development was 14,800 and was in good accord with the value of 15,000 obtained for the synthesized testosterone 3-isonicotinic acid hydrazone, whose spectrum was taken in the standard condensation medium. Therefore, it became apparent that testosterone reacted almost quantitatively with INH. The standard curves of testosterone. testosterone 17-glucosiduronate, and testosterone 17-sulfate are shown in Fig. 2. Linear proportionality
ASSAY
OF A4-3-OXOSTEROIDS
AND TABLE
RECOVERY OF TESTOSTERONE ADDED TO MICROSOMAL Microsomal
Compound Testosterone
THEIR
445
CONJUGATES
2
AND TESTOSTERONE CONJUGATES AND SOLUBLE FRACTIONS fraction
Soluble
fraction
Added (a)
Found ha
Recovery (96)
Added (PLg)
Found
128 253 433 866
122 272 432 878
95.3 108 99.8 101
119 236 510 800
II8 233 535 796
99.0 98.7 105 99.5
(/-a
Recovery (95)
Testosterone
17-sulfate
83.3 160 325 1140
91.0 158 334 1100
109 98.8 103 96.5
175 348 545 1090
178 363 518 1040
102 104 95.0 95.4
Testosterone siduronate
17-gluco-
195 399 660 1320
193 392 639 1270
99.0 98.2 96.8 96.2
186 369 680 1360
197 377 644 1320
106 102 94.7 97.1
between absorbance and concentration was obtained for these steroids at 15 to 260 nmol/3 ml. The color development was little affected by the assay buffer systems such as 0.2 M Tris-maleate buffer at pH 6.0 to 8.0 and 0.2~ Tris-HCl buffer at pH 7.2, which have been used as the assay medium for Ah?hydrogenases (1- 5). Furthermore, similar results were obtained when methanol solutions of steroids were processed with the INH reagent (Table 1). In the standard analysis, A”-3-oxosteroids such as testosterone, androstenedione, progesterone, corticosterone, and hydrocortisone gave molar extinction coefficients of about 15,000 (Table l), these values being about 25% higher than those reported by Umberger (7). Introduction of the 2- or 6-hydroxy (or 0x0) grouping into A4-3-oxosteroids resulted in somewhat lower color intensities. The high specificity for A”-3-0x0steroids was demonstrated by little or no color development of ring A saturated 3- or 17-oxosteroids and 3,17-dihydroxysteroids. Good recoveries of testosterone and testosterone conjugates added to the rat liver microsomal and soluble fractions proved the validity of this method (Table 2). DISCUSSION
Advantages of using the calorimetric assay of A4-3-oxosteroids and their conjugates described herein are simplicity and specificity. Following evaporation of the assay medium, the residue is condensed with the INH re-
446
MATSUI
AND TAKAHASHI
agent and the absorbance at 372 nm is determined. The color reaction is highly specific for A4-3-oxosteroids. Therefore, neither hydrolytic nor chromatographic procedures are necessary for the assay of these steroids. The catabolic sequence of A4-3-oxosteroids is well established and proceeds mainly via 4,5-dihydro-3-oxosteroids to saturated 3-hydroxysteroids by the consecutive action of A4-hydrogenases and 3-oxosteroidoxidoreductases (IO). Thus, this method is especially applicable to the kinetic studies of these enzyme systems, employing A4-3-oxosteroids and their conjugates as substrates. ACKNOWLEDGMENT This work was supported in part by a grant from the Ministry of Education of Japan.
REFERENCES 1. Matsui, M., Abe, F.. Kimura, K., and Okada, M. (1972) Chem. Pharm. Bull. (Tokyo) 20, 1913-1920. 2. Matsui, M., Abe, F., Kunikane, M., and Okada, M. (1973) Chem. Pharm. Bull. (Tokyo) 21, 558-564. 3. Matsui, M., Kinuyama, Y., Hakozaki, M., Abe, F., Kawase, M., and Okada, M. (1973) Chem. Pharm. Bull. (Tokyo) 21, 2764-2768. 4. Tomkins, G. M. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 499-503, Academic Press, New York. 5. Ringler, I., and Perrine, J. (1961) Endocrinology 69, 109% 1097. 6. Sondheimer, F., Kaufmann, S., Romo, J., Martinez, H., and Rosenkranz, G. (1953) J. Amer. Chem. Sot. 75,4712-4715. 7. Umberger, E. J. (1955) Anal. C/rem. 27, 768-773. 8. Matsui. M., Kawase, M.. and Okada, M. (1974) C/rem. Pharm. Bull. (Tokyo) X2,25302537. 9. Lowry, 0. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. Dorfman, R. I., and Ungar, F. (1965) Metabolism of Steroid Hormones, pp. 382424, Academic Press, New York.
75,441-446
BIOCHEMISTRY
(1976)
Assay of A”-3-Oxosteroids and Their Conjugates by the lsonicotinic Acid Hydrazide Method MICHIO Kyoritsu
College
MATSUI
of Pharmacy,
AND Shibakoen.
MIE
TAKAHASHI Minuto-ku,
TOXJY~ 105. Japan
Received April 26, 1976: accepted May 19, 1976 Enzyme-catalyzed hydrogenation of A”-3-oxosteroids and their conjugates can be assayed without hydrolytic and chromatographic procedures. The assay medium is mixed with methanol, followed by centrifugation. The resultant supematant is evaporated to dryness and subsequently condensed with the isonicotinic acid hydrazide reagent. The hydrazone of Al-3-oxosteroid thus formed is determined at 372 nm.
Previous studies with rat liver microsomal and soluble fractions demonstrated the presence of enzyme systems capable of reducing the A4 double bond of testosterone 17-glucosiduronate, testosterone 17-N-acetylglucosaminide, and testosterone 17-sulfate to the corresponding 5~ and S/3-reduced steroid conjugates (l-3). Following incubation of the radioactive conjugates with liver enzymes, the metabolites were separated and purified by a combination of column chromatography, hydrolysis of the conjugates, and identification of the liberated aglycones. For continuation of the enzymic studies, a simple and sensitive method for the analysis of Ad-3-oxosteroids and their conjugates is needed. The spectrophotometric methods for the assay of the A?-hydrogenase activity are based on the measurement ofthe absorption either of the A”-3-0~0 grouping (4) or of the hydrazone formed by condensation of the Ad-3-oxosteroid with isonicotinic acid hydrazide (INH) (5), after the steroid is extracted with organic solvents such as methylene chloride. However, these methods are not applicable to steroid conjugates, because the conjugates are not extracted with such solvents. This paper describes a specific, sensitive, and simple method for the quantitative estimation of A”-3-oxosteroids and their conjugates using the INH method. MATERIALS
AND METHODS
Steroids and reagents. 2a- and 2P-Hydroxytestosterones were prepared by the procedure described by Sondheimer et al. (6). Progesterone, corticosterone, and isonicotinic acid hydrazide were purchased from Tokyo Kasei Kogyo Co. Hydrocortisone and testosterone were obtained from Sigma Chemical Co. Testosterone 3-isonicotinic acid hydrazone (mp, 441 Copyright All rights
Q 1976 by Academic Press. Inc. of reproductmn in any form reserved.
442
MATSUI
0.2
AND TAKAHASHI
-
0.1-
0
’ 530
350
370
39” wAvEw.NGTI~
FIG. 1. Absorption standard analysis.
410
430
450
1 470
(nm)
spectrum of testosterone 3-isonicotinic
acid hydrazone formed in the
214-219°C; lit. (7), 216-218”(Z) was prepared by condensation of testosterone (100 mg) with INH (100 mg) in acetic acid-methanol solution (lo%, 2 ml). Other reference steroids and reagents used in this study were described in previous papers (1,2,8). Acetic acid-methanol reagent: 80 ml of acetic acid is adjusted to 100 ml with methanol. Isonicotinic acid hydrazide (INH) reagent: 200 mg of INH is dissolved in 100 ml of acetic acid-methanol reagent. Tissue preparation. Male rats of the Wistar strain, weighing 150-300 g were killed by decapitation. Liver microsomal and soluble fractions were prepared in ice-cold 0.25 M sucrose solution as previously described (l), and the protein concentrations were about 1.4 and 15 mg/ml, respectively, as determined by the procedure of Lowry et al. (9). Procedures. Standard curves were prepared in the following manner: To 2.0 ml of the assay medium, which consisted of 3.0 ml of 0.25 M sucrose solution and 3.5 ml of 0.2~ Tris-maleate buffer (pH 6.5), was added methanol solution of the steroid or steroid conjugate (15-260 nmol) and made up to 10 ml with methanol to afford the buffer-methanol solution. Then, 3.0 ml of this solution was evaporated to dryness in vacua. The residue was dissolved in 3.0 ml of the INH reagent and kept at 37°C for 2 hr. The absorbance at 372 nm was determined in a Hitachi 124 spectrophotometer using tubes with l.O-cm light path. The reference consisted of the test reagent with the solvent blank.
ASSAY OF A’-3-OXOSTEROIDS
AND THElR CONJUGATES
FIG. 2. Standard curves for testosterone (0). testosterone 17-glucosiduronate testosterone 17-sulfate (x ).
443
(0). and
Recoveries of the steroids from the incubation medium were carried out as follows: 3.0 ml of the microsomal or soluble fraction was added to 3.5 ml of 0.2 M Tris-maleate buffer (pH 6.5), containing an NADPH-generating system as described in the previous paper (1). To this enzyme medium, which was kept at 4°C in an ice bath, was added a methanol solution of the steroid or a 50% methanol solution of the steroid conjugate (0.2-3.0pmol. 100 pl), followed by thorough mixing. Two milliliters of this incubation medium was removed and immediately made up to 10 ml with methanol. The resultant mixture was allowed to stand at room temperature at least for 1 hr and centrifuged at 3000 rpm for 10 min. Three milliliters of the supernatant was evaporated to dryness in vacua. The residue was treated with the INH reagent, and the absorbance was determined as described above. The corrected absorbance was obtained by subtracting the absorbance of the supernatant blank from that of the supernatant described above, and the recovery was calculated from the standard curve. RESULTS
Condensation of testosterone with the INH-HCl reagent (5.7) was first attempted in the buffer-methanol solution. However, no more than 70% of the testosterone reacted with INH. In order to improve the yield. the condensation was carried out in an anhydrous medium. Thus,
444
MATSLJI AND TAKAHASHI TABLE
1
MOLAR EXTINCTION COEFFICIENTS (E) OF STEROIDS AND THEIR CONJUGATES
Compound Testosterone Testosterone 17-sulfate Testosterone 17-glucosiduronate Androstenedione Progesterone Corticosterone Hydrocortisone Za-Hydroxytestosterone Z/3-Hydroxytestosterone 6/3-Hydroxytestosterone 4-Androstene-3.6.17-trione 17/3-Hydroxy-So-androstan-3-one 17P-Hydroxy-Sp-androstan-3-one 3a-Hydroxy-Sa-androstan-17-one 3ol-Hydroxy-5P-androstan-17-one 3/3-Hydroxy-S-androsten-17-one 4-Androstene-3P. 17~diol Slu-Androstane3a. 17@diol 5@-Androstane-3a, 17P-diol
A”
B*
14,800 12.500 11.300 16,300 15.000 14,700 14,800 6,200 7.500 7,100 7.900 800 640 0 0 0 0 0 0
15,300 13,400 11,000 14,400 15,000 15,300 15,300 6.850 7,500 9.400 8,170 870 880 0 0 0 0 0 0
h max(4” 372 372 372 372 372 372 372 364 362 366 358
(7,250) (9,550) (7,400) (12,400)
n The buffer-methanol solution of steroid was evaporated to dryness and condensed with the INH reagent (standard procedure). * The methanol solution of steroid was evaporated to dryness and condensed with the INH reagent.
the buffer-methanol solution was evaporated to dryness and treated with the INH reagent. As a condensation solvent, acetic acid-methanol solution had advantages over the widely employed HCI-methanol (or ethanol) solution with respect to sensitivity and stability of the colored product. The greatest absorbance was obtained when testosterone was treated at 20 to 37°C for 2 hr with the acetic acid-methanol reagent containing 0.2 to 0.3% INH. The color intensity was stable for 24 hr. The standard procedure was established based on these observations. The absorption spectrum of testosterone 3-isonicotinic acid hydrazone formed in the standard analysis revealed an absorption maximum at 372 nm (Fig. 1). The maximal molar extinction coefficient of testosterone after color development was 14,800 and was in good accord with the value of 15,000 obtained for the synthesized testosterone 3-isonicotinic acid hydrazone, whose spectrum was taken in the standard condensation medium. Therefore, it became apparent that testosterone reacted almost quantitatively with INH. The standard curves of testosterone. testosterone 17-glucosiduronate, and testosterone 17-sulfate are shown in Fig. 2. Linear proportionality
ASSAY
OF A4-3-OXOSTEROIDS
AND TABLE
RECOVERY OF TESTOSTERONE ADDED TO MICROSOMAL Microsomal
Compound Testosterone
THEIR
445
CONJUGATES
2
AND TESTOSTERONE CONJUGATES AND SOLUBLE FRACTIONS fraction
Soluble
fraction
Added (a)
Found ha
Recovery (96)
Added (PLg)
Found
128 253 433 866
122 272 432 878
95.3 108 99.8 101
119 236 510 800
II8 233 535 796
99.0 98.7 105 99.5
(/-a
Recovery (95)
Testosterone
17-sulfate
83.3 160 325 1140
91.0 158 334 1100
109 98.8 103 96.5
175 348 545 1090
178 363 518 1040
102 104 95.0 95.4
Testosterone siduronate
17-gluco-
195 399 660 1320
193 392 639 1270
99.0 98.2 96.8 96.2
186 369 680 1360
197 377 644 1320
106 102 94.7 97.1
between absorbance and concentration was obtained for these steroids at 15 to 260 nmol/3 ml. The color development was little affected by the assay buffer systems such as 0.2 M Tris-maleate buffer at pH 6.0 to 8.0 and 0.2~ Tris-HCl buffer at pH 7.2, which have been used as the assay medium for Ah?hydrogenases (1- 5). Furthermore, similar results were obtained when methanol solutions of steroids were processed with the INH reagent (Table 1). In the standard analysis, A”-3-oxosteroids such as testosterone, androstenedione, progesterone, corticosterone, and hydrocortisone gave molar extinction coefficients of about 15,000 (Table l), these values being about 25% higher than those reported by Umberger (7). Introduction of the 2- or 6-hydroxy (or 0x0) grouping into A4-3-oxosteroids resulted in somewhat lower color intensities. The high specificity for A”-3-0x0steroids was demonstrated by little or no color development of ring A saturated 3- or 17-oxosteroids and 3,17-dihydroxysteroids. Good recoveries of testosterone and testosterone conjugates added to the rat liver microsomal and soluble fractions proved the validity of this method (Table 2). DISCUSSION
Advantages of using the calorimetric assay of A4-3-oxosteroids and their conjugates described herein are simplicity and specificity. Following evaporation of the assay medium, the residue is condensed with the INH re-
446
MATSUI
AND TAKAHASHI
agent and the absorbance at 372 nm is determined. The color reaction is highly specific for A4-3-oxosteroids. Therefore, neither hydrolytic nor chromatographic procedures are necessary for the assay of these steroids. The catabolic sequence of A4-3-oxosteroids is well established and proceeds mainly via 4,5-dihydro-3-oxosteroids to saturated 3-hydroxysteroids by the consecutive action of A4-hydrogenases and 3-oxosteroidoxidoreductases (IO). Thus, this method is especially applicable to the kinetic studies of these enzyme systems, employing A4-3-oxosteroids and their conjugates as substrates. ACKNOWLEDGMENT This work was supported in part by a grant from the Ministry of Education of Japan.
REFERENCES 1. Matsui, M., Abe, F.. Kimura, K., and Okada, M. (1972) Chem. Pharm. Bull. (Tokyo) 20, 1913-1920. 2. Matsui, M., Abe, F., Kunikane, M., and Okada, M. (1973) Chem. Pharm. Bull. (Tokyo) 21, 558-564. 3. Matsui, M., Kinuyama, Y., Hakozaki, M., Abe, F., Kawase, M., and Okada, M. (1973) Chem. Pharm. Bull. (Tokyo) 21, 2764-2768. 4. Tomkins, G. M. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 499-503, Academic Press, New York. 5. Ringler, I., and Perrine, J. (1961) Endocrinology 69, 109% 1097. 6. Sondheimer, F., Kaufmann, S., Romo, J., Martinez, H., and Rosenkranz, G. (1953) J. Amer. Chem. Sot. 75,4712-4715. 7. Umberger, E. J. (1955) Anal. C/rem. 27, 768-773. 8. Matsui. M., Kawase, M.. and Okada, M. (1974) C/rem. Pharm. Bull. (Tokyo) X2,25302537. 9. Lowry, 0. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. Dorfman, R. I., and Ungar, F. (1965) Metabolism of Steroid Hormones, pp. 382424, Academic Press, New York.
