557th MEETING, LIVERPOOL
677
degree of binding but that it may serve to orientate the steroid correctly relative to the active site concerned with oxidation. The very poor binding of 20,25-diazacholesterol to the enzyme is attributed to an adverse effect of the two basic centres: we have also noticed that the alkaloid solasodine is virtually unattacked by the enzyme, whereas its oxygen analogue, diosgenin, is a moderately good substrate. 5a-Cholestan-38-01 and 4-cholesten-3~-ol were comparable with cholesterol in their susceptibility to oxidation, but 5a-cholest-7-en-3/3-ol, 5acholest-8( 14)-en-38-01 and 5,7-cholestadien-3~-01were poorer substrates. 4,CDimethyl groups greatly decreased the rate of oxidation, and 4a-methylcholesterol was oxidized only oneeighth as fast as the 4 8 isomer, suggesting steric interference, particularly by the 4a-methyl group, in the removal of the 3a-hydrogen. (Relative-rate studies also showed that 48-hydroxycholestero1 was oxidized five times faster than the 4a-isomer.) Little or no oxidation was observed with 5~-cholestan-3~-ol or with 3a-hydroxy steroids. Sa-Cholestan-2&01 was not a substrate. Cholecalciferol has been reported to be a substrate for certain enzymes acting on cholesterol (Fraser & Kodicek, 1968), but was not oxidized by cholesterol oxidase. We thank the Medical Research Council for financial support (to C. J. W. B. and Professor W. A. Harland), Mr. D. Giles (Boehringer, London W.5, U.K.) for the gift ofcholesteroloxidase, Professor R. P. Cook, Dr. J. Gilmore,Dr. R.M. Moriarty, Dr. H. H. Rees, Dr. G. F. Woods and the M.R.C. Steroid Reference Collection for the gift of steroids, and Professor B. Capon for the use of his computer program. Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond,W. & Fu, P. C. (1974) Clin.Chem. (WinsfonSalem, N.C.) 20,470475
Eisenthal, R. & Cornish-Bowden, A. (1974) Biochem. J. 139,715-720 Flegg, H. M. (1973) Ann. Clin.Biochem. 10,79-94 Fraser, D. R. & Kodicek, E. (1968) Nafure (London) 220,1031-1032 Fukuda, H., Kawakami, Y. & Nakamura, S. (1973) Chem. Pharm. Bull. 21,2057-2060 Richmond, W. (1973) Clin.Chem. ( Winston-Salem,N.C.) 19,1350-1356 Smith, A. G. & Brooks, C. J. W. (1974) J. Chromatog. 101,373-378 Smith, A. G., Gilbert, J. D., Harland, W. A. & Brooks, C. J. W. (1974) Biochem. J. 139,793795
Tinder, P. (1969) Ann. Clin.Biochem. 6 , 2 4 2 7 Uwajima, T., Yagi, H., Nakamura, S. & Terada, 0. (1973) Agri. Biol. Chem. 37,2345-2350 Uwajima, T., Yagi, H. & Terada, 0. (1974) Agri. Biol. Chem. 38,1149-1156 Wentworth, W. E. (1965) J. Chem. Educ. 42,96-103
Testosterone Metabolism in Superfused Human Hyperplastic Prostate GRAHAM H. BEASTALL University Department of Steroid Biochemistry, Royal Infirmary, Glasgow G4 OSF, U.K. Since the observation by Bruchovsky & Wilson (1968) that testosterone was metabolized into dihydrotestosterone (178-hydroxy-5a-androstan-3-one)by rat ventral prostate, considerable attention has been focused on the metabolism of testosterone in the male accessory sex glands that are known target organs for the hormone. A wide distribution of testosterone 5a-reductase has been established (King & Mainwaring, 1974), and the almost universal presence of dihydrotestosterone in these tissues has led to the view that it is the active hormone for promoting androgen-induced growth (Robel, 1971; Wilson, 1972). However, the different techniques and experimental conditions used to study testosterone metabolism have led to inconsistencies as to the quantitative importance of dihydrotestosterone and the presence, or absence, of minor testosterone metabolites. Workers in this laboratory have used a superfusion technique to study steroid dynamics in slices of prostate, and conditions have been established, in which the tissue is in a steady state with respect to steroid uptake and metabolism
Vol. 3
BIOCHEMICAL SOCIETY TRANSACTIONS
678
hi"
A
5a-Androstane-3,17-dione Androst-4ene-j,17-dione
p
*4
3a-Hydroxy-5a-androstan-l7-one$ 17~-Hydroxy-5a-androstan-3-0ne$
**
3B-Hydroxy-5a-androstan- 1%one$
17b-Hydroxy-SB-androstan-3-one$ 17a-Hydroxyandrost-4-en-3-one$ 17b-Hydroxyandrost4en-3-one$
I Band C
6B-Hydroxyandrost4ene-3,17-dione 5a-Androstane-3a,17B-diol$ 5a-Androstane-3B-l7a-diol$ 5a-hdrostane-fB,17B-diol Androst&ne-3/3,17B-diol 5a-Androstane-3a,17a-diol
* Acetylation before t.1.c. t Saponification before t.1.c.
# Steroid isolated as acetates.
Scheme 1. Protocol for the separation on t.1.c. of the testosterone metabolites ofhuman hyperplastic prostate Where separation into more than one band occurs, the least polar is the upper on the scheme. T.1.c. systems: (1) silica gel H, chloroform-acetone (7:1, v/v); (2) silica gel H, cyclohexane-ethyl acetate (1 :1, v/v); (3) alumina P, chloroform, 4h, (4) alumina P, cyclohexane-ethyl acetate, (9:1, v/v), 16h; (5) alumina P, cyclohexane-ethyl acetate (20:1, v/v), 16h; (6) silica gel H, n-butyl acetate. (Giorgi et al., 1971, 1974). The steady state applies not only to testosterone but also to many of its metabolites, and the ability to study metabolism in a tissue where the concentrations of substrate and metabolites are relatively constant with time has obvious advantages over conventional techniques and provides more meaningful data in vitro. Fresh human hyperplastic prostatic tissue, removed by retropubic prostatectomy, was sliced with a Stadie-Riggs microtome. The slices (0.5g) were rinsed three times with 0.9% NaCI, transferred to the superfusion chamber (Giorgi et al., 1971) and superfused (25ml/h) with Krebs-Ringer bicarbonateglucose buffer, pH 7.4, containing 1.2ng of [1,2-3H]testosterone (56Ci/mmol)/ml. When the steady state had been achieved, the perfusate fraction was collected on ice for 30min, after which the tissue was removed, washed in 0.9% NaCl and transferred to 5ml of acetone containing known amounts (approx. 500pg) of each of the fourteen steroids listed in Scheme 1. The tissue was homogenized, the total lipids were extracted with 3 x 2 vol. of ether anda sample (1/100) was radioassayed. The dried extract and an equivalent extract from the perfusate were subjected to the t.1.c. separation described in Scheme 1. All the added marker steroids were completely resolved by this procedure, with the exception of 5a-androstane-3a,l7/lwhich were separated as the trimethylsilyl ethers by diol and 5a-androstane-3~,17a-diol, preparative g.l.c., on a 3 % OV-210 column at 220°C with a carrier gas flow of 4Omllmin. 1975
557th MEETING, LIVERPOOL
679
Table 1. Percentage composition and tissue concentration of testosterone and its metabolites after superfusion Perfusate Tissue Tissue concn. ( %) ( %) (ns/s) Steroid
---
Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2
5a-Androstane-3,17-dione Androst-4-ene-3,17-dione
0.9 0.3
3a-Hydroxy-5a-androstan-17-one 178-Hydroxy-5a-androstan-3-one 38-Hydroxy-5a-androstan-17-one
0.2
2.0 0.1 17/3-Hydroxy-5B-androstan-3-one
677
degree of binding but that it may serve to orientate the steroid correctly relative to the active site concerned with oxidation. The very poor binding of 20,25-diazacholesterol to the enzyme is attributed to an adverse effect of the two basic centres: we have also noticed that the alkaloid solasodine is virtually unattacked by the enzyme, whereas its oxygen analogue, diosgenin, is a moderately good substrate. 5a-Cholestan-38-01 and 4-cholesten-3~-ol were comparable with cholesterol in their susceptibility to oxidation, but 5a-cholest-7-en-3/3-ol, 5acholest-8( 14)-en-38-01 and 5,7-cholestadien-3~-01were poorer substrates. 4,CDimethyl groups greatly decreased the rate of oxidation, and 4a-methylcholesterol was oxidized only oneeighth as fast as the 4 8 isomer, suggesting steric interference, particularly by the 4a-methyl group, in the removal of the 3a-hydrogen. (Relative-rate studies also showed that 48-hydroxycholestero1 was oxidized five times faster than the 4a-isomer.) Little or no oxidation was observed with 5~-cholestan-3~-ol or with 3a-hydroxy steroids. Sa-Cholestan-2&01 was not a substrate. Cholecalciferol has been reported to be a substrate for certain enzymes acting on cholesterol (Fraser & Kodicek, 1968), but was not oxidized by cholesterol oxidase. We thank the Medical Research Council for financial support (to C. J. W. B. and Professor W. A. Harland), Mr. D. Giles (Boehringer, London W.5, U.K.) for the gift ofcholesteroloxidase, Professor R. P. Cook, Dr. J. Gilmore,Dr. R.M. Moriarty, Dr. H. H. Rees, Dr. G. F. Woods and the M.R.C. Steroid Reference Collection for the gift of steroids, and Professor B. Capon for the use of his computer program. Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond,W. & Fu, P. C. (1974) Clin.Chem. (WinsfonSalem, N.C.) 20,470475
Eisenthal, R. & Cornish-Bowden, A. (1974) Biochem. J. 139,715-720 Flegg, H. M. (1973) Ann. Clin.Biochem. 10,79-94 Fraser, D. R. & Kodicek, E. (1968) Nafure (London) 220,1031-1032 Fukuda, H., Kawakami, Y. & Nakamura, S. (1973) Chem. Pharm. Bull. 21,2057-2060 Richmond, W. (1973) Clin.Chem. ( Winston-Salem,N.C.) 19,1350-1356 Smith, A. G. & Brooks, C. J. W. (1974) J. Chromatog. 101,373-378 Smith, A. G., Gilbert, J. D., Harland, W. A. & Brooks, C. J. W. (1974) Biochem. J. 139,793795
Tinder, P. (1969) Ann. Clin.Biochem. 6 , 2 4 2 7 Uwajima, T., Yagi, H., Nakamura, S. & Terada, 0. (1973) Agri. Biol. Chem. 37,2345-2350 Uwajima, T., Yagi, H. & Terada, 0. (1974) Agri. Biol. Chem. 38,1149-1156 Wentworth, W. E. (1965) J. Chem. Educ. 42,96-103
Testosterone Metabolism in Superfused Human Hyperplastic Prostate GRAHAM H. BEASTALL University Department of Steroid Biochemistry, Royal Infirmary, Glasgow G4 OSF, U.K. Since the observation by Bruchovsky & Wilson (1968) that testosterone was metabolized into dihydrotestosterone (178-hydroxy-5a-androstan-3-one)by rat ventral prostate, considerable attention has been focused on the metabolism of testosterone in the male accessory sex glands that are known target organs for the hormone. A wide distribution of testosterone 5a-reductase has been established (King & Mainwaring, 1974), and the almost universal presence of dihydrotestosterone in these tissues has led to the view that it is the active hormone for promoting androgen-induced growth (Robel, 1971; Wilson, 1972). However, the different techniques and experimental conditions used to study testosterone metabolism have led to inconsistencies as to the quantitative importance of dihydrotestosterone and the presence, or absence, of minor testosterone metabolites. Workers in this laboratory have used a superfusion technique to study steroid dynamics in slices of prostate, and conditions have been established, in which the tissue is in a steady state with respect to steroid uptake and metabolism
Vol. 3
BIOCHEMICAL SOCIETY TRANSACTIONS
678
hi"
A
5a-Androstane-3,17-dione Androst-4ene-j,17-dione
p
*4
3a-Hydroxy-5a-androstan-l7-one$ 17~-Hydroxy-5a-androstan-3-0ne$
**
3B-Hydroxy-5a-androstan- 1%one$
17b-Hydroxy-SB-androstan-3-one$ 17a-Hydroxyandrost-4-en-3-one$ 17b-Hydroxyandrost4en-3-one$
I Band C
6B-Hydroxyandrost4ene-3,17-dione 5a-Androstane-3a,17B-diol$ 5a-Androstane-3B-l7a-diol$ 5a-hdrostane-fB,17B-diol Androst&ne-3/3,17B-diol 5a-Androstane-3a,17a-diol
* Acetylation before t.1.c. t Saponification before t.1.c.
# Steroid isolated as acetates.
Scheme 1. Protocol for the separation on t.1.c. of the testosterone metabolites ofhuman hyperplastic prostate Where separation into more than one band occurs, the least polar is the upper on the scheme. T.1.c. systems: (1) silica gel H, chloroform-acetone (7:1, v/v); (2) silica gel H, cyclohexane-ethyl acetate (1 :1, v/v); (3) alumina P, chloroform, 4h, (4) alumina P, cyclohexane-ethyl acetate, (9:1, v/v), 16h; (5) alumina P, cyclohexane-ethyl acetate (20:1, v/v), 16h; (6) silica gel H, n-butyl acetate. (Giorgi et al., 1971, 1974). The steady state applies not only to testosterone but also to many of its metabolites, and the ability to study metabolism in a tissue where the concentrations of substrate and metabolites are relatively constant with time has obvious advantages over conventional techniques and provides more meaningful data in vitro. Fresh human hyperplastic prostatic tissue, removed by retropubic prostatectomy, was sliced with a Stadie-Riggs microtome. The slices (0.5g) were rinsed three times with 0.9% NaCI, transferred to the superfusion chamber (Giorgi et al., 1971) and superfused (25ml/h) with Krebs-Ringer bicarbonateglucose buffer, pH 7.4, containing 1.2ng of [1,2-3H]testosterone (56Ci/mmol)/ml. When the steady state had been achieved, the perfusate fraction was collected on ice for 30min, after which the tissue was removed, washed in 0.9% NaCl and transferred to 5ml of acetone containing known amounts (approx. 500pg) of each of the fourteen steroids listed in Scheme 1. The tissue was homogenized, the total lipids were extracted with 3 x 2 vol. of ether anda sample (1/100) was radioassayed. The dried extract and an equivalent extract from the perfusate were subjected to the t.1.c. separation described in Scheme 1. All the added marker steroids were completely resolved by this procedure, with the exception of 5a-androstane-3a,l7/lwhich were separated as the trimethylsilyl ethers by diol and 5a-androstane-3~,17a-diol, preparative g.l.c., on a 3 % OV-210 column at 220°C with a carrier gas flow of 4Omllmin. 1975
557th MEETING, LIVERPOOL
679
Table 1. Percentage composition and tissue concentration of testosterone and its metabolites after superfusion Perfusate Tissue Tissue concn. ( %) ( %) (ns/s) Steroid
---
Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2
5a-Androstane-3,17-dione Androst-4-ene-3,17-dione
0.9 0.3
3a-Hydroxy-5a-androstan-17-one 178-Hydroxy-5a-androstan-3-one 38-Hydroxy-5a-androstan-17-one
0.2
2.0 0.1 17/3-Hydroxy-5B-androstan-3-one
