Laboratory, Urological Unit, Royal Postgraduate Medical School,
Prostate Research
Du Cane Road, London, W12 OHS
SERUM 5\g=a\-DIHYDROTESTOSTERONE AND TESTOSTERONE CHANGES WITH AGE IN MAN
By
J. G. Lewis, R. Ghanadian
and G. D. Chisholm
ABSTRACT Serum 5\g=a\-dihydrotestosterone (DHT) and testosterone (T) were measured in 98 normal adult men aged between 20\p=n-\80 years, separating T and DHT by thin layer chromatography and using a sensitive and reliable radioimmunoassay. In three age groups between 20\p=n-\40, 40\p=n-\60 and 60\p=n-\80, the means \m=+-\ sem for serum DHT were 84 \m=+-\ 4, 79 \m=+-\ 3 and 67 \m=+-\ 3 (ng/ 100 ml) respectively. The corresponding values for testosterone were 559 \m=+-\25, 491 \m=+-\25 and 475 \m=+-\28 (ng/100 ml). A significant decrease was observed in the DHT level of the 60\p=n-\80 years age group compared to either the 20\p=n-\40 (P < 0.01) or the 40\p=n-\60 (P < 0.02) age groups. There was a moderate decline in the testosterone level of the 60\p=n-\80 years age group compared to 20\p=n-\40 years (P < 0.05) but there were no significant changes in the testosterone levels between other groups.
Much attention has been focused on the changes with age of androgens in man. Some reports have suggested that there is no decline of total uncorjjugated testosterone until the 9th decade (Kent 8c Acone 1966; Frick 8c Kind 1969) whereas others have indicated a moderate decline with age (Kirschner 8c Coffman 1968; Vermeulen et al. 1972; Pirke 8c Doerr 1973; Sterns et al. 1974). A more significant decline has been demonstrated in the apparent free testo¬ sterone concentration (AFTC) after the age of 50 (Sterns et al. 1974; Vermeu¬ len et al. 1971) and this seems to represent a more important parameter in assessing the active testosterone level in plasma. The changes of other andro¬ gens particularly 5a-dihydrotestosterone (DHT), the most potent endogenous androgen, remains uncertain. The present study was designed to measure both 444
5a-dihydrotestosterone and testosterone in a large population, using a reliable radioimmunoassay (Ghanadian et al. 1975e) which allows the measurement of both 5«-dihydrotestosterone and testosterone in the same serum sample.
MATERIALS AND METHODS
Ninety-eight subjects were
normal men, aged between 20-80 years were studied. All of these in good health and there were no relevant general medical or urological symptoms or signs. Peripheral venous blood was drawn between 10—12 a.m. For each decade between 18-20 samples were collected exept for the group over 70 where only 4 cases fulfilled our normal criteria. The serum was separated and stored at -20° C until required for assay. Radioimmunoassay of serum samples was performed by the method described by Ghanadian et al. (1975b). Serum samples (1.0 ml), blanks and known pools were equilibrated with 1000 cpm of each steroid, to monitor procedural losses, prior to ether extraction and thin layer chromatography. After chromatography the T and DHT sample zones were eluted and aliquots taken for duplicate assay. The dried assay aliquots and 8 duplicates of T and DHT (10-400 pg) standards were equilibrated with antisera prior to the addition of tritiated testosterone for an overnight incubation at 4° C. Dextran coated charcoal was used to separate the bound from free steroid and after centrifugation the resulting supernatant was transferred to 10 ml of scintillation fluid and the radioactivity counted in a scintillation spectrometer. The amount of T and DHT in aliquots of the samples was interpolated on their respective standard curves and expressed as ng/100 ml serum after correction for assay aliquots and recovery. The within-assay variance evaluated by 6 duplicate determinations on human pooled serum gave coefficients of variation of 11.6%) and 6.5 %> for T and DHT respectively. The between-assay variation for 8 determinations was found to be 9.2 %> and 6.5 °/o for T and DHT. The mean recovery of T and DHT was 62 + 1 %>. The sensitivity of the RIA method was 5 and 10 pg for T and DHT, respectively. When increasing known amounts of both T and DHT were added to 1 ml of pooled female serum and 1 ml of buffer, the T and DHT curves were found to be within the coefficient of variation as determined by precision experiments. Blanks were always undetectable and if known pools of human serum read more than 2 standard deviations from the mean as determined by precision experiments the assay was repeated. The assay was further monitored by performing the measurement without chromatography.
RESULTS
The results of serum 5«-dihydrotestosterone and testosterone for each indi¬ vidual were plotted against age. These showed a wide variation from one individual to another in the same age group (Figs. 1 and 2). When the whole population was grouped into three age periods 20-40, 40-60 and 60-80 the mean ± sem for 5a-dihydrotestosterone were 84 ± 4, 79 ± 3 and 67 ± 3 respectively. Statistical analysis of the results (paired /-test) de445
140 120
100
80 .
• '
•
•
.
•.
60
•
•
•
•
•»I
•
m
•
40
20
t-r
10
0
Serum
1000
20
30
5a-dihydrotestosterone
40
~60
50
Age (years) Fig- L in normal
men
aged
70-
80
between 20-80 years.
i
800
600 ••
1
.S
400
200
10
20
30
i
i
40
50
-1-r-
60
70
Age (years) Serum
Fig. testosterone in normal
2.
men
446
aged
between 20-80 years.
80
90
a significant difference between 20—40 and 60-80 age group and between the 40-60 and 60-80 age groups (P < 0.02). (P P > 0.05). The mean of T : DHT ratio for the whole population was 6.7 ± 0.1. The mean + sem for unchromatographed measurement (T plus DHT) for the whole population was 568 ± 15. When T and DHT were separated by chro¬ matography the mean ± sem for T and DHT was 512 ± 16 and 78 ± 2 respectively. Since the antisera cross-reacted with DHT to the extent of 60 °/o, then only 60 °/o of the DHT will be measured when the serum sample is not chromatographed. Considering the coefficient variation of the method this indicates a good agreement in the results with and without chromatography. The results also suggested a good correlation between the circulating level of dihydrotestosterone and testosterone (r 0.747).
monstrated
=
DISCUSSION
Neither the normal circulating level, nor the change with age has been estab¬ lished for 5a-dihydrotestosterone. The reported values for the circulating level of this steroid differ significantly (Barbería 8c Thorneycroft 1974; Coyolupa et al. 1972; Baiiminger et al. 1974; Pazzagli et al. 1974; Mahoudeau et al. 1974). These differences may be due to the wide range of age together with the limited population in these studies. 5u-dihydrotestosterone has been found to be the most potent endogenous androgen and has been reported to be involved in pathological conditions of the prostate (Siiteri 8c Wilson 1970; Ghanadian et al. 1975a). The low level of this steroid in peripheral blood, required a highly sensitive and reliable technique for its measurement. These criteria have been furnished in the described method and have enabled us to measure even the much lower concentration of dihydrotestosterone in rat peripheral blood (Ghanadian et al. 1975e). The results suggest that although there is a wide variation in the concentration of 5a-DHT in the individuals of each group, a decline in the level of dihydrotestosterone with age was ap¬ parent. This decline was significant when the 20-40 or 40-60 age groups were compared to 60-80 years. The decrease in the concentration of circulating dihydrotestosterone with age is in accordance with the findings of Vermeulen et al. (1972) who showed a relative increase of 5ß over 5a metabolites of testosterone with age. 447
The overall mean value for circulating testosterone reported in this paper is in agreement with the well established value for this hormone. The finding that testosterone moderately declines, particularly after 60 years of age, has been reported by other investigators (Vermeiden et al. 1972; Pirke 8c Doerr 1973; Sterns et al. 1974) who have also studied large populations. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) are now known to increase with age in normal men (Sterns et al. 1974; Isurugi et al. 1974) and this correlates with the observation of a decrease in testosterone with age. Since this work was submitted, Pirke 8c Doerr (1975) reported no change in plasma dihydrotestosterone with age, but a fall by 20.6 °/o of testosterone in a limited study of 45 normal subjects.
ACKNOWLEDGMENT We wish to thank Dr. A. Bailey and Miss S. Waller of the Medical Centre, Research for providing us with the serum samples.
Department (B.U.P.A.)
REFERENCES
J. M. &• Thorneycroft I. H.: Steroids 23 (1974) 757. Bauminger S., Kohen F., Lindner H. R. Se Weinstein A.: Steroids 24 (1974) 477. Coyotupa J'., Parlow A. F. 8e Abraham G. E.: Analyt. Letters 5(6) (1972) 329. Frick J. Se Kind F. A.: Steroids 13 (1969) 495. Ghanadian R., Chisholm G. D. Se Ansel! I. D.: J. Endocr. 65 (1975a) 253. Ghanadian R., Lewis J. G. Se Chisholm G. D.: Steroids 25 (19756) 753. Isurugi K., Fukutani K., Takayasu H., Wakabayashi K. Se Tamaoki B.: J. clin. Endocr. 39 (1974) 955. Kent J. R. Se Acone A. B.: Excerpta med. (Amst.) Intern. Congr. Ser. 101 (1966) 31. Kirschner M. A. Se Coffman G. R.: J. clin. Endocr. 28 (1968) 1347. Mahoudeau J. A., Delassalle A. 8c Bricaire H.: Acta endocr. (Kbh.) 77 (1974) 401. Pazzagli M., Borrelli D., Forti G. Se Serio M.: Acta endocr. (Kbh.) 76 (1974) 388. Pirke K. M. 8c Doerr P.: Acta endocr. (Kbh.) 74 (1973) 792. Pirke K. M. Se Doerr P.: Acta endocr. (Kbh.) 79 (1975) 357. Siiteri P. K. Se Wilson J. D.: J. clin. Invest. 49 (1970) 1737. Stems E. L., MacDonnell J. A., Kauffman B. J., PaduaR., Lucman T. S., Winter J. S. D. Se Faiman C. A.: Amer J. Med. 57 (1974) 761. Vermeulen A., Rubens R. Se Verdonck L.: J. clin. Endocr. 34 (1972) 730. Vermeulen A., Stoica T. Se Verdonck L.: J. clin. Endocr. 33 (1971) 759. Barberia
Received
on
July 1st,
1975.
448
Prostate Research
Du Cane Road, London, W12 OHS
SERUM 5\g=a\-DIHYDROTESTOSTERONE AND TESTOSTERONE CHANGES WITH AGE IN MAN
By
J. G. Lewis, R. Ghanadian
and G. D. Chisholm
ABSTRACT Serum 5\g=a\-dihydrotestosterone (DHT) and testosterone (T) were measured in 98 normal adult men aged between 20\p=n-\80 years, separating T and DHT by thin layer chromatography and using a sensitive and reliable radioimmunoassay. In three age groups between 20\p=n-\40, 40\p=n-\60 and 60\p=n-\80, the means \m=+-\ sem for serum DHT were 84 \m=+-\ 4, 79 \m=+-\ 3 and 67 \m=+-\ 3 (ng/ 100 ml) respectively. The corresponding values for testosterone were 559 \m=+-\25, 491 \m=+-\25 and 475 \m=+-\28 (ng/100 ml). A significant decrease was observed in the DHT level of the 60\p=n-\80 years age group compared to either the 20\p=n-\40 (P < 0.01) or the 40\p=n-\60 (P < 0.02) age groups. There was a moderate decline in the testosterone level of the 60\p=n-\80 years age group compared to 20\p=n-\40 years (P < 0.05) but there were no significant changes in the testosterone levels between other groups.
Much attention has been focused on the changes with age of androgens in man. Some reports have suggested that there is no decline of total uncorjjugated testosterone until the 9th decade (Kent 8c Acone 1966; Frick 8c Kind 1969) whereas others have indicated a moderate decline with age (Kirschner 8c Coffman 1968; Vermeulen et al. 1972; Pirke 8c Doerr 1973; Sterns et al. 1974). A more significant decline has been demonstrated in the apparent free testo¬ sterone concentration (AFTC) after the age of 50 (Sterns et al. 1974; Vermeu¬ len et al. 1971) and this seems to represent a more important parameter in assessing the active testosterone level in plasma. The changes of other andro¬ gens particularly 5a-dihydrotestosterone (DHT), the most potent endogenous androgen, remains uncertain. The present study was designed to measure both 444
5a-dihydrotestosterone and testosterone in a large population, using a reliable radioimmunoassay (Ghanadian et al. 1975e) which allows the measurement of both 5«-dihydrotestosterone and testosterone in the same serum sample.
MATERIALS AND METHODS
Ninety-eight subjects were
normal men, aged between 20-80 years were studied. All of these in good health and there were no relevant general medical or urological symptoms or signs. Peripheral venous blood was drawn between 10—12 a.m. For each decade between 18-20 samples were collected exept for the group over 70 where only 4 cases fulfilled our normal criteria. The serum was separated and stored at -20° C until required for assay. Radioimmunoassay of serum samples was performed by the method described by Ghanadian et al. (1975b). Serum samples (1.0 ml), blanks and known pools were equilibrated with 1000 cpm of each steroid, to monitor procedural losses, prior to ether extraction and thin layer chromatography. After chromatography the T and DHT sample zones were eluted and aliquots taken for duplicate assay. The dried assay aliquots and 8 duplicates of T and DHT (10-400 pg) standards were equilibrated with antisera prior to the addition of tritiated testosterone for an overnight incubation at 4° C. Dextran coated charcoal was used to separate the bound from free steroid and after centrifugation the resulting supernatant was transferred to 10 ml of scintillation fluid and the radioactivity counted in a scintillation spectrometer. The amount of T and DHT in aliquots of the samples was interpolated on their respective standard curves and expressed as ng/100 ml serum after correction for assay aliquots and recovery. The within-assay variance evaluated by 6 duplicate determinations on human pooled serum gave coefficients of variation of 11.6%) and 6.5 %> for T and DHT respectively. The between-assay variation for 8 determinations was found to be 9.2 %> and 6.5 °/o for T and DHT. The mean recovery of T and DHT was 62 + 1 %>. The sensitivity of the RIA method was 5 and 10 pg for T and DHT, respectively. When increasing known amounts of both T and DHT were added to 1 ml of pooled female serum and 1 ml of buffer, the T and DHT curves were found to be within the coefficient of variation as determined by precision experiments. Blanks were always undetectable and if known pools of human serum read more than 2 standard deviations from the mean as determined by precision experiments the assay was repeated. The assay was further monitored by performing the measurement without chromatography.
RESULTS
The results of serum 5«-dihydrotestosterone and testosterone for each indi¬ vidual were plotted against age. These showed a wide variation from one individual to another in the same age group (Figs. 1 and 2). When the whole population was grouped into three age periods 20-40, 40-60 and 60-80 the mean ± sem for 5a-dihydrotestosterone were 84 ± 4, 79 ± 3 and 67 ± 3 respectively. Statistical analysis of the results (paired /-test) de445
140 120
100
80 .
• '
•
•
.
•.
60
•
•
•
•
•»I
•
m
•
40
20
t-r
10
0
Serum
1000
20
30
5a-dihydrotestosterone
40
~60
50
Age (years) Fig- L in normal
men
aged
70-
80
between 20-80 years.
i
800
600 ••
1
.S
400
200
10
20
30
i
i
40
50
-1-r-
60
70
Age (years) Serum
Fig. testosterone in normal
2.
men
446
aged
between 20-80 years.
80
90
a significant difference between 20—40 and 60-80 age group and between the 40-60 and 60-80 age groups (P < 0.02). (P P > 0.05). The mean of T : DHT ratio for the whole population was 6.7 ± 0.1. The mean + sem for unchromatographed measurement (T plus DHT) for the whole population was 568 ± 15. When T and DHT were separated by chro¬ matography the mean ± sem for T and DHT was 512 ± 16 and 78 ± 2 respectively. Since the antisera cross-reacted with DHT to the extent of 60 °/o, then only 60 °/o of the DHT will be measured when the serum sample is not chromatographed. Considering the coefficient variation of the method this indicates a good agreement in the results with and without chromatography. The results also suggested a good correlation between the circulating level of dihydrotestosterone and testosterone (r 0.747).
monstrated
=
DISCUSSION
Neither the normal circulating level, nor the change with age has been estab¬ lished for 5a-dihydrotestosterone. The reported values for the circulating level of this steroid differ significantly (Barbería 8c Thorneycroft 1974; Coyolupa et al. 1972; Baiiminger et al. 1974; Pazzagli et al. 1974; Mahoudeau et al. 1974). These differences may be due to the wide range of age together with the limited population in these studies. 5u-dihydrotestosterone has been found to be the most potent endogenous androgen and has been reported to be involved in pathological conditions of the prostate (Siiteri 8c Wilson 1970; Ghanadian et al. 1975a). The low level of this steroid in peripheral blood, required a highly sensitive and reliable technique for its measurement. These criteria have been furnished in the described method and have enabled us to measure even the much lower concentration of dihydrotestosterone in rat peripheral blood (Ghanadian et al. 1975e). The results suggest that although there is a wide variation in the concentration of 5a-DHT in the individuals of each group, a decline in the level of dihydrotestosterone with age was ap¬ parent. This decline was significant when the 20-40 or 40-60 age groups were compared to 60-80 years. The decrease in the concentration of circulating dihydrotestosterone with age is in accordance with the findings of Vermeulen et al. (1972) who showed a relative increase of 5ß over 5a metabolites of testosterone with age. 447
The overall mean value for circulating testosterone reported in this paper is in agreement with the well established value for this hormone. The finding that testosterone moderately declines, particularly after 60 years of age, has been reported by other investigators (Vermeiden et al. 1972; Pirke 8c Doerr 1973; Sterns et al. 1974) who have also studied large populations. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) are now known to increase with age in normal men (Sterns et al. 1974; Isurugi et al. 1974) and this correlates with the observation of a decrease in testosterone with age. Since this work was submitted, Pirke 8c Doerr (1975) reported no change in plasma dihydrotestosterone with age, but a fall by 20.6 °/o of testosterone in a limited study of 45 normal subjects.
ACKNOWLEDGMENT We wish to thank Dr. A. Bailey and Miss S. Waller of the Medical Centre, Research for providing us with the serum samples.
Department (B.U.P.A.)
REFERENCES
J. M. &• Thorneycroft I. H.: Steroids 23 (1974) 757. Bauminger S., Kohen F., Lindner H. R. Se Weinstein A.: Steroids 24 (1974) 477. Coyotupa J'., Parlow A. F. 8e Abraham G. E.: Analyt. Letters 5(6) (1972) 329. Frick J. Se Kind F. A.: Steroids 13 (1969) 495. Ghanadian R., Chisholm G. D. Se Ansel! I. D.: J. Endocr. 65 (1975a) 253. Ghanadian R., Lewis J. G. Se Chisholm G. D.: Steroids 25 (19756) 753. Isurugi K., Fukutani K., Takayasu H., Wakabayashi K. Se Tamaoki B.: J. clin. Endocr. 39 (1974) 955. Kent J. R. Se Acone A. B.: Excerpta med. (Amst.) Intern. Congr. Ser. 101 (1966) 31. Kirschner M. A. Se Coffman G. R.: J. clin. Endocr. 28 (1968) 1347. Mahoudeau J. A., Delassalle A. 8c Bricaire H.: Acta endocr. (Kbh.) 77 (1974) 401. Pazzagli M., Borrelli D., Forti G. Se Serio M.: Acta endocr. (Kbh.) 76 (1974) 388. Pirke K. M. 8c Doerr P.: Acta endocr. (Kbh.) 74 (1973) 792. Pirke K. M. Se Doerr P.: Acta endocr. (Kbh.) 79 (1975) 357. Siiteri P. K. Se Wilson J. D.: J. clin. Invest. 49 (1970) 1737. Stems E. L., MacDonnell J. A., Kauffman B. J., PaduaR., Lucman T. S., Winter J. S. D. Se Faiman C. A.: Amer J. Med. 57 (1974) 761. Vermeulen A., Rubens R. Se Verdonck L.: J. clin. Endocr. 34 (1972) 730. Vermeulen A., Stoica T. Se Verdonck L.: J. clin. Endocr. 33 (1971) 759. Barberia
Received
on
July 1st,
1975.
448
