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Division of Endocrinology and Metabolism, Departments of Internal Medicine III (H.V., P.N., W.W.) and Dermatology (H.M.), University of Vienna, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: H. Vierhapper, M.D., Clinical Division of Endocrinology and Metabolism, Department of Internal Medicine III, Währinger Gürtel 18-20, A-1090 Wien, Austria. E-mail: h.vierhapper{at}akh-wien.qc.at
Abstract
Production rates of dihydrotestosterone (DHT) were determined in
healthy men (n = 8), in healthy women during the follicular phase
of their menstrual cycle (n = 7), and in young men with male
pattern baldness (n = 8) using the stable isotope dilution
technique and mass spectrometry. [2,3,4-13C]DHT was
infused for 10 h at doses of 15 µg/h (men) and 2 µg/h (women),
and blood samples were obtained at 20-min intervals during the last
4 h of the observation period. Production rates estimated between
April and June were 2.9 ± 1.1 µg/h (women) and 17.8 ± 6.2
µg/h (men). In men production rates of DHT were similar (16.2 ±
7.7 µg/h) when the investigation was repeated between October and
December. Mean production rates of DHT in young men with male pattern
baldness (60 ± 50 µg/h) were higher than those in healthy men
(P < 0.005). Although this group included two
individuals with normal production rates of DHT, the production rate of
DHT was markedly elevated (range, 32.0–161.0 µg/h) in the remaining
patients. Stable isotope-labeled infusions of DHT are suitable for
clinical use in a routine setting to obtain analytically correct
estimates of DHT production in vivo. In the majority of
men with male pattern baldness endogenous production of DHT is markedly
increased, providing a rationale for therapeutic 5
-reductase
inhibition in this disorder.
GAS CHROMATOGRAPHY/mass spectrometry (GC/MS) and stable isotope-labeled analogs of steroid hormones have been used to determine production rates of T (1) and the conversion of T into its main metabolic products (2, 3). In the present series of experiments we first determined production rates of dihydrotestosterone (DHT) by GC/MS in healthy individuals. Subsequently, we used the same protocol in young men with male pattern baldness, as we hypothesized that these patients might present abnormally high DHT production rates.
Materials and Methods
Experimental protocol
Eight healthy nonobese men (aged 19–35 yr), 7 healthy nonobese women (aged 22–38 yr; in the follicular phase of the menstrual cycle), and 8 men (aged 20–32 yr) with male pattern baldness who had been carefully informed about the aims and the possible risks of the study gave their written consent to participate. Men with male pattern baldness had a normal body mass index (22.1 ± 2.2 kg/m2; range, 19.0–24.5 kg/m2) and presented marked fronto-parietal and/or vertex loss of hair (4), but were otherwise healthy and not taking any medication that could interfere with the study results.
On the day of the experiments an indwelling catheter was inserted at
0800 h into an antecubital vein. Subsequently women received an iv
continuous (Infusomat, Braun-Melsungen, Melsungen, Germany; 40
ml/h, 10 h) infusion of [2,3,4-13C]DHT (25
µg in 500 ml 0.9% saline also containing 2 ml of the individual’s
own blood). Healthy men and men with male pattern baldness were given a
higher dose (250 µg in 500 ml 0.9% saline) of
[2,3,4-13C]DHT together with 250 µg
1,2-D-T (CIL Isotopes, Andover, MA). To correct for
losses by adsorption, samples of the infusate were obtained at the
beginning and end of each infusion from the end of the infusion line.
Actual infusion rates of [2,3,4-13C]DHT
corrected for losses by adsorption were 14.9 ± 1.5 µg/h (men)
and 2.2 ± 0.1 µg/h (women). After an equilibration period of
6 h (at 1400 h) a second indwelling catheter was inserted
into the contralateral arm, and blood samples (5 ml) were obtained at
20-min intervals for 4 h (i.e. until 1800 h).
Blood samples were subsequently pooled for the whole 4-h period. These
pooled samples were used for the determination of SHBG and
androstenedione and for analysis by GC/MS.
The initial experiments in healthy women and men were performed between April and August. The group of healthy men was studied again between October and December.
Materials
All organic solvents were of HPLC grade and purchased from Baker Chemicals (Phillipsburg, NJ). Nonactive T (4-androsten-17ß-ol-3-one) and DHT (4-androstan-17ß-ol-3-one) were obtained from Steraloids, Inc. (Wilton, NH). Radioactive [1,2,6,7-3H]T (SA, 95 Ci/mmol) and radioactive [1,2,4,5,6,7-3H]DHT (SA, 110 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). Stable isotope-labeled 1,2-D-T (isotopic enrichment, 99.0%) was purchased from CIL (Andover, MA). Stable isotope-labeled [2,3,4-13C]DHT (isotopic enrichment, 99.0%) (5, 6) was obtained from Steroko Chemicals (Vienna, Austria).
Sample preparation and analysis by GC-MS
The sample preparation for the GC/MS analysis of T has been described previously (1). For the estimation of DHT, 40,000 dpm [3H]DHT were added to 5.0 ml plasma for determination of recovery. After an incubation period of 10 min (room temperature) samples were diluted (20.0 ml water/0.25% trifluoroacetic acid and applied to a Sep-Pak C18 cartridges (500 mg; Waters/Millipore Corp., Milford, MA) pretreated with successive application of 5.0 ml methanol, 5.0 ml ethyl acetate, 20 ml water, and 5.0 ml trifluoroacetic acid (0.25%, wt/vol). This column was washed three times with 5 ml water. Steroids were eluted by ethyl acetate (3 x 1.0 ml), dried under a stream of nitrogen at 37 C, reconstituted in 100 µl CH2Cl2, and further prepurified by TLC (liquid phase, cyclohexane-ethylacetate, 60:40). The zone containing DHT was eluted (twice, 2.5 ml methanol) and supplemented with 1 ng (women) and 5 ng (men) DHT as an internal standard for GC/MS analysis. The samples were then brought to dryness and derivatized at room temperature (60 min) using a mixture (250 µl) of heptafluorobutyric anhydride/acetone (1:4). Subsequently the samples were diluted in benzene, and recovery was determined. The recovery of [3H]DHT from the derivatized samples was 47 ± 7% (n = 40). Finally, the samples were analyzed by GC/MS (Finnigan MAT95 equipped with a CB5 fused silica column; 25 m, 0.25 mm, 0.12 µm) using S.I.M. mode and electric ionization (resolution: samples of men, 6000; samples of women, 2600). The tracer ions were m/e 486 for (native) DHT, m/e 489 for labeled DHT, and m/e 678 for internal standard DHT. The sensitivity at a peak to noise ratio of 10:1 was less than 100 fg.
Calculation of DHT and T production rates (PRs)
The PRs of DHT and of T were calculated from the product of the known infusion rate (Rt) and the ratio of tracer infusate enrichment (Et) to tracer dilution in the plasma (Es): (PR = Rt x (Et/Es - 1) (7)
RIAs
SHBG concentrations were determined by RIA as reported previously (8), and those of androstenedione were determined radioimmunologically after prepurification by TLC (benzene/acetone, 75:25) using a commercially available method (Diagnostics Systems Laboratories, Inc., Webster, TX). The intra- and interassay coefficients of variation of these methods were less than 10%.
Statistics
Data in the text in the tables are given as the mean ± SD. A t test for matched and unmatched pairs was used for statistical evaluation.
Results
In healthy women plasma SHBG concentrations were 59.6 ± 12.6 nmol/liter. Similar plasma SHBG concentrations were seen in healthy men during the summer (25.6 ± 7.4 nmol/liter) and winter (24.2 ± 5.4 nmol/liter) as well as in patients with male pattern baldness (26.0 ± 7.7 nmol/liter). Plasma androstenedione concentrations were similar in healthy women (177 ± 38 ng/dl), healthy men (summer, 121 ± 25 ng/dl; winter, 153 ± 55 ng/dl), and patients with male pattern baldness (126 ± 42 ng/dl).
The plasma concentrations of unlabeled (native) T and the DHT PRs in
healthy women and men are summarized in Table 1
. In men, PRs of DHT were similar for
the period from April to June (17.8 ± 6.2 µg/h) and from
October to December (16.2 ± 7.7 µg/h). PRs of DHT in healthy
women (determined between April and June) were 2.9 ± 1.1
µg/h.
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contains the individual
production rates of T and DHT and plasma concentrations of T and DHT as
determined by GC/MS of men with male pattern baldness. T PRs were
elevated in three of these individuals compared with a control group of
similar age, but the mean value of 278 ± 105 µg/h was not
different from that of the control group (262 ± 60 µg/h). On
the other hand, elevated production rates of DHT were seen in six of
the eight patients with baldness, whereas two patients had normal DHT
PRs. This resulted in a large variability within the group of patients.
Nevertheless, mean PRs of DHT in patients with male pattern baldness
(60 ± 50 µg/h) were markedly higher (P <
0.005) than those in healthy men (17 ± 7 µg/h). A similar
picture emerged when PRs of T were divided by those of DHT, as in five
patients with male pattern baldness this ratio was below the range
calculated in healthy men (15 ± 7). The increased PRs in these
individuals corresponded to an increase in their MCRs of DHT. No
correlation was seen between the latter and the patient’s body mass
index or with plasma concentrations of either DHT or SHBG.
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Androgens determine the development of the male phenotype. The
importance of the conversion from T into DHT by the enzyme
5-reductase is apparent in males with a congenital deficiency of
5-reductase. These patients are characterized by a rudimentary,
clitoral-like penis, a bifid scrotum, and scarce body hair
(9), whereas their scalp hair is ample and shows no signs
of male-type of baldness. They do not develop prostatic
hyperplasia.
Hair follicles are active sites of the 5-reduction of T to DHT
(10). T is believed to be responsible for follicle
priming, whereas DHT may regulate linear growth in activated follicles
(11). Men with male pattern baldness present an enhanced
local activity of 5-reductase in their hair follicles
(11). The role of 5-reductase in the development of
prostatic hyperplasia has led to the development of drugs
(12) that improve this disorder by inhibiting the activity
of 5-reductase (13). More recently, these drugs have
also been used to treat male pattern baldness (13, 14, 15).
Scalp DHT concentrations are elevated in these patients
(15), but this information is not routinely available.
Hence, therapy with 5-reductase inhibitors is instituted without the
benefit of biochemical parameters that might help to preselect
potential responders.
Before evaluating patients with male pattern baldness, it was necessary first to establish the normal range of DHT PRs in healthy individuals. In the past this has been done by several investigators using the radiotracer technique (16, 17, 18, 19). DHT PRs have been reported (19) to be 674 µg/d (28 µg/h in men) and 55 µg/d (2.3 µg/h in women). Our own results in women confirm these data, whereas the estimated mean values in men were somewhat lower. However, our investigation was performed only during a 4-h period (from 1400–1800 h, after a 6-h equilibration period) and should not be extrapolated to calculate daily PRs. It was not our intention to study daily PRs of DHT and/or their potential circadian rhythm. We therefore cannot exclude that the PRs of DHT might vary throughout a 24-h period. Similar to our previous investigations with T (1, 20) and cortisol (21, 22, 23), we deliberately chose the present protocol because it can be done on a routine, out-patient basis. In regard to the use of stable isotope-labeled tracers, their advantages compared with radioactive materials include the possibility of long-term infusions to achieve steady state conditions, the avoidance of incomplete recovery of the tracer and the tracee from biological materials, and the fact that both labeled and endogenous materials are simultaneously analyzed using an identical technology. The infused amount of stable isotope-labeled DHT (men, 15 µg/h; women, 2.0 µg/h) is on the same order as the calculated endogenous production of DHT. However, as even the administration of 500 µg DHT/d failed to suppress the secretion of gonadotropins (24), it is unlikely that the amount of DHT infused influenced its endogenous PR. Furthermore, our data do not suggest seasonal variability as a potential bias when comparing the data obtained in patients with those in healthy controls.
In summary, a large subgroup, possibly the majority, of male patients
with male pattern baldness have increased PRs of DHT. The number of
investigated patients was small. To determine the actual share of
individuals with increased DHT PRs among patients with male pattern
baldness a larger group of patients must be studied. The determination
of DHT PRs may then prove to be a useful tool to select patients for
therapy with 5-reductase inhibitors.
Acknowledgments
The technical assistance of Ms. A. Fürst, Ms. A. Hofer, and Ms. E. Nowotny is gratefully acknowledged.
Footnotes
Abbreviations: DHT, Dihydrotestosterone; GC/MS, gas chromatography/mass spectrometry; PR, production rate.
Received April 19, 2001.
Accepted August 31, 2001.
References
-androstandediol
during/after i.v. administration of 13C-labelled
testosterone in man. J Steroid Biochem 29:105–109[CrossRef][Medline]
-reductase 2 deficiency. Endocr Rev 14:577–593[CrossRef][Medline]
-reductase inihitor, on circulating androgens in male
volunteers. J Clin Endocrinol Metab 70:1136–1141[Abstract]
-reductase inhibitor, on scalp skin testosterone and
dihydrotestosterone concentrations in patients with male pattern
baldness. J Clin Endocrinol Metab 79:703–706[Abstract]
-androstanediols. J Clin Invest 50:1338–1344
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