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Testosterone Metabolic Clearance and Production Rates Determined by Stable Isotope Dilution/Tandem Mass Spectrometry in Normal Men: Influence of Ethnicity and Age

Division of Endocrinology, Department of Medicine (C.W., A.L., G.L., L.H., R.S.S.), Harbor-University of California Los Angeles (UCLA) Medical Center and Research and Education Institute, Torrance, California 90509; and Olympic Analytical Laboratory, Department of Molecular and Medical Pharmacology (D.H.C., B.S., E.D.), UCLA, Los Angeles, California 90025

Address all correspondence and requests for reprints to: Christina Wang, M.D., General Clinical Research Center, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, California 90509. E-mail: wang{at}gcrc.rei.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The metabolic clearance rate (MCR_T) and production rate (PR_T) of testosterone (T) were measured using constant infusion of trideuterated (d₃) T and quantitating serum d₃T by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Serum unlabeled T (d₀T) was measured by LC-MS-MS, and serum total T (d₃T + d₀T) was measured by RIA. Mean MCR_T (measured by LC-MS-MS) in young white men (1272 ± 168 liters/d) was not significantly different from young Asian men (1070 ± 166 liters/d). Mean PR_T was also not significantly different between the two ethnic groups (whites, 9.11 ± 1.11 mg/d; Asians, 7.22 ± 1.15 mg/d; P = 0.19 using d₀T data). Both the mean MCR_T (812 ± 64 liters/d; P < 0.01) and the PR_T (3.88 ± 0.27 mg/d; P < 0.001) were significantly lower in middle-aged white men when compared with their younger counterparts. The mean MCR_T and PR_T calculated using serum total T or d₀T data showed a diurnal variation, with levels at midday significantly higher than those measured in the evening in the young (MCR_T, P < 0.01; PR_T, P < 0.001) and to a lesser extent in the older men (MCR_T, P < 0.05; PR_T, P < 0.05 using total T and P < 0.001 using d₀T data). We conclude that using LC-MS-MS to detect d₃T in serum after constant infusion of stable isotope-labeled T allows the measurements of MCR_T and PR_T, which can be used to study androgen metabolism repeatedly after physiological or pharmacological interventions.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
RECENT STUDIES HAVE used liquid chromatography (LC)- or gas chromatography (GC)-mass spectrometry (MS) and stable isotope-labeled steroids to determine the concentration, metabolic clearance rates (MCRs), and production rates (PRs) of a number of steroid hormones (1, 2, 3, 4, 5, 6, 7). Using LC-MS analyses and constant infusion of trideuterated (d₃) cortisol, Esteban et al. (8) determined that the daily PR of cortisol (average, 10 mg/d) was lower than previously reported using isotopic methods. This is physiologically and clinically important because it implies that the standard replacement doses of cortisol (20 mg in the morning and 10 mg in the evening) may be over the physiological range. Although other factors, including first-pass clearance effects, must be considered in deciding on optimal replacement of cortisol, an overestimation of cortisol PR may explain why replacement doses of cortisol are commonly associated with signs and symptoms of glucocorticoid excess. Esteban et al. (8) also demonstrated that the PR of cortisol showed a diurnal variation and was lowest between 2000 and 0400 h. The PR_T was also determined in healthy men and women using GC-MS on samples collected during constant infusion of dideuterated T. In men, the PR_T averaged 3.7 ± 2.2 mg/d and in women 0.04 ± 0.01 mg/d (9). There was no diurnal rhythm of PR_T, and the reported PR_T in men using stable isotope dilution was lower than that reported previously using radioactive isotopes (10, 11). The reason for the lower reported PR_T was not clear. In a subsequent report studying the production rate of dihydrotestosterone in men, Vierhapper et al. (12) reported a mean PR_T of 6.29 mg/d in eight nonobese healthy men using GC-MS. In this report, we determined the PR_T and MCR_T using a constant infusion for 12 h of trideuterated T (d₃T) in healthy young men from different ethnic groups and in middle-aged men. Labeled T (d₃T) was measured by a sensitive and specific assay using LC-tandem MS (LC-MS-MS). Serum total T (d₃T + d₀T) was measured by RIA and unlabeled T (d₀T) by LC-MS-MS.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Nine young Asian and 11 young and 18 middle-aged white, healthy volunteers were recruited into the study after screening by medical and social history and physical examination. They had normal blood counts, urinalysis, and serum biochemistry, as well as normal baseline serum LH, FSH, and T levels. The young Asian and white subjects were recruited for a study to determine the differences in responsiveness of serum LH and FSH to graded T infusions. The middle-aged men were recruited for a study to determine the effects of altering dietary fat on androgen metabolism. All subjects were studied at basal condition before any intervention. To reduce the ethnic variability of the subjects, we selected subjects whose parents and grandparents were from the same ethnic group. Asians were recruited from subjects of Chinese, Korean, or Japanese descent, and the white men were recruited from families of European descent. Of the Asian subjects, three were born in Asia, whereas the remaining six were first-generation Asian-Americans. All of the white subjects were born in the United States. The mean age of the young Asians was 27 ± 1.8 yr (mean ± SE), which was not much different from that of the young whites (33 ± 2.5 yr). The Asians were shorter in height (171.7 ± 2.1 cm) than the whites (180.7 ± 2.4 cm; P < 0.05), but their weight was not significantly different (Asians, 71.4 ± 3.7 kg; whites, 80.3 ± 4.6 kg). The mean combined testes volume was significantly lower in Asians (39.8 ± 2.6 ml), compared with the whites (47.0 ± 1.9 ml; P < 0.05). The mean age of the middle-aged white men was 55 ± 0.9 yr, and their height (176 ± 13.5 cm) and weight (84.5 ± 3.6 kg) were not significantly different from their younger counterparts. The studies were approved by the Institutional Review Board of the Harbor-UCLA Medical Center including the use of d₃T in human subjects, and all subjects gave written informed consent.

Preparation of d₃T infusion

d₃T (16,16,17-²H₃T) was obtained from Cambridge Isotope Laboratories (Andover, MA); 13.7 mg of d₃T was dissolved in 6.86 ml ethanol and then diluted with 1363 ml normal saline to make a stock solution (10 µg/ml) by the institutional research pharmacist using aseptic techniques. This stock solution was aliquoted into 30-ml sterile vials and then tested for sterility and pyrogenicity. The volume of d₃T was calculated based on the dose required for infusion to achieve approximately 10% enrichment of d₃T and on body surface area of subjects, and it was determined for each subject for each 12-h period. It should be noted that although this amount of d₃T was not anticipated to suppress the hypothalamic-pituitary axis in normal men, the amount of labeled T infused might have to be reduced in studies in hypogonadal men. Ten percent of the total dose was injected as an iv bolus. The remaining dose was diluted with saline for the 12-h infusion. The appropriate volume of d₃T was added to 250 ml of normal saline after the same volume of saline was removed from the infusion bag for constant infusion. The infusion solution was used to purge the infusion tubings and allowed to stand for at least 30 min before infusion. The solution was infused over 12 h by an infusion pump (Flo-Gard-200, Baxter, Deerfield, IL) at approximately 20 ml/h. The rate of the infusion was checked by weighing the infusion bag and the tubing before and immediately after the completion of each infusion. Aliquots of the infusate were collected at the end of the infusion from the infusion tubing to correct for losses by adsorption to the infusion bag and the tubing and for the determination of d₃T concentrations in the infusate used for the calculation of the amount of d₃T infused. About 40% of the d₃T was adsorbed to the bag and tubing. Because the infusates were collected from the end-infusion tubing, this relatively high adsorption to the bag would not affect the results. The concentrations of the d₃T in the infusates were measured by RIA as total T or by LC-MS-MS as d₃T. The average d₃T concentration in the infusates was 2.2 ± 0.09 µmol/liter (637 ± 26 ng/ml; 12.7 µg/h), which gave an average of 0.15 mg of T infused in 12 h and was less than 2.5% of the estimated daily T production in a healthy adult male.

Study design

Each subject was admitted to the General Clinical Research Center at Harbor-University of California Los Angeles Medical Center on the evening before the start of the infusion study. The subjects remained recumbent overnight and throughout the infusion period. At 0800 h the next day, the subjects were administered the bolus dose of d₃T, and the 12-h infusion was started immediately thereafter. Blood samples (25 ml) were drawn in serum collection tubes before and then at 3, 4, 5, and 12 h from the arm not receiving the infusion. Serum was obtained, and individual samples were saved at –20 C for total T (by RIA), d₀T (by LC-MS-MS), and d₃T (labeled with stable isotope by LC-MS-MS) determinations. All samples from any one subject were analyzed in the same assay.

Serum T assay by RIA

Serum T levels were determined as previously described by specific RIA (13, 14) using reagents obtained from ICN Pharmaceuticals (Costa Mesa, CA). The serum was extracted by ethyl-acetate and hexane before assay. The cross-reactivities of the antiserum used in the T RIA were 2.0% for dihydrotestosterone, 2.3% for androstenedione, 0.8% for 3 -5 androstanediol, 0.6% for etiocholanolone, and less than 0.01% for all other steroids tested. The lower limit of quantitation of serum T measured by this assay is 0.87 nmol/liter (0.25 ng/ml). This is the lowest concentration of T measured in serum that can be accurately distinguished from steroid free serum with 12% coefficient of variation (CV). The accuracy (recovery) of the T assay, determined by spiking steroid free serum with 0.87, 1.73, 3.47, 11, 34.7, and 52 nmol/liter of T, was 114, 118, 109, 94, 92, and 92%, respectively (mean, 104%). The within-assay precision (CV) at a serum T concentration of 22.4 nmol/liter was 5.9%. The between-assay precision (CV) for low, medium, and high serum T concentrations of 4.7, 18.4, and 51.2 nmol/liter was 12.4, 9.3, and 12.5%, respectively. The normal adult male range in this laboratory was 10.33 to 36.17 nmol/liter (2.98–10.43 ng/ml). Any duplicate counts showing more than 10% CV were repeated. This assay measured both d₀T and d₃T after the d₃T infusion. This RIA was also used to estimate the concentration of d₃T in the infusate because the mass of the T was of sufficient quantity to be measured by the RIA for MCR calculated using RIA values.

Serum d₀T and d₃T measurements by LC-MS-MS

We developed a stable isotope method designed to quantitate d₀T and d₃T in serum with a low limit of detection suitable for the estimation of MCR_T and PR_T after constant infusion of d₃T (15). The advantages of the LC-MS-MS approach include simplified sample preparation (underivatized steroids can be analyzed directly), high recovery, improved signal-to-noise ratio, and less difficulty with interferences due to MS-MS technology (16). An LC-10A binary pump LC (Shimadzu Scientific Instruments, Columbia, MD) equipped with a Series 200 autosampler (Applied Biosystems, Foster City, CA) and coupled to a Sciex API 300 triple quadrupole mass spectrometer (Applied Biosystems-Sciex, Thornhill, Ontario, Canada), and equipped with an APCI interface, was used to perform the analysis. The LC-MS-MS was operated in the positive ion mode. After adding internal standard (19-nortestosterone, 50 µl 0.2 ng/µl), 2 ml sodium acetate buffer, and 5 ml diethyl ether to a 2-ml serum aliquot, the mixture was shaken and centrifuged for 10 min, and the ether layer was transferred to a clean tube and dried. The extract was reconstituted in 100 µl methanol, and 15 µl was injected into the LC-MS-MS. The column was a 3-µm Hypersil BDS C18 (Keystone, Bellefonte, PA) (150 x 2.1 mm). Gradient elution was used at room temperature. Solvent A was 0.1% formic acid, and solvent B was methanol. The gradient program began with 50% B for 0.5 min, ramped to 90% B at 9 min, returned to 50% of B in 1 min, and was held for 6 min. The flow rate was 0.2 ml/min. The LC-MS-MS was operated in the positive ion mode using a corona charge current of 2 mA. Gas and energy were nitrogen and 28 eV, respectively. The temperature of the heated nebulizer was 350 C, and the protonated molecular ions [M-H⁺] were used as parent ions. d₀T, d₃T, and the internal standard were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0–20 ng/ml for d₀T and 0–2.0 ng/ml for d₃T. The lower limits of quantitation for d₀T and d₃T were 0.5 and 0.05 ng/ml. The 10 times lower limit of detection for d₃T is explained by significantly less interference for the m/z 292 to m/z 97 transition compared with the m/z 289 to m/z 97 transition of d₀T. The absence of interferences in serum for m/z 292 to m/z 97 transition gave significantly better signal-to-noise ratio for d₃T peak transition of d₀T. The absence of interferences in serum for m/z 292 to m/z 97 transition gave significantly better signal-to-noise ratio for d₃T peak. The recoveries for d₀T and d₃T were 91.5 and 96.4%. For d₀T at 1.25 and 4.0 ng/ml, the intraday precision was 3.9 and 4.3%; the intraday accuracy was 0.01 and 4.5%, respectively. The interday precision at these levels was 5.3 and 5.4%, and the interday accuracy was 1.9 and 0.3%. For d₃T at 0.125 and 0.4 ng/ml, the intraday precision was 2.8 and 8.3%, and the intraday accuracy was 1.8 and 5.6%. The interday precision at these levels was 10.0 and 7.6%, and the interday accuracy was 5.7 and 3.4%. The concentrations of d₀T in the 38 healthy subjects ranged from 8.6 to 48.6 nmol/liter (2.5–14.0 ng/ml) with a mean of 21.5 nmol/liter (6.2 ng/ml). The details of the LC-MS-MS for d₀T and d₃T are described elsewhere (15).

The serum d₀T and d₃T concentrations measured in a representative subject before and during the 12-h d₃T infusion are shown in Fig. 1. The infusion rate and the concentration of d₃T in the infusates collected at the end of the infusion were used to calculate the amount of T infused per hour. MCR_T was calculated by the formula: MCR_T = amount of d₃T infused per hour (measured as total T by RIA and d₃T by LC-MS-MS)/ concentration of d₃T (measured by LC-MS-MS) in the serum and multiplied by 24 h to express as liters per day (per body surface area as liters per day per meter²). PR_T was then calculated from the formula: PR_T = MCR_T x serum T (measured as total T by RIA and d₀T by LC-MS-MS) concentration, expressed in milligrams per day and milligrams per day per meter². The average serum d₃T and d₀T or T concentrations at 4 and 5 h after the start of the infusion were used to calculate the MCR_T and PR_T during the day, and the d₃T and d₀T or T levels at 12 h after infusion were used to calculate the evening MCR_T and PR_T, respectively.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Serum d0T (solid line) and d3T (dotted line) levels during d3T infusion in a subject. Note the achievement of steady concentrations of serum d3T at 4 h after the start of infusion in this subject.

Because serum T and MCR_T values are not normally distributed, all data underwent logarithmic transformation before statistical analyses. Group comparisons were done using Student’s t tests for independent groups (Asian vs. whites, young vs. middle-aged) and paired t tests for within-group (day vs. evening) comparisons. Two-tailed comparisons with P values < 0.05 were considered statistically significant. A one-tailed comparison was used for the estimation of diurnal variation, assuming that the day values were higher than those from midday or evening. For simplicity of presentation, all data in the table and figures are given as mean ± SEM without logarithmic transformation.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The enrichment of d₃T in the serum was about 7 to 8% in all three groups. Serum d₃T concentrations were not significantly different for samples drawn between 4 and 5 h in all subjects after the start of the infusion presumably reaching steady state (Fig. 1). Thus, the PR_T and MCR_T during daytime were calculated using the average serum T and d₃T concentrations drawn between 4 and 5 h after the start of infusion (usually between 1200 and 1300 h).

The data for MCR_T and PR_T, calculated by using total T measured by RIA or d₀T by LC-MS-MS, are shown in Table 1. Both MCR_T and PR_T were not significantly different between Asian and white young men, calculated using data from either method (Table 1). Serum T levels (LC-MS-MS) were lower (P < 0.05) in middle-aged white men compared with young white men. Using RIA data, the difference was not significant. The MCR_T calculated with LC-MS-MS data as liters per day (P < 0.01) or as liters per day per meter² (P < 0.01) were lower in middle-aged white men vs. young white men; using RIA data only, MCR_T calculated as liters per day per meter² (P < 0.05) was significant. The PR_T values calculated with LC-MS-MS data were lower in middle-aged men compared with young white men whether the data were calculated as milligrams per day or milligrams per day per meter² (both P < 0.001), whereas using the RIA data, both PR_T calculations were lower with P < 0.01.

View larger version (57K): [in this window] [in a new window]   FIG. 2. Diurnal variation of serum T, MCRT, and PRT in healthy young and middle-aged men. Data calculated from serum T and infusate T measured by RIA are shown in panel A, and those from d0T levels measured by LC-MS-MS are shown in panel B. Serum concentrations were obtained at 0800 h (morning), 1200–1300 h (midday), and 2000 h (evening). MCRT and PRT during the day and evening were calculated using serum levels of labeled T (d3T) and unlabeled T (d0T) at 4–5 h (day, 1200–1300 h) and 12 h (evening, 2000 h) after the start of d3T infusion. a, P < 0.05; aa, P < 0.01; and aaa, P < 0.005 when compared with evening values. b, P < 0.05 when compared with midday values.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In this study, we used LC-MS-MS to measure d₃T and d₀T precisely and accurately in the serum (15). Serum total T was measured by a validated RIA (13, 14). During a constant infusion of d₃T and using LC-MS-MS to quantitate d₃T at steady state, we showed that the mean MCR_T and PR_T of healthy young men were similar to those previously reported in studies using isotopic T infusions (10, 11, 17, 18, 19, 20, 21). The PR_T determined in young white men in this study (9.11 ± 1.11 mg/d using d₀T data) is considerably higher than the PR_T reported by Vierhapper et al. (9) using GC-MS (3.7 ± 2.2 mg/d). The infusion rate of 20 ml/h was similar in the two studies. There are differences between the methods used by Vierhapper et al. (9) and this study. First, the d₃T was infused at approximately 12 µg/h in this study. Vierhapper et al. (9) infused dideuterated T at 70 µg/h (1.68 mg/d) and found that the PR_T was very low (1.85 mg/d) due to the suppression of the hypothalamic-pituitary testicular axis. When the amount of labeled T administered was reduced to 15 µg/h, which was about 5% of the estimated daily production rate, the PR_T obtained remained lower compared with prior studies and showed large between-subject variation from 1.67 to 7.44 mg/d. Second, Vierhapper et al. (9) used GC-MS, which requires derivatization to quantitate serum total T and labeled T. Our studies used LC-MS-MS without derivatization. Third, in this study, the subjects were administered labeled T according to body surface area, whereas Vierhapper et al. (9) gave the same dose to subjects participating in the same experiment. Both studies had similar losses of labeled T due to adsorption to the infusion bag and tubing.

In this study based on a very limited number of Asian and white young men, the serum T, MCR_T, and PR_T levels were not significantly different between Asian and white men (P = 0.68, 0.38, and 0.19, respectively). It has been shown in prior studies that whites and Asians have similar serum T levels, but 5-reduced androgens were lower in Asians (22, 23). Santner et al. (24) compared MCR_T and PR_T in three groups of subjects, i.e. Asians in Asia, Asians in the United States, and whites in the United States, and found the MCR_T measurements were not significantly different between the three groups, but the mean PR_T in Chinese living in Beijing, China, was lower when compared with Chinese living in Hershey, Pennsylvania. Our studies on MCR_T and PR_T in Asians and whites living in Los Angeles, California, showed no significant difference between the two ethnic groups, possibly because of the small sample size.

In middle-aged white men (mean age, 55 yr), serum d₀T concentrations, MCR_T, and PR_T measured at midday were significantly lower compared with their younger counterparts. The results were in the same trend when serum and infusate T were measured by RIA, where the MCR_T corrected for body surface area and PR_T were also lower in the middle-aged men. The results in the middle-aged men were about 20% greater than those reported in healthy, middle-aged men by Meikle et al. (25) (MCR_T, 346 ± 20 liters/d/m²; and PR_T, 1.70 ± 0.11 mg/d/m²). Significant decreases in MCR_T and PR_T with aging had been reported previously (26, 27, 28). Lower clearance of androgens in older men may be related to the increase in the SHBG-bound T fraction found in older men (29). Lower MCR_T is compounded by the lower serum T levels, resulting in more marked decrease in PR_T in middle-aged men as shown in this study.

We demonstrated significantly lower MCR_T and even more significantly lower PR_T in young men in the evening compared with values obtained at midday. No diurnal variation of MCR_T was reported in prior studies using the isotope dilution method, but PR_T showed a diurnal variation because of the differences in serum T concentrations measured at 0900, 1700, and 2200 h (11). Using GC-MS analyses and stable isotope dilution methods during constant deuterated T infusion for 24 h, Vierhapper et al. (9) showed no significant decrease in serum T concentrations or PR_T in the evening. The failure to demonstrate significant diurnal variation could be due to the large variation in PR_T measured in the seven healthy men studied by Vierhapper et al. (9) (PR_T at 0800–1200 h, 3.96 ± 0.91 mg/d; at 1200–1600 h, 3.70 ± 0.35 mg/d; and 1600–2000 h, 3.48 ± 0.83 mg/d, respectively).

The diurnal variation of serum T was not evident in the middle-aged men when measured by RIA because the RIA measured both the labeled T (d₃T) and d₀T in the serum samples. Thus, samples collected midday and in the evening contained both species of T, whereas the morning sample collected before the start of labeled T infusion contains only d₀T. When we corrected the total serum levels by subtracting the d₃T concentrations, the serum T concentrations were significantly lower (P < 0.05) both at midday and in the evening. Significant diurnal variation was evident in the middle-aged men when d₀T was measured by LC-MS-MS. Recently, it has been shown that healthy middle-aged men had significant diurnal rhythm in serum total, free, and bioavailable T (30). The loss of diurnal variation in serum T concentration in elderly men had been demonstrated previously, but this loss could be affected by the health of the elderly men (31, 32, 33, 34, 35, 36). Because of the lower MCR_T in the evening, we demonstrated in this report that the mean PR_T in middle-aged men was significantly decreased when compared with that quantitated at midday. The experimental design did not allow studying the clearance and production rates earlier in the day.

The use of LC-MS-MS to quantitate specifically labeled vs. unlabeled steroids and applied for clearance studies have not been previously reported. We have shown that using stable isotope-labeled T via a constant infusion and analyses of serum samples collected for labeled T by LC-MS-MS allow the quantitation of PR_T and MCR_T in healthy young and middle-aged men. The amount of d₃T infused was small, and the LC-MS-MS method has sufficient sensitivity to allow these parameters to be measured without perturbation of the endogenous production of T. In the small sample of men studied, the MCR_T and PR_T were not different between Asian and white young men. Middle-aged men had lower MCR_T and PR_T compared with young men. Diurnal variations were observed for serum T concentration, MCR_T, and PR_T in younger men but to a lesser extent in middle-aged men. We conclude that stable isotope infusion and LC-MS-MS measurements of labeled T allow accurate and specific measurements of PR_T and MCR_T, which can be used to study androgen metabolism repeatedly in men undergoing physiological or pharmacological interventions.

	Acknowledgments

 
We thank Sally Avancena, M.A., for her expert assistance in preparation of the manuscript; Behrouz Salehian, M.D., Veronica McDonald, R.N., and the General Clinical Research Center nurses at Harbor-UCLA Medical Center for their skilled assistance with the conduct of the study. We thank C. K. Hatton, Ph.D., for her valuable advice on analytical issues and K. M. Schramm for coordinating and managing the complex protocols.

	Footnotes

 
This work was supported by National Institutes of Health Grants RO1 CA-71053 and RO1 DK-61006 (to C.W., D.H.C., and R.S.S.); M01 RR00425 to the General Clinical Research Center at Harbor-UCLA Medical Center; and by the United States Anti-Doping Agency and the National Collegiate Athletic Association (to D.H.C.).

Abbreviations: CV, Coefficient of variation; d₀T, unlabeled T; d₃, trideuterated; GC, gas chromatography; LC, liquid chromatography; LC-MS-MS, LC-tandem MS; MCR, metabolic clearance rate; MCR_T, metabolic clearance rate of T; MS, mass spectrometry; PR, production rate; PR_T, production rate of T; T, testosterone.

Received October 16, 2003.

Accepted February 23, 2004.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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