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The Journal of Clinical Endocrinology & Metabolism 89(6):2936 –2941
Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2003-031802
Testosterone Metabolic Clearance and Production Rates
Determined by Stable Isotope Dilution/Tandem Mass
Spectrometry in Normal Men: Influence of Ethnicity
and Age
CHRISTINA WANG, DON H. CATLIN, BORISLAV STARCEVIC, ANDREW LEUNG,
EMMA DISTEFANO, GERALDINE LUCAS, LAURA HULL, AND RONALD S. SWERDLOFF
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
The metabolic clearance rate (MCRT) and production rate
(PRT) of testosterone (T) were measured using constant infusion of trideuterated (d3) T and quantitating serum d3T by
liquid chromatography-tandem mass spectrometry (LC-MSMS). Serum unlabeled T (d0T) was measured by LC-MS-MS,
and serum total T (d3T ⴙ d0T) was measured by RIA. Mean
MCRT (measured by LC-MS-MS) in young white men (1272 ⴞ
168 liters/d) was not significantly different from young Asian
men (1070 ⴞ 166 liters/d). Mean PRT was also not significantly
different between the two ethnic groups (whites, 9.11 ⴞ 1.11
mg/d; Asians, 7.22 ⴞ 1.15 mg/d; P ⴝ 0.19 using d0T data). Both
the mean MCRT (812 ⴞ 64 liters/d; P < 0.01) and the PRT (3.88 ⴞ
0.27 mg/d; P < 0.001) were significantly lower in middle-aged
R
ECENT 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 (1–7).
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
PRT was also determined in healthy men and women using
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; MCRT, metabolic clearance rate of T; MS, mass spectrometry; PR, production rate;
PRT, production rate of T; T, testosterone.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
white men when compared with their younger counterparts.
The mean MCRT and PRT 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 (MCRT, P < 0.01; PRT, P < 0.001) and to a lesser extent
in the older men (MCRT, P < 0.05; PRT, P < 0.05 using total T
and P < 0.001 using d0T data). We conclude that using LCMS-MS to detect d3T in serum after constant infusion of stable
isotope-labeled T allows the measurements of MCRT and PRT,
which can be used to study androgen metabolism repeatedly
after physiological or pharmacological interventions. (J Clin
Endocrinol Metab 89: 2936 –2941, 2004)
GC-MS on samples collected during constant infusion of
dideuterated T. In men, the PRT averaged 3.7 ⫾ 2.2 mg/d and
in women 0.04 ⫾ 0.01 mg/d (9). There was no diurnal rhythm
of PRT, and the reported PRT in men using stable isotope
dilution was lower than that reported previously using radioactive isotopes (10, 11). The reason for the lower reported
PRT was not clear. In a subsequent report studying the production rate of dihydrotestosterone in men, Vierhapper et al.
(12) reported a mean PRT of 6.29 mg/d in eight nonobese
healthy men using GC-MS. In this report, we determined the
PRT and MCRT 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
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
2936
Wang et al. • Testosterone Production Rates
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 HarborUCLA 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 ␮g/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 ␮mol/liter (637 ⫾ 26 ng/ml; 12.7 ␮g/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
J Clin Endocrinol Metab, June 2004, 89(6):2936 –2941 2937
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
MCRT and PRT 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 ␮l 0.2
ng/␮l), 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 ␮l methanol, and 15 ␮l was injected into the LCMS-MS. The column was a 3-␮m Hypersil BDS C18 (Keystone, Bellefonte, PA) (150 ⫻ 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 signalto-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
2938
J Clin Endocrinol Metab, June 2004, 89(6):2936 –2941
Wang et al. • Testosterone Production Rates
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. MCRT was calculated by the formula: MCRT ⫽ 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). PRT was then calculated from the formula: PRT ⫽
MCRT ⫻ 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 MCRT and
PRT during the day, and the d3T and d0T or T levels at 12 h after infusion
were used to calculate the evening MCRT and PRT, respectively.
Statistical analyses
Because serum T and MCRT 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 onetailed 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.
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.
Results
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 PRT and MCRT 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 MCRT and PRT, calculated by using total T
measured by RIA or d0T by LC-MS-MS, are shown in Table
1. Both MCRT and PRT 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 MCRT 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, MCRT calculated as liters per day
per meter2 (P ⬍ 0.05) was significant. The PRT 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
PRT calculations were lower with P ⬍ 0.01.
Figure 2 shows the differences between the day and
evening serum T (left panels), MCRT (middle panels), and PRT
(right panels) measured by both assay methods (top panel,
using serum total T by RIA; bottom panel, using d0T by LCMS-MS) in young and middle-aged white men. In the
younger subjects, serum T concentrations by both methods
were significantly higher in the morning (P ⬍ 0.001) and
midday (P ⬍ 0.01) when compared with the evening,
whereas the morning serum T concentrations were higher
than those at midday by LC-MS-MS but not by RIA. The
mean serum T concentrations in the middle-aged men were
not different during the day if measured by RIA, whereas
mean serum T concentrations measured by LC-MS-MS were
significantly lower in the evening (14.5 ⫾ 1.4 nmol/liter) in
the middle-aged men compared with those in the morning
(21.8 ⫾ 2.0 nmol/liter; P ⬍ 0.001) and midday (17.7 ⫾ 1.3
nmol/liter; P ⬍ 0.01).
TABLE 1. MCRT and PRT in Asian and white men
n
Asian
RIA
LC-MS-MS
White
Young
RIA
LC-MS-MS
Middle-aged
RIA
LC-MS-MS
Serum Ta (nmol/liter)
MCRT
PRT
(liters/d)
(liters/m 䡠d)
(mg/d)
(mg/m2䡠d)
2
9
9
22.8 ⫾ 2.7
24.1 ⫾ 2.8
1082 ⫾ 170
1071 ⫾ 166
579 ⫾ 83
584 ⫾ 81
6.73 ⫾ 1.03
7.22 ⫾ 1.15
3.61 ⫾ 0.50
3.97 ⫾ 0.61
11
10
25.3 ⫾ 3.1
25.5 ⫾ 2.9
1219 ⫾ 160
1272 ⫾ 168
604 ⫾ 69
636 ⫾ 72
8.45 ⫾ 1.14
9.11 ⫾ 1.11
4.25 ⫾ 0.55
4.72 ⫾ 0.56
18
18
19.8 ⫾ 1.3
17.7 ⫾ 1.3b
934 ⫾ 65
812 ⫾ 64c
463 ⫾ 33b
404 ⫾ 34c
5.12 ⫾ 0.36c
3.88 ⫾ 0.27d
2.54 ⫾ 0.18c
1.93 ⫾ 0.13d
a
The average serum T (d0T ⫹ d3T measured by RIA) or d0T (measured by LC-MS-MS) was used together with d3T at 4 and 5 h to calculate
the PRT.
b
P ⬍ 0.05; c P ⬍ 0.01; d P ⬍ 0.001 when compared with young white men.
Wang et al. • Testosterone Production Rates
J Clin Endocrinol Metab, June 2004, 89(6):2936 –2941 2939
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 LCMS-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.
Mean MCRT was lower in the evening in both the young
(P ⬍ 0.01) and middle-aged men (P ⬍ 0.05). The mean PRT
was significantly lower in the evening (P ⬍ 0.01) by both RIA
and LC-MS-MS in the young and also in the middle-aged
men using total serum T levels by RIA (P ⬍ 0.05) or serum
d0T measured by LC-MS-MS (P ⬍ 0.001).
Discussion
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 MCRT and PRT of
healthy young men were similar to those previously reported
in studies using isotopic T infusions (10 –11, 17–21). The PRT
determined in young white men in this study (9.11 ⫾ 1.11
mg/d using d0T data) is considerably higher than the PRT
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 ␮g/h in this study. Vierhapper et al. (9)
infused dideuterated T at 70 ␮g/h (1.68 mg/d) and found
that the PRT 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 ␮g/h,
which was about 5% of the estimated daily production rate,
the PRT 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, MCRT, and PRT 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 MCRT and PRT in three groups
of subjects, i.e. Asians in Asia, Asians in the United States,
and whites in the United States, and found the MCRT measurements were not significantly different between the three
groups, but the mean PRT in Chinese living in Beijing, China,
was lower when compared with Chinese living in Hershey,
Pennsylvania. Our studies on MCRT and PRT 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, MCRT, and PRT 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 MCRT corrected
for body surface area and PRT were also lower in the middleaged 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) (MCRT, 346 ⫾ 20 liters/d/m2; and PRT,
1.70 ⫾ 0.11 mg/d/m2). Significant decreases in MCRT and
PRT with aging had been reported previously (26 –28). Lower
clearance of androgens in older men may be related to the
increase in the SHBG-bound T fraction found in older men
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J Clin Endocrinol Metab, June 2004, 89(6):2936 –2941
(29). Lower MCRT is compounded by the lower serum T
levels, resulting in more marked decrease in PRT in middleaged men as shown in this study.
We demonstrated significantly lower MCRT and even
more significantly lower PRT in young men in the evening
compared with values obtained at midday. No diurnal variation of MCRT was reported in prior studies using the isotope
dilution method, but PRT 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 PRT in the evening.
The failure to demonstrate significant diurnal variation
could be due to the large variation in PRT measured in the
seven healthy men studied by Vierhapper et al. (9) (PRT 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 (31–36). Because of the lower MCRT in the
evening, we demonstrated in this report that the mean PRT
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 PRT and MCRT 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 MCRT and PRT were not different between Asian
and white young men. Middle-aged men had lower MCRT
and PRT compared with young men. Diurnal variations were
observed for serum T concentration, MCRT, and PRT 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 PRT and MCRT, which can be used to study
androgen metabolism repeatedly in men undergoing physiological or pharmacological interventions.
Wang et al. • Testosterone Production Rates
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.
Received October 16, 2003. Accepted February 23, 2004.
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:
[email protected].
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.).
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