Validity of Free Testosterone and Free Estradiol Determinations
in Serum Samples from Postmenopausal Women by
Theoretical Calculations
Sabina Rinaldi, Annabelle Geay, Henri De ´chaud,
Carine Biessy, Anne Zeleniuch-Jacquotte,
Arslan Akhmedkhanov, Roy E. Shore, Elio Riboli,
Paolo Toniolo, and Rudolf Kaaks
1
IARC, Unit of Nutrition and Cancer, 69500 Lyon, France [S. R., C. B., E. R.,
R. K.]; Service de Radioanalyse et Radiopharmacie, Ho ˆpital Neuro-
cardiologique, Hospices Civils de Lyon, Lyon, France [A. G., H. D.]; and
Departments of Environmental Medicine [A. Z-J., A. A., R. E. S., P. T.] and
Obstetrics and Gynecology [A. A., P. T.], New York University School of
Medicine, New York, New York 10010
Abstract
In this study, we validated measurements of free
testosterone (fT) and free estradiol (fE
2
) concentrations
calculated from total serum concentrations of testosterone
(T), estradiol (E
2
), and sex hormone-binding globulin
(SHBG), measured by direct, commercial
radioimmunoassays, by comparison with reference
measurements obtained by dialysis plus an in-house
radioimmunoassay after extraction and chromatographic
purification. The study was conducted in serum samples
from 19 postmenopausal women who were part of an
ongoing prospective cohort study. We also performed
sensitivity analyses to examine the robustness of the
theoretical calculations. Sensitivity analyses showed
that in this population, competitive binding of
dihydrotestosterone and total T could be ignored in
the calculation of fE
2
, and competitive binding by
dihydrotestosterone does not need to be taken into
account for calculation of fT. Furthermore, variations in
albumin and SHBG concentrations had negligible effects
on fT and fE
2
calculations. Values of fT and fE
2,
calculated from total T and E
2
concentrations obtained
by the same in-house radioimmunoassay used for the
dialysis method, correlated highly with the measurements
by dialysis (Pearson’s coefficients of correlation above
0.97). When calculating fT and fE
2
using total T and
total E
2
concentrations obtained by different direct
radioimmunoassays, almost all kits gave good correlations
with the reference method for fT (Pearson’s r > 0.83),
but only a few gave good correlations for fE
2
(Diagnostic
System Laboratories and DiaSorin; r > 0.80). The direct
radioimmunoassays giving the best correlation for fT and
fE
2
with the dialysis method were those that best
measured total concentrations of T and E
2
. Furthermore,
mean values of fT and fE
2
corresponded well to mean
values by the reference method if SHBG measurements
were also well calibrated. We conclude that in
postmenopausal women, theoretical calculations are
valid for the determination of fT and fE
2
concentrations
and can give reliable estimation of cancer risk in
epidemiological studies when the total concentrations
of T, E
2
, and SHBG are measured accurately.
Introduction
Epidemiological studies have shown relationships of breast and
endometrial cancers with concentrations of T
2
and E
2
in blood
among postmenopausal women (1–3). Both T and E
2
are trans-
ported in blood bound to proteins (4, 5), of which the most
important are albumin and SHBG. Together, these proteins
bind 97% of T and E
2
circulating in blood (6). The percentage
of T and E
2
that circulates either free (i.e., unbound to any
protein) or bound to albumin is defined as the “bioavailable
fraction” because only this fraction can potentially cross cellu-
lar membranes and bind to the nuclear steroid receptors (7). The
bioavailable fraction is virtually equal to the fraction not linked
to SHBG (8) and represents 50% of total E
2
and 30% of
total T in normal women and men (9). fT and fE
2
are most
directly available to tissues under physiological conditions and
generally correlate strongly with concentrations of T and E
2
unbound to SHBG (8).
To clarify the roles of fT and fE
2
(and therefore of their
bioavailable fractions) in the development of cancer in post-
menopausal women, it is important to have precise and inex-
pensive methods for their measurement that can be easily
applied to large-scale epidemiological studies. Several methods
have been set up for the measurement of fT and fE
2
rather than
for the measurement of the bioavailable fractions because in
normal subjects, the free and bioavailable fractions are very
highly correlated (8), and the methods for the measurement of
the T and E
2
bioavailable fractions require a large amount of
biological sample (10 –12). The most common methods for
measurement of fT and fE
2
are based on dialysis (13, 14),
ultrafiltration (15–18), and gel filtration (19, 20). These meth-
ods do not measure the absolute concentrations of fT and fE
2
directly but measure fT and fE
2
as percentages of the total
circulating T and E
2
concentrations. Absolute levels of fT and
fE
2
are then determined by multiplying the percentage of fT or
fE
2
with measurements of total T and E
2
, respectively (13). Gel
Received 2/4/02; revised 5/17/02; accepted 5/29/02.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom requests for reprints should be addressed, at IARC, Unit of Nutrition
and Cancer, 150 Cours Albert Thomas, 69372 Lyon, France. Fax: 33-04-72-73-
85-75.
2
The abbreviations used are: T, testosterone; fT, free testosterone; E
2
, estradiol;
fE
2
, free estradiol; SHBG, sex hormone-binding globulin; DHT, dihydrotestos-
terone; fDHT, free dihydrotestosterone; CI, confidence interval.
1065 Vol. 11, 1065–1071, October 2002 Cancer Epidemiology, Biomarkers & Prevention
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