New quantification method for estradiol in the prostatic tissues of benign prostatic hyperplasia using liquid chromatography-tandem mass spectrometry

Article abstract of DOI:10.1016/j.steroids.2009.09.004

Estrogen is suspected to play a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer. To clarify the role of estradiol (E2) in the prostatic tissues (prostatic tissue E2) during the development of prostatic disorders, we developed a new sensitive and specific quantification method for prostatic tissue E2 using liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the solid-phase extraction, E2 was purified by anion-exchange through an Oasis MAX cartridge. In addition, after the formation of 3-pentaflurobenzyl-17β-pyridinium-estradiol derivative (E2-PFBPY), E2-PFBPY was purified by cation-exchange through an Oasis WCX cartridge. These processes in the LC-MS/MS method improved the specificity and sensitivity for prostatic tissue E2 measurement, compared to the radioimmunoassay (RIA) method. The validation tests showed that intra-day and inter-day precisions were both within ±15% (except for 15.5% of the inter-day precision of the lowest concentration), with the accuracy ranging from 88 to 110%. The quantification limit of this assay was 0.15 pg/tube in our method, which was 80-fold more sensitive than that of the RIA method. With the use of our present method, the median E2 levels in the prostatic tissues in patients with BPH (n = 20, median age: 71 years) were 12.0 pg/g tissue (95% confidence interval = 9.1-22.6 pg/g tissue). Furthermore, the E2 levels increased significantly with aging. These results showed that our present method would be useful for elucidating the role of prostatic tissue E2 in the development of prostatic disorders with a small amount of tissue samples.

Full text of DOI:10.1016/j.steroids.2009.09.004

Steroids 75 (2010) 13–19
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
New quantification method for estradiol in the prostatic tissues of benign
prostatic hyperplasia using liquid chromatography–tandem mass spectrometry
Seiji Araia,∗, Yoshimichi Miyashirob, Yasuhiro Shibataa, Bunzo Kashiwagic, Yukio Tomaruc,
Mikio Kobayashid, Yoko Watanabeb, Seijiro Honmab, Kazuhiro Suzukia
a Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan
b Aska Pharma Medical Co. Ltd., 1604 Shimo-sakunobe, Takatsu-ku, Kawasaki 213-8522, Japan
c Division of Urology, Kiryu Kosei General Hospital, 6-3 Orihime-machi, Kiryu 376-0024, Japan
d Division of Urology, Isesaki Municipal Hospital, 12-1 Tsunatorimoto-machi, Isesaki 372-0817, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2009
Received in revised form 6 September 2009
Accepted 10 September 2009
Available online 18 September 2009
Estrogen is suspected to play a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate
cancer. To clarify the role of estradiol (E2) in the prostatic tissues (prostatic tissue E2) during the develop-
ment of prostatic disorders, we developed a new sensitive and specific quantification method for prostatic
tissue E2 using liquid chromatography–tandem mass spectrometry (LC–MS/MS). For the solid-phase
extraction, E2 was purified by anion-exchange through an Oasis MAX cartridge. In addition, after the for-
mation of 3-pentaflurobenzyl-17␤-pyridinium-estradiol derivative (E2-PFBPY), E2-PFBPY was purified
by cation-exchange through an Oasis WCX cartridge. These processes in the LC-MS/MS method improved
the specificity and sensitivity for prostatic tissue E2 measurement, compared to the radioimmunoassay
(RIA) method. The validation tests showed that intra-day and inter-day precisions were both within 15%
(except for 15.5% of the inter-day precision of the lowest concentration), with the accuracy ranging from
88 to 110%. The quantification limit of this assay was 0.15 pg/tube in our method, which was 80-fold
more sensitive than that of the RIA method. With the use of our present method, the median E2 levels
in the prostatic tissues in patients with BPH (n = 20, median age: 71 years) were 12.0 pg/g tissue (95%
confidence interval = 9.1–22.6 pg/g tissue). Furthermore, the E2 levels increased significantly with aging.
These results showed that our present method would be useful for elucidating the role of prostatic tissue
E2 in the development of prostatic disorders with a small amount of tissue samples.
Keywords:
Liquid chromatography–tandem mass
spectrometry
Estrogen
Benign prostatic hyperplasia
Solid-phase extraction
Age
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
the development of epithelial dysplasia and adenocarcinoma in
the dorsolateral prostate [8], whereas the development of E2- and
The role of estrogen in the development of prostatic disor-
ders, including benign prostatic hyperplasia (BPH) and prostate
cancer (PCa), is poorly understood. It is known that estrogen
can indirectly reduce serum androgen levels via the suppression
of hypothalamic–pituitary–gonadal axis [1,2], whereas the direct
effects of estrogen on the prostatic tissues are still equivocal. Sev-
eral reports have revealed that the aromatase enzyme is expressed
in both BPH and PCa tissues [3]. Two subtypes of estrogen recep-
tor are also expressed in the prostatic tissues; estrogen receptor
␣ in the stroma and estrogen receptor ␤ predominantly in the
epithelium [4,5]. In human BPH tissues, increased accumulation
of estradiol (E2) is observed in the nuclei of stromal cells [6], and
stromal E2 levels increase with aging [7]. Moreover, in the Noble
rat model, the co-administration of E2 and testosterone induces
testosterone-related dysplasia is completely prevented by a syn-
thetic estrogen receptor antagonist [9]. These findings evidence the
synthesis of estrogen in the prostatic tissues, and suggest that E2
may contribute to the pathogenesis of BPH and PCa in collabora-
tion with the estrogen receptors. Therefore, the measurement of E2
levels in the prostatic tissues is essential for the elucidation of the
direct effect of E2 on the development of BPH and PCa.
Despite this, only a few reports have investigated E2 levels in
the prostatic tissues [6,7,10,11]. In their methods, the levels of pro-
static tissue E2 are measured using radioimmunoassay (RIA), and
the validation of their methods has not been performed sufficiently
below the level of 10 pg. We had previously developed and vali-
dated a precise method for the prostatic tissue E2 measurements
by RIA [12]. The sensitivity of our method using RIA was low, and
over 1 g of the prostatic tissue was required for a precise analysis.
Liquid
chromatography–tandem
mass
spectrometry
(LC–MS/MS) has recently considered to be a useful method
for quantification of steroid hormones because of its accuracy
∗
Corresponding author. Tel.: +81 27 220 8322; fax: +81 27 220 8318.
E-mail address: seiji@showa.gunma-u.ac.jp (S. Arai).
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.09.004

14
S. Arai et al. / Steroids 75 (2010) 13–19
and sensitivity [13], but no investigators have performed the pre-
cise measurement of prostatic tissue E2 levels using LC–MS/MS.
Solid-phase extraction is widely used for the purification of steroid
hormone before LC–MS/MS analysis. Oasis MAX cartridges have
the ability of anion-exchange, which is one of the retention meth-
ods of solid-phase extraction and a selective isolation procedure
for acidic compounds such as phenols, including E2 and other
estrogens [14–16]. Therefore, an Oasis MAX cartridge might be
useful for the specific purification of prostatic tissue E2. Moreover,
we had recently developed a new derivatization method for E2
(Fig. 1) [17,18] that might improve the sensitivity of quantification
method for prostatic tissue E2 using LC–MS/MS compared to that
of the previous RIA method.
In this study, we developed and validated a new determination
method for prostatic tissue E2 levels using Oasis MAX extraction,
and a new derivatization method for E2 followed by LC–MS/MS
analysis. Furthermore, we investigated age-related changes of E2
levels in the prostatic tissues with BPH, to elucidate the role of E2
in the development of BPH.
source (Applied Biosystems Inc., Foster City, CA, USA) and a Shi-
madzu HPLC system (SCL-10Avp system controller, LC-20AD pump,
SIL-HTc column oven, CTO-20A auto-sampler; Shimadzu Co. Ltd.,
Kyoto, Japan) was employed. The column was Xterra MS-C18 col-
umn (100 mm × 2.1 mm i.d., 3.5 ␮m, Waters Co., Milford, MA, USA)
and used at 40 ◦C. The mobile phase consisting of 0.1% HCOOH (Sol-
vent A) and CH3CN/CH3OH (50:50) (Solvent B) was used with a
gradient elution of A:B = 50:50–40:60 (0.0–3.0 min), 40:60–20:80
(3.0–7.0 min), 0:100 (7.0–9.0 min), and 50:50 (9.0–12.0 min) at
a flow rate of 0.4 ml/min. E2 and E2-C4 were analyzed as their
3-pentaflurobenzyl-17␤-pyridiniums (PFBPY derivatives) by ESI-
MS/MS. The ESI-MS/MS conditions were as follows: declustering
potential, 100 V; spray voltage, 5500 V; collision gas, nitrogen, 6 psi
(gas pressure); collision energy, 40 eV; curtain gas, nitrogen, 12 psi
(gas pressure); and ion source temperature, 550 ◦C. The precursor
and product ions of PFBPY derivatives were as follows: m/z 544.2
and 339.0 for E2-PFBPY; and m/z 548.2 and 343.2 for E2-C4-PFBPY.
2.3. Analytical methods of estrogen
2.3.1. Prostatic tissues
2. Experimental
Prostatic tissues were obtained from 20 patients to whom open
or transurethral surgery for BPH without hormonal therapy had
been performed at Gunma University and its affiliate hospitals.
The prostatic tissues were excised immediately after collection
and stored at −80 ◦C until analysis. All tissues were obtained in
accordance with the requirements and approval of the Commit-
tee for Human Ethics and Experimentation at Gunma University,
Isesaki Municipal Hospital, and Kiryu Kosei General Hospital. Writ-
ten informed consent was obtained from all participants.
2.1. Materials and reagents
Standard E2 was purchased from National Institute of Health
Sciences (Tokyo, Japan). [1,2, 3, 4-13C4]-E2 (E2-C4) was purchased
from Hayashi Pure Chemical Ind. Ltd. (Osaka, Japan). Pentafluroben-
zyl bromide and 2-fluoro-1-methylpyridinium p-tolensulfonate
were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo,
Japan). Triethylamine was purchased from Wako Pure Chemical
Industries Ltd. (Osaka, Japan). Oasis MAX (60 mg, 3 ml) and Oasis
WCX (60 mg, 3 ml) cartridges were purchased from Waters Co.
(Milford, MA, USA). An InertSep SI cartridge (500 mg, 3 ml) was pur-
chased from GL sciences Inc. (Tokyo, Japan). All solvents were of
analytical grade.
2.3.2. Sample preparation (Fig. 2)
The frozen prostatic tissues were pulverized in liquid nitrogen
to powder, and then homogenized in 10 times volume of distilled
water using an Ultra-Turrax homogenizer (IKA, Staufen, Germany)
in an ice bath. The homogenized sample (50–100 mg) was trans-
ferred to a test tube, and 100 pg of E2-C4 was added as an internal
standard to the homogenized sample, which was then extracted
with 3 ml of ethyl acetate. After evaporation, the extract was dis-
solved in 0.25 ml of CH3OH, diluted with 1 ml of distilled water, and
2.2. LC–MS/MS
For LC–MS/MS, an API-5000 triple stage quadrupole mass spec-
trometer equipped with a positive electrospray ionization (ESI)
Fig. 1. Derivatization of E2.

S. Arai et al. / Steroids 75 (2010) 13–19
15
standard tube as internal standards. E2 was isolated from each solu-
tion, and then E2 was derivatized as described above and subjected
to an LC–MS/MS analysis. The calibration curve was constructed
by plotting the peak area ratio (analyte steroid/internal standard)
against the amount of analyte steroid.
2.4. Validation of the analytical method
2.4.1. Assay accuracy
The prostatic tissue samples (9 mg) collected from 5 individuals,
which were spiked with (spiked samples) or without (un-spiked
samples) E2, were prepared. An internal standard was added to
each sample, and the resulting sample was then measured as
described above. The assay accuracy was evaluated by analyzing
the recovery rate, which was defined as Fs/F0 × 100%, where Fs and
F0 were the measured amounts of E2 in the spiked samples and
those of un-spiked samples and the spiked amount, respectively.
The spiked amounts of E2 were 0.5 pg and 10 pg.
2.4.2. Assay precision
The prostatic tissues (3 mg), which were spiked with various
amounts of E2, were used for the determination of the assay preci-
sion. Internal standard was added to each sample, and the resulting
sample was then measured as described above. The intra-day pre-
cision was evaluated by measuring the resulting samples (n = 5) on
the same day. The inter-day precision was evaluated by measuring
the resulting samples for 3 days (n = 5, each day). The recovery rate
was also measured as described above. The spiked amounts of E2
were 0.15 pg, 0.25 pg, 5 pg, and 50 pg.
Fig. 2. Flow sheet of the analytical method.
then applied to an Oasis MAX cartridge which had been successively
conditioned with 3 ml of CH3OH, 3 ml of distilled water, 1 ml of
0.2 M NaOH, and 3 ml of distilled water. The cartridge was washed
with 3 ml of CH3OH and 2 ml of HCOOH/50% CH3OH (2:98), and
then E2 was eluted with 3 ml of HCOOH/80% CH3CN (2:98). After
evaporation, the residue was subjected to derivatization described
below.
2.5. Statistical analysis
The correlation between the prostatic tissue E2 levels and age in
patients with BPH was analyzed using Spearman rank correlation
coefficients. E2 levels below the quantification limit were set equal
to the lower limit of quantification for statistical analysis; which
might have introduced bias in favor of not finding a significant dif-
ference. All data were analyzed using SPSS software v.11.0 (SPSS,
Inc., Chicago, IL). Tests were two-sided, and P values of less than
0.05 were considered to be statistically significant.
2.3.3. Derivatization of E2 with pentaflurobenzyl bromide and
2-fluoro-1-methylpyridinium p-tolensulfonate
E2 was dried perfectly using a vacuum desiccator at 40 ◦C for
1 h. The dried E2 was reacted with 0.1 ml of pentaflurobenzyl
bromide/CH3CN (1:40) and 50 ␮l of 0.8% KOH at 53 ◦C for 1 h using a
thermostat, and then 3-pentafluorobenzyl-estradiol (E2-PFB) was
dried under a steam of nitrogen. The residue was dissolved in
0.25 ml of ethyl acetate, diluted with 1 ml of hexane, and then
applied to an InertSep SI cartridge, which had been successively
conditioned with 3 ml of ethyl acetate and 3 ml of hexane. The
cartridge was washed with 1 ml of hexane and 2.5 ml of ethyl
acetate/hexane (10:90), and then E2-PFB was eluted with 2.5 ml
of ethyl acetate/hexane (50:50). E2-PFB was evaporated to dry-
ness using a centrifugal evaporator, and then dried perfectly using
a vacuum desiccator at 40 ◦C for 1 h. E2-PFB was then reacted with
0.2 ml of mixed solution (20 mg of 2-fluoro-1-methylpyridinium
p-tolensulfonate in 1 ml of dichloromethane) and 30 ␮l of tri-
ethylamine/dichloromethane (1:10) at room temperature for 1.5 h.
After reaction, the sample was dried under a stream of nitrogen. E2-
PFBPY was dissolved in 0.5 ml of CH3OH and diluted with 0.5 ml of
distilled water, and then the mixture was applied to an Oasis WCX
cartridge which had been successively conditioned with 3 ml of
CH3CN, 3 ml of CH3OH and 3 ml of distilled water. The cartridge was
washed successively with 1 ml of 0.3% ammonia solution, 3 ml of
CH3CN and 2 ml of HCOOH/50% CH3OH (2:98), and then E2-PFBPY
was eluted with 2 ml of HCOOH/80% CH3CN (2:98). After evapora-
tion, the residue was dissolved in 0.1 ml of the mobile phase, and
20 ␮l of the solution was subjected to an LC–MS/MS analysis, as
described above.
3. Results
3.1. Recovery of E2 during purification with Oasis MAX and Oasis
WCX cartridges
After conditioning an Oasis MAX cartridge with NaOH solu-
tion, E2 was retained to the solid-phase, whereas other neutral
steroids (e.g., testosterone) were recovered from the liquid-phase
that passed though the solid-phase (Fig. 3). The mean recovery rates
of E2 and testosterone were 83.4% (n = 2) and 98.6% (n = 2), respec-
tively. After conditioning an Oasis WCX cartridge with ammonia
solution, E2-PFBPY was retained to the solid-phase. The mean
recovery rate of E2-PFBPY from an Oasis WCX cartridge was 88%
(n = 2).
3.2. Development of method of quantitative analysis
3.2.1. LC–MS/MS
The molecular cation was only observed at m/z 544.2 for E2-
PFBPY as a base peak (Fig. 4a). Collision of the molecular ion of
E2-PFBPY (Fig. 4b) gave the characteristic product ions at m/z 339.0
(ring A moiety of E2-PFBPY which cleaved ring C moiety), m/z 181.0
(the pentaflurobenzyl moiety), and m/z 110.0 (the pyridinium moi-
ety). MRM transition of m/z 544.2 to m/z 339.0 was chosen by means
2.3.4. Calibration of standard curves
Seven concentrations of standard E2 (0.15–100 pg/tube) were
put into each tube. Subsequently, 100 pg of E2-C4 was added to each

16
S. Arai et al. / Steroids 75 (2010) 13–19
3.3. Assay accuracy and precision
The assay accuracy for E2 was defined as the recovery rate
described above. Mean (SD) recoveries in E2 assays were 104.1%
(9.3%) for 0.5 pg of spiked amount and 98.6% (5.1%) for 10 pg
(Table 1). All the assay accuracies ranged from 85 to 115%. The
intra-day assay precision (relative standard deviation, RSD) and the
inter-day assay precision (RSD) for E2 were 1.3–6.9% and 3.1–15.5%,
respectively (Table 2). All the assay precisions were within 15%,
except for 15.5% of the inter-day assay precision at the lowest
spiked concentration.
3.4. The E2 levels in BPH
Twenty patients at 58–82 years of age (median, 71 years) under-
went open or transurethral surgery for BPH. The median prostatic
tissue E2 levels in patients with BPH were 12.0 pg/g tissue (95%
confidence interval = 9.1–22.6 pg/g tissue). The prostatic tissue E2
levels increased significantly with aging (rs = 0.51, P = 0.02; Fig. 6).
Fig. 3. Recovery of E2 and testosterone from the solid-phase and the liquid-phase
that passed through an Oasis MAX cartridge.
4. Discussion
of providing the highest signal to the noise ratio and lowest back-
ground peak, whereas the highest ESI responses were obtained by
using MRM transition of m/z 544.2 to m/z 110.0. The typical MRM
chromatogram of E2 was shown in Fig. 5. The resolution of the peak
demonstrated no interfering peak.
In this study, we developed a sensitive and specific analyt-
ical method for quantifying E2 in the prostatic tissue by using
LC–MS/MS for the first time. Several reports have suggested that
local E2 may contribute to the pathogenesis of BPH and PCa [19,20],
but only a few groups have reported the details of prostatic tissue
E2 [6,7,10–12]. All of these previous reports have determined pro-
static tissue E2 levels by RIA methods. However, it is well known
that the dose–response curves using RIA data are non-linear, and
consequently various curve-fitting methods have been used for the
determination by RIA [21,22]. Despite use of various curve-fitting
methods, only a segment of the standard curve is linear. Thus, low
levels of the steroid hormones are usually determined at a portion
of the calibration curve where the variance is large. These findings
have been previously confirmed by various reports [23–25]. There-
fore, measurement of prostatic tissue E2 levels by RIA may lead to
false results, since the prostatic tissue E2 levels are known to be very
low [12]. In contrast, the calibration curves of LC–MS/MS are lin-
ear, and consequently LC–MS/MS avoids the bias at low levels of E2
within the range of the calibration curves. Hsing et al. report the low
reproducibility of the RIA method as compared to the LC–MS/MS
3.2.2. Calibration curve and limit of quantification
Standard calibration curve for E2 was linear, and coefficient of
correlation was beyond 0.999. Regression analysis of five standard
calibration curves showed a mean (SD) slope of 0.0042 (0.0001) and
a mean y intercept of 0.00028 pg (0.00013 pg) within the range of
0.15–100 pg/tube. Relative error (n = 5) and coefficient of variation
(n = 5) at the lowest level on the standard curve were −6.7% and
13.4%, respectively. Signal to the noise ratio at the lowest level on
the standard curve was more than 10. The limit of quantification
was defined as the lowest level on the standard curve of the analyte
with an acceptable accuracy (relative error < 20%) and precision
(coefficient of variation < 20%), and with signal to the noise ratio
of not less than 5. According to this definition, the lower limit of
quantification for E2 was 0.15 pg/tube.
Fig. 4. Mass spectra of (a) E2-PFBPY and (b) its product ion.

S. Arai et al. / Steroids 75 (2010) 13–19
17
Fig. 5. Typical LC–MS/MS chromatograms of PFBPY derivatives obtained from (a) standard E2 (0.5 pg) + internal standard and (b) prostatic tissue (100 mg) + internal standard.
method for quantifying serum E2 [13]. Nelson et al. report that RIA
and LC–MS/MS give different measurement results for serum E2
below the level of 13.5 pg/ml [26]. These findings demonstrate the
usefulness of LC–MS/MS method for the determination of low levels
of prostatic tissue steroid hormones, such as E2. Furthermore, when
using LC–MS/MS method, the tandem MS can increase specificity
of analytical steroid hormones, since the molecular ion is cleaved
twice and there is little chance for contamination by impurities
(Fig. 4).
The derivatization of steroid hormones is essential for improv-
ing the detection sensitivity of LC–MS/MS due to the low ionization
of steroid hormones [16]. We had previously measured the serum
E2 levels by LC–MS/MS using reversed-phase extraction and deriva-
tization procedure to form E2-PFBPY, and the quantification was
Table 1
Assay accuracy.
Patient
1
Matrices
Spiked (pg)
Measured (pg)
Expected (pg)a
Recovery rate (%)b
0
Not detected
0.52
9.76
0.13
0.54
10.61
0.09
0.55
9.21
0.18
0.75
9.90
Not detected
0.44
9.98
–
–
104.0
97.6
–
85.7
104.7
–
93.2
91.3
–
110.3
97.2
–
0.5
10.0
0
0.5
10.0
0
0.5
10.0
0
0.5
10.0
0
0.50
10.00
–
0.63
10.13
–
0.59
10.09
–
0.68
10.18
–
2
3
4
5
Prostatic tissue (9 mg)
0.5
10.0
0.50
10.50
88.0
99.8
a
Values were obtained by addition of spiked amount and measured amount of un-spiked sample.
Recovery rate: measured value/expected value × 100.
b
Table 2
Intra-day and inter-day assay precision.
Matrices
Spiked (pg)
Intra-day assay
Measured (pg)a
Inter-day assay
RSD (pg)b
Recovery rate (pg)c
Measured (pg)a
RSD (pg)b
Recovery rate (%)c
0
0.15 0.02
0.30 0.02
0.41 0.02
5.06 0.15
50.24 0.65
–
–
0.11 0.05
0.25 0.04
0.36 0.05
4.84 0.30
49.34 1.51
–
–
0.15
0.25
5.00
50.00
6.9
4.4
2.9
1.3
100.0
102.5
98.3
15.5
12.5
6.3
97.4
99.3
94.7
98.5
Prostatic tissue (3 mg)
100.2
3.1
a
Mean SD for the intra-day (n = 5) and inter-day (3 days, n = 5 for each day) assay variation tests.
Relative standard deviation.
Recovery rate: measured value/expected value × 100. Expected values were obtained by addition of spiked amount and measured amount of un-spiked sample for each
b
c
case.

18
S. Arai et al. / Steroids 75 (2010) 13–19
As the incidence of BPH increases with aging, age-related
changes in the endocrine environment in the prostatic tissue must
be associated with the pathogenesis of BPH. Our present results
showed the prostatic tissue E2 levels increased significantly with
aging, which were consistent with the results of Krieg et al. [7].
They report that age-related increase of E2 levels in the prostatic
stroma and age-related decrease of 5-alpha dihydrotestosterone
(DHT) in the prostatic epithelium lead to the dramatic increase
of the ratio of estrogen to androgen in the prostatic stroma with
age. We had also previously reported that the ratio of E2 to DHT
level in the prostatic tissue increased with age and showed a sig-
nificant positive correlation with the proportion of the prostatic
stroma [12]. These findings suggest that the prostatic tissue E2 has
a synergistic role with prostatic tissue DHT in the pathogenesis of
BPH. Furthermore, in contrast with the age-related increase of the
prostatic tissue E2, the serum E2 levels remained unchanged with
aging [28]. This discrepancy suggests the increase of E2 synthesis
in the prostatic tissues with age, but no further reports are avail-
able about the changes of aromatase expression and activity in BPH
with aging. Therefore aromatase in BPH should be investigated in
the future to elucidate the relationship between aging and devel-
opment of BPH. Moreover, Hiramatsu et al. report that aromatase
expression and activity are identified in both BPH and PCa tissues
[3]. Ellem et al. report that aromatase expression is observed in PCa
cell lines and microdissected PCa cells [29]. Tsuchiya et al. report
that the aromatase gene polymorphism may be a prognostic pre-
dictor of metastatic PCa [30]. These reports suggest that estrogen
has a significant role in the development of PCa, as well as BPH.
Therefore, measurement of estrogen levels in the prostatic tissue
is necessary for elucidating the mechanism of both BPH and PCa
development. Furthermore, our sensitive method would provide
reliable results about the prostatic tissue E2 levels with a small
amount of tissue sample, such as a biopsy sample, and consequently
would be a powerful tool for future studies on the role of prostatic
tissue E2 in development of BPH and PCa.
Fig. 6. Relation of the prostatic tissue E2 with ages.
possible at the 0.1 pg/ml levels (un-published date). We had also
previously measured the tissue E2 levels by the same method,
but the inadequate purification resulted in low sensitivity of E2
(data not shown). Thus, in this study, we improved the purification
method of E2.
Oasis MAX cartridges are made of divinylbenzene-N-
vinylpyrrolidone copolymer backbone, on which quaternary
amine groups are covalently bonded [15]. Due to the presence of
these quaternary amine groups, Oasis MAX cartridges can retain
acidic compounds, such as E2, through anion-exchange, which
is a subtype of ion-exchanges [14]. Thus, after conditioning an
Oasis MAX cartridge with NaOH solution, E2 was dissociated
from proton and consequently could be efficiently retained to the
cartridge, whereas other steroids could not be retained (Fig. 2).
Cation-exchange is also a subtype of ion-exchanges used for
retention on the solid-phase [27]. Oasis WCX cartridges are made
of divinylbenzene-N-vinylpyrrolidone copolymer backbone, on
which carboxylic acid groups are covalently bonded. Due to the
presence of these carboxylic acid groups, Oasis WCX cartridges
can retain strong basic compounds such as quaternary amine.
Interestingly, E2 has changed to have a basic feature after the
formation of E2-PFBPY because of the presence of quaternary
amine on pyridinium. Thus, after conditioning an Oasis WCX
cartridge with ammonia solution, proton in the cartridge was
dissociated from carboxylic acid and consequently E2-PFBPY could
be efficiently retained to the cartridge.
These purification methods using solid-phase extraction and
derivatization procedure to form E2-PFBPY improved the speci-
ficity and sensitivity of prostatic tissue E2 by LC–MS/MS. Our
validation studies demonstrated that both intra-day and inter-day
precisions were less than 20% and yielded mean E2 recoveries of
104% with calibration curve linearity of 0.999. Moreover, we con-
firmed a good linearity for each prostatic tissue (n = 5, 10–100 mg)
in the evaluation of the additivity of prostatic tissues (data not
shown). The quantification limit (0.15 pg/tube) for the prostatic
tissue E2 in our present study by LC–MS/MS was 80-fold more sen-
sitive than that of our previous method by RIA [12], and our new
method enabled the quantification of prostatic tissue E2 with only
a small amount of tissue sample (16 mg). Our results on the quan-
tification of prostatic tissue E2 in BPH generally resembled those
in the previous reports, except for a study by Belis et al. [6,10,12].
In the RIA method by Belis et al., 500 mg of prostatic tissues are
used for the measurement of tissue E2 level, and the validation of
this method has not been performed below the level of 20 pg. Thus,
the measurements of prostatic tissue E2 below the levels of 40 pg/g
tissue may lead to false results, which may be the reason why their
results are higher than those of other reports.
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