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-Hydroxyestrone, and Their Ratio: Reproducibility, Validity, and Assay Performance after Freeze-Thaw Cycling and Preservation by Boric Acid
Division of Cancer Epidemiology and Genetics [R. T. F., T. R. F., J. D., R. G. Z.] and Office of Science Policy [S. C. R.], National Cancer Institute, Bethesda, Maryland 20892; Strang Cancer Research Laboratory, New York, New York 10021 [D. W. S., A. M., H. L. B.]; and Folkhälsan Research Center and Department of Clinical Chemistry, University of Helsinki, FIN-00014, Finland [H. A.]
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxyestrone
(16-OHE1) metabolites as low as 2 ng/ml and produce
consistent results in premenopausal urines. However, reproducibility
was problematic in postmenopausal urines where concentrations of these
compounds are much lower. In response to our concern, a new ELISA was
developed with a sensitivity of 0.625 ng/ml, which we evaluated using
the same pre- and postmenopausal urine samples analyzed in the earlier
ELISA. In this report, we present findings on the new kit with regard
to reproducibility of the 2-OHE1 and 16-OHE1
measurements, comparability of results with gas chromatography-mass
spectroscopy values, and with regard to the stability of the
metabolites after repeated freeze-thaw cycles and after preservation by
boric acid. For the most part, we found the new ELISA to be
reproducible, with assay coefficients of variation ranging from 10 to
20%, and intraclass correlation coefficients (ICCs) ranging from 80 to
95% in both the pre- and postmenopausal urines. ELISA results for
16-OHE1 differed from 1 day (i.e., batch)
to the next, and the absolute values of the metabolites obtained by the
ELISA were consistently lower than but well correlated with those
obtained by gas chromatography-mass spectroscopy. Values of the
2-OHE1:16-OHE1 ratio also differed
between the methods, but because the range of values was not large, the
magnitude of these differences was not as great. For the ratio, the
correlation between methods was excellent, and the ICCs were high for
both groups of women. After preservation by boric acid, values of the
ratio varied according to acid concentration but not in a linear
fashion. Ratio values were similar in urine samples exposed to four
different freeze-thaw cycle treatments, although values for all
treatments were consistently lower in one batch. Because batch-to-batch
variability was not negligible, it is advisable that matched cases and
controls be analyzed in the same batch. Provided this is done, the
relatively low assay coefficient of variation and high ICC demonstrate
that the new ELISA kit can reliably measure the
2-OHE1:16-OHE1 ratio and detect small
case-control differences in large population-based studies, where rapid
and relatively easy laboratory methods are critical. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHE1 and
estriol. In some animal models or cell cultures, the
16-OHE1 metabolite is a potent estrogen with
genotoxic and tumorigenic properties, and the
2-OHE1 metabolite shows little estrogenic or
antiestrogenic activity. Although there is no experimental support for
a carcinogenic role for 2-OHE1, abundant evidence
exists for such a role for 4-OHE1, particularly
in the male Syrian hamster kidney model (7
, 8)
. It has
been proposed that metabolism favoring the
16-OHE1 pathway and a lower
2-OHE1:16-OHE1 ratio is
a risk factor for breast cancer (9)
. Results from clinical
studies are mixed, with some showing lower
2-OHE1:16-OHE1 ratios in
urine of postmenopausal breast cancer cases versus controls
(4
, 10, 11, 12, 13)
, and others showing high levels of the
2-OHE1 metabolite alone or a high
2-OHE1:16-OHE1 ratio in
premenopausal breast cancer cases or women at high risk for the disease
(6
, 14
, 15)
. We note that levels of estrogen metabolites
in urine most likely reflect metabolic activity in the liver, and
associations between these metabolites and cancer risk in extrahepatic
tissues where the expression of P-450 isozymes may be quite
different must be interpreted with caution.
Because the 2-OHE1 and
16-OHE1 metabolites are present in very low
concentrations in the blood, laboratories have focused on detection in
urine; until recently, these efforts were laborious, requiring GC-MS
methods (16)
. The development of a relatively rapid and
inexpensive ELISA kit to measure 2-OHE1 and
16-OHE1 in unextracted urine has made the
study of these markers in large epidemiological studies possible
(17)
. As part of a large methodological study to evaluate
steroid sex hormone assay reproducibility and validity, we found that
reproducibility of an earlier ELISA kit was problematic with urines
from postmenopausal women (18)
, where analyte
concentrations were at or near the limit of assay detection,
i.e., 2 ng/ml; in response to our concerns, a new kit was
developed with a sensitivity of 0.625 ng/ml (19)
. This
report presents findings on the reproducibility of this new ELISA kit
for 2-OHE1 and 16-OHE1,
using urines collected for the original methodological study. We
compare these findings to GC-MS values and also investigate the
stability of the 2-OHE1 and
16-OHE1 metabolites after repeated freeze-thaw
cycles and preservation by boric acid.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reproducibility Study
Urine samples used were from five premenopausal women in the
mid-luteal phase of the menstrual cycle (four to six days prior to the
estimated start of the next menses; mean age, 39 years) and five
postmenopausal women (at least 3 years since their last menstrual
cycle; mean age, 56 years). To confirm a menstrual phase, premenopausal
women were contacted regarding the date of their subsequent menses. The
ELISA laboratory was sent 10 batches of urine at one time consisting of
five batches from premenopausal women and five batches from
postmenopausal women and was told the menopausal status of the batch.
In each batch, we shipped two blinded samples from each woman, for a
total of 10 tubes/batch. Tubes were placed randomly and in a different
order in each batch. The laboratory was instructed to assay one
batch/day, with premenopausal urines analyzed one week and
postmenopausal urines the next. Each aliquot was assayed in triplicate,
and the results were averaged.
Validity Study
The GC-MS laboratory received a single batch of 10 urines,
consisting of one aliquot from each of the five pre- and five
postmenopausal women participating in the reproducibility study. The
laboratory was told whether the sample came from a pre- or
postmenopausal woman.
Borate Study
An overnight 12-h urine was obtained from a female volunteer in
mid-luteal phase. After decanting into 25-ml conical tubes, boric acid
was added in the following amounts: 0, 1.25, 2.5, 3.75, 5, 7.5, 10, 20,
and 40 mg, with two tubes prepared at each concentration. The ELISA
laboratory received a total of 18 blinded aliquots in random order. All
samples were assayed on the same day. The assays were done in
triplicate (for a total of 54 measurements for each metabolite), with
the 2-OHE1 and 16-OHE1
metabolites from a particular aliquot measured on parallel plates.
Freeze-Thaw Study
An overnight 12-h urine sample was obtained from a female
volunteer in the mid-luteal phase of the menstrual cycle. The urine was
collected in a half-gallon container containing 1 teaspoon of boric
acid and then decanted into 25-ml conical tubes. Tubes were assigned
one of the following freeze-thaw treatments: zero, one, two, or three
cycles of thawing at room temperature and refreezing at -70°C. The
ELISA laboratory was sent two batches, each containing four frozen
aliquots of urine that received one of the four freeze-thaw treatments.
Aliquots were shipped in random order and assayed on the same day on
parallel plates in triplicate. For both 2-OHE1
and 16-OHE1, a total of 24 measurements was
obtained (two batches x four freeze-thaw treatments x three
measurements/treatment).
Laboratory Methods
ELISA.
The new ESTRAMET 2/16 kit measures 2-OHE1 and
16-OHE1 in urine using a modified version of
the recently developed competitive-inhibition ELISA kit
(19)
. The modified kit was made more sensitive than the
original by decreasing the relative amounts of specific antibodies and
increasing the enzyme activity of alkaline phosphatase in the
estrogen:enzyme conjugates. With the exception of modified antibody
concentrations, enzyme conjugates, and standards (0.625, 1.25, 2.5, 5,
10, and 20 ng/ml), all kit components and assay procedures in the new
kit are equivalent to the original kit. Urine samples, controls, and
standards were pipetted into individual microtubes in triplicate, and
190 µl of hydrolysis buffer containing ß-glucuronidase and
arylsulfatase from Helix pomatia were added to each tube,
followed by incubation for 2 h at room temperature. After this
time, the enzyme digest was neutralized using 200 µl of
neutralization buffer to bring the final pH to 7.0, and 75 µl of the
deconjugated urine were added to a 96-well plate coated with polyclonal
rabbit antimouse immunoglobulin. A conjugate buffer containing
metabolite-specific murine monoclonal antibodies freshly mixed with
either 2-OHE1:alkaline phosphatase or
16-OHE1:alkaline phosphatase was then added to
the respective assay plates. These wells were incubated for 3 h at
room temperature, the time required for maximum binding of the
estrogen:enzyme conjugates, following which, the plates were washed,
and enzyme substrate para-nitrophenylphosphate was added. The antibody
was thus captured on the solid phase with the antigen (estrogen
metabolite) labeled with the enzyme alkaline phosphatase, and the
enzyme product (a color dye) was inversely proportional to the
concentration of the free antigen. The absorbance in the wells was read
kinetically with a Ceres 900 HDI plate reader (Biotek Instruments,
Winooski, VT) at a wavelength of 410 nm, and the data were reduced
using Kineticale EIA Application software (Biotek Instruments). A
four-parameter fit of the log concentration versus
absorbance at 410 nm yielded a sigmoidal curve with a linear range of 1
to 15 ng/ml. Initially, all assays were carried out without dilution,
but when values did not fall within the linear part of the curve,
urines were diluted and reanalyzed. Both the
2-OHE1 and 16-OHE1
assays demonstrated 100% recovery of metabolites with serial dilution
and the addition of exogenous estrogens into urine samples. For
both the 2-OHE1 and
16-OHE1 metabolite, values were normalized per
mg creatinine; the laboratory-reported variabilities within and between
assays were similar for both metabolites, with within and between
assays CV of 6 and 10%, respectively. The new kit is currently
available for research use (Immuna Care Corp., Bethlehem, PA).
GC-MS Method.
After the hydrolysis of conjugates, isotope dilution GS-MS in the
selected ion monitoring mode was used to identify urinary
estrogens (16)
. A total of 14 estrogens, including
estrone, estradiol, estriol, 2-OHE1, 2-OHE2,
2-MeOHE1, 2-MeOHE2, 4-OHE1,
15-OHE1, 16-OHE1, 16-ketoestradiol
16-epiestriol, and 17-epiestriol, was measured. Estrogen conjugates
were extracted on Sep-PakC18 cartridges and
purified on the acetate form of DEAE-Sephadex. The samples were then
hydrolyzed using H. pomatia juice and purified on the
acetate form of QAE-Sephadex. Recovery after hydrolysis was estimated
to be 75–82% based on the addition of deuterated
(d5-)-ethoxime derivatives of all ketonic
estrogens as internal standards (20)
; these deuterated
estrogens were later used to correct for these losses. Estrogens with
vicinal cis-hydroxyls and diphenolic compounds were
fractionated on the borate and bicarbonate forms of QAE-Sephadex,
respectively. Neutral steroids were removed by the free base form of
DEAE-Sephadex, after which estrogens were separated into two groups
using Lipidx 5000 in a straight phase system. After trimethylsilyl
ether derivatization, estrogens were analyzed by capillary GC with
stable isotope dilution MS. Deuterated internal standards were
available for all of the estrogens except 16ß-hydroxyestrone and
17-epiestriol and were used to correct for losses after the hydrolysis
step. However, prior to the introduction of the deuterated internal
standards, it is estimated that 5–10% of the hormones may be lost
because of incomplete hydrolysis, and this loss cannot be quantified.
The laboratory was shipped 10 aliquots of urines and analyzed each
sample in duplicate. These duplicate runs were not blinded. For this
analysis, the duplicate results were averaged. For all of the
estrogens, the limit of detection varied from 0.5 to 3 nmol/l. The CVs
in premenopausal urine samples for the 10 major estrogens were reported
to range between 4 and 7%.
Statistical Methods
For the reproducibility, freeze-thaw, and borate concentration
components of this study, the means of triplicate readings were
analyzed on the logarithmic scale (log base 10) to reduce the
dependence of the SD of the response on the mean to satisfy assumptions
of parametric statistical models. All analyzes were performed
separately for 2-OHE1 and
16-OHE1 as well as on the ratio of the
metabolites. Metabolite values were adjusted for creatinine levels.
Reproducibility Study.
A nested, within-person ANOVA was used to test for assay
reproducibility. Variance components methods were used to model the
total variability in the laboratory measurements. Estimates of
variability among women in a given menstrual group
(
2
a), among batches for a
given women (2
b) and among
tubes for a given batch (2
) were estimated by
the SAS procedure VARCOMP (21)
. With
yij denoting the natural log of the
mean assay measurement over triplicates for woman I =
1, 2, 3, 4, 5 at batch j(I)= 1, 2, 3, 4, 5 on tube
k(ij), the model is:
 |
2
a,
2
b, and
2
. The variance components were used to
estimate the assay CV and the ICC =
2
a,/(2
a,+
2
b +
2
), i.e., the percentage of the
total variability explained by hormonal differences among the women. A
large ICC indicates that the range of hormone values in the population
is large relative to the assay variability and assures that the assay
can detect small but possibly relevant case-control differences in
hormone levels.
Validity Study.
Spearman correlation coefficients were used to compare the rank order
of urine values between the ELISA and GC-MS laboratories, and Wilcoxon
signed-rank tests were used to compare means.
Boric Acid Study.
Standard ANOVA methods were used to examine the natural logarithm of
the 2-OHE1:16-OHE1 ratio
by nine boric acid concentrations. Boric acid levels were regarded as
having fixed effects, the aliquot effects nested within boric acid
level, and replicate effects nested within the aliquot.
Freeze-Thaw Study.
Standard regression and ANOVA methods were used. The natural logarithms
of the metabolites or their ratio were considered the dependent
variables, with the number of freeze thaw cycles as the independent
variable. Analyzes were conducted within batch.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHE1 and their ratio are presented in Figs. 1
2
3
. For illustrative purposes, Fig. 1A
presents
2-OHE1 levels in premenopausal women, with the
leftmost symbol being the mean of the duplicates obtained by GC-MS, and
the values plotted at days 1–5 being the means of the triplicates
obtained by the ELISA kit each day. For each woman, two blinded
aliquots were analyzed on each of the five days. Each symbol represents
a different woman. These figures graphically display hormone
differences among women, measurement variability over 5 days for a
given woman, and variability among aliquots on a given day.
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). All assays
for postmenopausal women were successful with no dilution.
Urine specimens from one premenopausal woman had to be diluted in three
of five batches. Laboratory variability was somewhat lower in
postmenopausal urines, where the CV was 10% compared with 16% for
premenopausal samples (Table 1)
. In both groups, the range of 2-OHE1 values was
large, and the resulting ICCs were high.
|
-OHE1.-OHE1 were
observed (Fig. 2
); however, measurements were not consistent from one day to the next.
In both the pre- and postmenopausal urines, the variance component for
the batch was significant, and the CVs were >15% (Table 1)
.
2-OHE1:16-OHE1 Ratio.
No significant trends were observed (Fig. 3
) and the range of values for the ratio was much smaller than the
ranges of the metabolites. The ratio was consistently measured in
premenopausal samples, where the CV was 12% (Table 1)
. In
postmenopausal samples, values of the ratio were not consistent from
day to day, and the variance component for the batch was significant.
For both groups of women, differences among women accounted for most of
the variability in measurements of the ratio, and ICCs were 
90%.
Validity Study
The laboratory reported duplicate results from the single aliquot
provided for each woman, and for all analyses, we used the mean of
these duplicate values. Table 2
reports the geometric mean levels of the urinary metabolites and their
ratio according to menstrual group for both laboratories. The Spearman
rho indicates the correlation between laboratories, and P
from the signed-rank test indicates differences in absolute values.
|
. Among postmenopausal women, the
2-OHE1 values from the ELISA and GC-MS
laboratories were similar and highly correlated.
16-OHE1.
The ELISA values were lower than the GC-MS findings in all instances
and significant for premenopausal urines (Table 2)
. For all women,
16-OHE1 levels from both laboratories were
well correlated.
2-OHE1:16-OHE1 Ratio.
The 2-OHE1:16-OHE1 ratio
was higher using the ELISA kit (Table 1)
, particularly for
postmenopausal women. Despite differences in the absolute values,
the ratios were well correlated. Tests of the new ELISA kit found that
cross-reactivity was absent or minimal for both antibodies, with the
possible exception of the 16-OHE1 antibody
with 16-ketoestradiol. GC-MS values of this ligand were low, and
combining these values with the 16-OHE1
measurement did not alter the conclusions regarding ELISA
versus GC-MS estimates of the
2-OHE1:16-OHE1 ratio.
Borate Study
Overall, the total variability in these data is very small. The
ANOVA showed significant variation in the
2-OHE1:16-OHE1 by boric
acid level, but no linear dose-response pattern was evident. Fig. 4
plots the ratio values by boric acid concentration; the line connects
the mean of the blinded duplicates at each concentration. Analyzes of
2-OHE1 or 16-OHE1
measurements alone showed no significant difference in metabolite
according to level of boric acid. It is unlikely that these
concentrations of boric acid affect the ratio values because no trend
with level is apparent. Rather, we believe the significant findings are
a consequence of analyzing a ratio of measurements; this substantially
reduces the error variance and increases the power to detect aliquot
differences.
|
plots the logarithm of the
2-OHE1:16-OHE1 ratio
according to freeze-thaw treatment and batch. To distinguish the
batches, results are indicated by open diamonds or asterisks. The ANOVA
showed no effect of freeze-thaw cycling on measurement of the ratio of
the metabolites. However, a significant difference was observed between
the batches for all freeze-thaw treatments, with values from one batch
consistently lower than the other (P = 0.008; the
dashed line represents the fitted regression for one batch; the
straight line, for the other). No freeze-thaw cycling or batch effect
was observed for 2-OHE1 or
16-OHE1 alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHE1 shows that the kit
performs adequately with regard to reproducibility, validity, stability
after several freeze-thaw cycles, and after the addition of relatively
low concentrations of borate as a preservative. Results were for the
most part reproducible, with laboratory assay CVs between 10 and 20%
and ICCs ranging from 80 to 95%. Although the absolute values of the
metabolites differed by laboratory method, results were well
correlated.
Assessment of the earlier ELISA kit suggested that it was not
performing adequately in postmenopausal women, for whom CVs were close
to 20% for both the 2-OHE1 and the
16-OHE1 metabolites (18)
. Using
the new kit, 2-OHE1 and
16-OHE1 levels were more consistently measured
in these women, with CVs of 10 and 17%, respectively, and the CV for
the ratio was 18%. We note that these reproducibility measures are not
entirely comparable because the assays were performed 1 month apart in
the earlier study, whereas in this study, assays were performed 1 day
apart over the course of a week. Thus, we could not detect variability
that may arise in assays conducted over several months, such as
storage effects. Nevertheless, results for the new ELISA are
encouraging for studies in which assays can be completed in a
relatively short period of time. The relatively high ICCs ensure that
case-control differences in the
2-OHE1:16-OHE1 ratio can
be detected in large population-based studies. However, it is advisable
that matched cases and controls be batched for the assays, because
batch-to-batch variability was not negligible, particularly in
postmenopausal urines. Moreover, a batch effect was observed in the
freeze-thaw experiment.
Both the ELISA and GC-MS method have inherent strengths and
limitations. MS analysis furnishes quite accurate identification of
compounds, although losses of hormone metabolites that occur in the
steps before hydrolysis may have introduced quantitation problems. The
ELISA analysis involves substantially less sample preparation, but a
number of other constituents found in urine can interfere with its
binding activity; these compounds can vary from person-to-person based
on differences in metabolism as well as differences in other factors
such as diet. Because of these issues, one cannot designate either
method as representing "truth." We found the absolute values of
2-OHE1 and 16-OHE1, and
the ratio of the metabolites tended to be much lower when measured by
the new ELISA kit than when measured by GC-MS, but the correlations
between methods were generally high. Although it is highly desirable
that the new kit measures the metabolites accurately, the high
correlation between values from GC-MS and the new kit ensures that
individuals can be ranked comparably by both methods, which is critical
for large population-based research. Furthermore, the relative low cost
and ease of implementing the ELISA kit makes it amenable to the high
laboratory throughput required in such large-scale studies.
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Footnotes |
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1 To whom requests for reprints should be
addressed, at Environmental Epidemiology Branch, National Cancer
Institute, 6120 Executive Boulevard, MSC 7234, Suite 7070, Bethesda, MD
20892. 
2 The abbreviations used are: 2-OHE1,
2-hydroxyestrone; 4-OHE1, 4-hydroxyestrone;
2-OHE2, 2-hydroxyestradiol; 4-OHE2,
4-hydroxyestradiol; 2-MeOHE1, 2-methoxyestrone;
4-MeOHE1, 4-methoxyestrone; 2-MeOHE2,
2-methoxyestradiol; 4-MeOHE2, 4-methoxyestradiol;
16-OHE1, 16-hydroxyestrone; GC-MS, gas
chromatography-mass spectroscopy; CV, coefficient of variation; ICC,
intraclass correlation coefficient.
Received 7/13/99; revised 10/ 1/99; accepted 10/25/99.
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References |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxylase: a risk marker for breast cancer. Cancer Surv., 5: 574-583, 1986.
-hydroxylation in human breast tissue: a potential biomarker of breast cancer risk. J. Natl. Cancer Inst., 85: 1917-1920, 1993.-hydroxyestrone is lower in breast cancer patients that controls. Cancer Epidemiol. Biomark. Prev., 7: 85-86, 1998.[Medline]
-hydroxyestrone ratio: correlation with serum insulin-like growth factor binding protein-3 and a potential biomarker of breast cancer risk. Ann. Acad. Med. Singapore, 27: 294-299, 1998.[Medline]
-hydroxyestrone in urine. Steroids, 58: 648-654, 1994.
-hydroxyestrone in urine. Environ. Health Perspect., 105(Suppl.3): 607-614, 1997.
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. Straight line, the
slope from the regression of results from one batch; dashed
line, the slope from the other batch.