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Experimental Studies |
Department of Internal Medicine, Yonsei University College of Medicine (S.K.L., Y.J.W., J.H.L., S.H.K., E.J.L., K.R.K., H.C.L., K.B.H.); and the Doping Control Center, Korean Institute of Science and Technology (B.C.C.), Seoul, Korea
Address all correspondence and requests for reprints to: Sung Kil Lim, M.D., Department of Internal Medicine, Yonsei University College of Medicine, 134 Shin-chon Dong Sue Dae Moon Gu, 120–752 Seoul, Korea.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxyestrone [16-(OH)E1]
level was significantly lower in patients with spinal osteopenia
(P < 0.001). Among the 18 urinary metabolites of
estrogen, the 16-(OH)E1 and
16-(OH)E1/2-hydroxyestrone
[2-(OH)E1) ratio showed positive correlations
with spinal BMD (P < 0.05), whereas
2-(OH)E2 showed a negative correlation with
femoral neck BMD (P < 0.05). The urinary
16-(OH)E1 level also revealed a positive
correlation with the age-matched z score of BMD in the spine
(P < 0.05). In multiple stepwise regression
analysis, weight, 16-(OH)E1, interaction
between 16-(OH)E1 and
2-(OH)E2, 2-(OH)E2, and
years after menopause were statistically significant for spinal BMD
(r2 = 0.4968). For femoral neck BMD and weight,
16-(OH)E1 and 2-(OH)E2
were the independent determinants (r2 = 0.3369). In
conclusion, the activity of estrogen 16-hydroxylase was decreased
and/or the activity of estrogen 2-hydroxylase was enhanced in
postmenopausal osteopenia. We speculated that these derangements may
serve as contributing factors for the acceleration of bone loss in
postmenopausal osteoporosis. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Direct catheterization of the ovarian circulation has shown little contribution of estradiol (E2) from the ovary in postmenopausal women (5). Also, the rate of bone loss in patients with bilateral oophorectomy at premenopausal age is accelerated compared to that in women with natural menopause (2, 3). Women lose bone at different rates from 5–10 yr after menopause, even though there is no significant difference in their total serum E2 levels (6). These findings suggest that ovarian androgens as well as adrenal androgens may play an important role in the maintenance of bone after menopause, and conversely, that their low serum concentrations or the derangement of peripheral conversion to estrone (E1) might accelerate bone loss and could contribute to the development of osteoporosis (5). Recently, most studies have focused on the rate of conversion of androgens to E1, because the extent of aromatization of androstenedione to E1 is determined by differences in the rates of conversion rather than by differences in the production of the androgen precursor from the adrenal gland or ovary (7).
Although E1 is less potent, it is the major circulating estrogen in postmenopausal women (8). It is now well established that androstenedione is the major precursor of E1 and that the conversion is carried out by the aromatase of the adipose tissue (9). The adrenal androgen dehydroepiandrostenedione can also be converted to estrone by the aromatase cytochrome P450 of the osteoblast (10). E1 is further oxidized by microsomal enzyme into intermediate metabolites, including E2, which then react with cellular macromolecules and have many biological effects (11). Hence, the plasma E1 concentration may not accurately reflect the biological estrogen effect in postmenopausal women (12). However, there have been few studies about estrogen metabolism and urinary excretion of the metabolites in postmenopausal osteoporosis (13).
The two main pathways of estrogen metabolism consist of the
2-hydroxylation and the 16-hydroxylation pathways (14).
2-Hydroxyestrogens have been shown to have little estrogenic activity,
and in some experimental systems they have even been shown to have some
antagonistic activity (15). We hypothesized that there was a difference
among individuals in the ratio between the 2- and 16-hydroxylation
pathways, and that this difference may lead to the variation in the
development of postmenopausal osteopenia. This hypothesis was made
based on the findings that 1) the direct ovarian contribution to
circulating E1 is minuscule in postmenopausal women; 2) the
adrenal gland does not secrete estrogens; 3) the conversion of androgen
precursors to E1 by peripheral fat cells or osteoblasts
might contribute to maintain bone mineral density (BMD) (9, 16); and 4)
E1 is further oxidized into active or inactive metabolites
by microsomal enzymes.
In this study, we quantified the urinary metabolites of estrogen by
using the highly sensitive gas chromatography-mass spectrometry (GC-MS)
system. We found a decrease in the 16-hydroxylation of estrogen
and/or an increase in the 2-hydroxylation of estrogen in Korean women
with postmenopausal osteopenia.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Fifty-nine women were recruited for the measurement of 18 urinary metabolites of estrogen. All subjects studied were of Korean ethnic background, and all resided in Seoul, Korea, with ages ranging from 55–60 yr (mean age, 57.0 ± 0.5). The mean years after menopause was 6.6 ± 0.5. All of these women visited Severance Hospital for a general physical checkup and were in good health. The subjects were screened by a detailed questionnaire. Patients with a history of renal or liver disease, surgical menopause, cancer, heart disease, arthritis, hypertension, or diabetes mellitus; those who reported receiving estrogen replacement treatment or regularly consuming alcohol; or those who had surgical menopause or any other diseases were excluded. None of the subjects was a cigarette smoker, and those who exercised excessively were also excluded from this study. Thyroid function was evaluated by TSH immunoradiometric assay, and any subject with deviations from the normal value (0.35 < normal value < 3.5 IU/mL) was excluded. Osteopenia was defined as a BMD value more than 1 SD below the young adult mean value for Koreans. The study was approved by the research ethics committee of Severance Hospital, Yonsei University, and all subjects gave informed consent.
Measurement of BMD, serum E2, and E1
BMD was measured by a Lunar DPX machine (Lunar Radiation, Madison, WI). Serum E2 (Diagnostic Products Corp., Los Angeles, CA) and E1 (Diagnostic Systems Laboratories, Webster, TX) were measured by RIA.
Measurement of urinary estrogen metabolites
Chemicals. Estrogen standards were purchased from Sigma Chemical Co. (St. Louis, MO). d2-E2 used as an internal standard was purchased from MSD isotope (Montreal, Canada). Serdolit AD-2 resin (particle size, 0.1–0.2 mm) was purchased from Serva (Heidelberg, Germany). ß-Glucuronidase/arylsulfatase from Helix pomatia were purchased from Boehringer Mannheim (Mannheim, Germany); ß-glucuronidase activity was 5.5 U/mL (at 39 C), and arylsulfatase activity was 2.6 U/mL (at 38 C). Deionized water was distilled before use. Silylating reagents, N-methyl-N-trimethylsilytrifluoroacetamide and trimethylsilychloride were purchased from Sigma.
GC-MS. The Hewlett-Packard GC-MS system (model 5890A gas chromatography, model 5970B mass-selective detector) equipped with a cross-linked 5% phenylmethylsiloxane-fused silica capillary column Ultra-2 (id, 25 m x 0.2 mm; 0.33 µm) was used. The carrier gas was helium at a flow rate of 0.85 mL/min. The injection port, transfer line, and ion source were kept at 300, 300, and 200 C, respectively. The temperature program was set at 180 C, raised at 20 C/min to 260 C and kept constant for 6 min, then raised at 2 C/min to 275 C and kept constant for 8 min, and finally raised at 15 C/min to 300 C and kept constant for 10 min. The ionized energy was 70 eV.
Sample preparation. Each sample included overnight
collections of urine (from 2000–0800 h the next day). All women
totally fasted after dinner. In the present study, the period of urine
collection was 10 days. The samples were frozen with ascorbic acid
(
0.1–0.2%) and sodium azide (0.1%), and then stored.
Sample extraction. The urine sample (3 mL) and internal standard (d2-E2; 0.5 µg) were applied to the column of preconditioned Serodolit AD-2 resin. After washing with 3 mL water, the free and conjugated estrogens were eluted three times with 3 mL methanol. The eluate was evaporated until dry. To carry out enzyme hydrolysis, the residue was dissolved in 1 mL acetate buffer (0.2 N; pH 5.0) containing 50 µL ß-glucuronidase/arylsulfatase (from Helix pomatia) and ascorbic acid (1 mg/mL). The sample was incubated overnight at 37 C or for 3 h at 55 C. After the hydrolysis, 100 mg potassium carbonate were added, and the mixture was extracted with 5 mL ethyl acetate, then the organic layer was evaporated until dry.
Derivatization. The residue was dissolved in 50 µL of the reagent mixture (N-methyl-N-trimethylsilytrifluoroacetamide-trimethylsilychloride, 100:1 volume ratio) and heated for 30 min at 60 C. After heating, 2-µL aliquots were injected into the GC column by an autosampler.
Assay. The following estrogens were determined:
E1, 17ß-E2, 2-hydroxyestrone
[2-(OH)E1], 2-hydroxyestradiol [2-(OH)E2],
2-methoxyestrone, 17-E2, 6-dehydroxyestrone,
6-hydroxyestradiol, 4-methoxyestradiol, estriol, 16-epiestriol,
16,17-epiestriol, 16-hydro-xyestrone
[16-(OH)E1], 17-epiestriol, 6-ketoestriol,
2-methoxyestriol, 6-hydroxyestriol, and 16-ketoestradiol. All values
were corrected by the concentration of urinary creatinine.
All urine samples were analyzed in separate batches for the two groups within a 1-month period together with one duplicate quality control sample for each batch. The quality control samples used were pooled urine samples from normal individuals.
The recovery range of this method was 80.97–97.81%. It was found to be reproducible and quantitative. The relative SD range of intraday analysis was 0.24–10.52%, and that of interday analysis was 1.05–10.24%.
Statistical analysis
The statistical significance of differences between groups was determined by the Kruskal-Wallis test. The Pearson correlation coefficient was used to analyze the univariate relationship between urinary metabolites and BMD. Factors related to BMD were analyzed using stepwise multiple regression. P < 0.05 was accepted as the level of significance.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). There
was no significant difference in mean age (56.8 and 57.2 yr) or years
after menopause (6.8 and 6.4 yr) between the groups.
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). Among the 18 estrogen metabolites,
16-(OH)E1 and the
16-(OH)E1/2-(OH)E1 ratio
showed positive correlations with spinal BMD, whereas
2-(OH)E2 showed a negative correlation with BMD
of the femoral neck (Table 3 and Fig. 1). There were no
significant differences in serum E1 and
E2 levels between the patients with osteopenia
and the nonosteopenic subjects (Table 1
). In patients with spinal and
femoral osteopenia, urinary excretions of total estrogen were not
significantly different from those in the nonosteopenic subjects (Table 2). However, the
16-(OH)E1 level was significantly lower in
patients with spinal osteopenia than that in the nonosteopenic subjects
(P < 0.05), and 2-(OH)E1 was
significantly higher in patients with femoral osteopenia than that in
the nonoteopenic subjects (P < 0.05).
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-(OH)E1 showed a positive correlation with
the age-matched z score (Table 4). However, there were
no correlations between the estrogen metabolites and the age-matched z
score in the femoral neck.
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-(OH)E1, interaction between
16-(OH)E1 and 2-(OH)E2,
2-(OH)E2, and years after menopause were
statistically significant for spinal BMD (r2 = 0.4968). As
for femoral neck BMD, weight, 16-(OH)E1, and
2-(OH)E2 were the independent determinants
(r2 = 0.3369; Tables 5 and 6). From these results, we speculated that a low
conversion to 16-(OH)E1 and/or a high
conversion to 2-(OH)E2 may serve as independent
risk factors for the development of osteopenia.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylation of estrogen
in patients with osteopenia, may aid in developing a better
understanding of the pathogenesis of postmenopausal osteopenia. It is widely believed that there is a strong acceleration of bone loss that begins before or as menopause commences (3). Pouilles et al. reported that spinal bone loss is approximately 3% in the first year after menopause, but rapidly decreases to 2% in the second year and to less than 1% by the fourth year after menopause, and there was no evidence of further acceleration of bone loss from their longitudinal study (17). There is, however, a considerable individual variation and debate with regard to the course of bone loss associated with aging (18). There are those who lose bone more quickly than others and those who show more positive responses to estrogen replacement than other individuals (6, 19). However, we do not yet know the nature of these variations.
Direct catheterization of the ovarian circulation has shown little contribution of E2 from the postmenopausal ovary, with the remainder from the conversion from E1, which, in turn, is derived from the aromatization of androstenedione (9, 10). E1, a less potent estrogen than E2, can be further metabolized to E2 at the cellular level in estrogen-sensitive target tissue, so that the plasma E1 concentration may not accurately reflect the biological estrogen effect in postmenopausal women (12). Urinary assays continue to be of value in clinical practice because the changes in estrogen metabolites in urinary excretion may represent metabolic, rather than secretory, changes (11, 20), and urinary steroid profiles have been investigated for their potential usefulness as biochemical markers of diseases (13, 15, 21). To overcome this limitation of the measurement of serum estrogens in postmenopausal women, we determined the levels of urinary metabolites of estrogen simultaneously by using a sensitive GC-MS system. In this study, the sample preparation step was improved by the extraction of steroids with Serdolit AD-2 resin and the derivatization of steroids by trimethysilyation to enhance their specificity on gas chromatography.
All subjects were slim, and adjustments for BMI had, in general, only a
negligible effect on any of the estrogen results. Also, BMI was not a
determinant of BMD in multiple regression analysis (results not shown).
Even though a significant association was found between height and
fecal plus urinary estrogen excretion in a previous study (21), we did
not find any association between height and urinary estrogen
metabolites in our subjects (data not shown). Through multiple
stepwise regression analysis, 16-(OH)E1 was
found to be the important determinant of spinal BMD along with body
weight and years after menopause despite the interaction between
16-(OH)E1 and 2-(OH)E2.
16-(OH)E1 and 2-(OH)E2
were also the determinants of femoral neck BMD with body weight. The
above findings clearly reflected that the alteration of hydroxylation
of estrogen could be important for determination of BMD in
postmenopausal osteopenia.
The two main pathways of estrogen metabolism are the
2-hydroxylation and 16-hydroxylation pathways (14). To determine the
relationship between the rate of change in estrogen metabolism and the
development of breast cancer, a Finnish group studied the ratio of
2-hydroxylated to 16-hydroxylated estrogens in Finnish women (11).
16-Hydroxyestrone binds covalently to primary amino groups in
proteins, more specifically, the estrogen receptor, which can prolong
its effect in target organs such as the breast (22). There is also good
evidence that 2-hydroxyestrogens have little estrogenic activity and
that they may even be antagonistic in some experiment systems (15).
Catecholestrogens have a t1/2 in the circulation of about
17 s, so what is measured in the urine are catecholestrogens
formed in the liver and immediately conjugated, and hence they are
inactive (23). Thus, any shift in the ratio of 2-hydroxylated to
16-hydroxylated estrogen is biologically significant. In our study,
16-hydroxylation of estrogen was significantly decreased in spinal
osteoporotics and 2-hydroxylation of E2 was
enhanced in femoral osteoporotics.
We do not know whether such changes in estrogen metabolism have any
direct effects on bone. When we recruited the patients, we screened out
those with the known enhancing factors of 2-hydroxylation of estrogen,
such as cigarette smoking, heavy exercise, and hypothyroidism (23).
However, it is possible that some unknown acquired factors that exert
influence on bone directly or indirectly might enhance the rate of
2-hydroxylation and decrease the rate of 16-hydroxylation of
estrogen and lead to low bone mass. We also do not know whether this
change in estrogen metabolism influences bone mass directly or
indirectly through action on the extra-bony target organs of estrogen
such as intestine and kidney. Another plausible explanation is that
this difference in estrogen metabolism may be one of genetic origins.
Genetic factors affecting enzyme activities are frequently important
determinants in the disposition of drugs including exogenous and
endogenous hormones as well as their efficacy and toxicity (24). Racial
patterns based on the genetic polymorphism of the cytochrome P450
enzyme have been proposed as the causes of some observed differences in
estrogen metabolism between premenopausal Orientals and Caucasians
(25). Further studies to answer these issues are still pending.
In conclusion, the rate of 16-hydroxylation and/or 2-hydroxylation
of estrogen metabolism was deranged in Korean postmenopausal
osteoporotics, and the decreased rate of 16-hydroxylation and/or the
increased rate of 2-hydroxylation of estrogen metabolism could serve as
contributing factors for the acceleration of bone loss.
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Acknowledgments |
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Received September 25, 1996.
Revised January 3, 1997.
Accepted January 10, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylase activity in women with breast
and endometrial cancer. J Steroid Biochem. 20:1077–1081.[CrossRef][Medline]
-hydroxyestrone to estradiol receptor in human
breast cancer cells: characterization and intranuclear localization. Proc Natl Acad Sci USA. 85:7831–7835.This article has been cited by other articles:
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