Estrogen Metabolite Ratios

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Estrogen Metabolite Ratios – A Naturopathic Perspective Essay

Over the years, scientists have conducted extensive research on the causative agents of cancer. One such research set out to find the causative agents of breast cancer. From research, estrogen compounds have been associated with breast cancer. The two key determinants have been found to be 2- hydroxyesterone and 16α-hydroxyestrone. It was found that 2- hydroxyesterone was a beneficial compound in the body as its high percentage in the breast lowers the prevalence of breast cancer while 16α-hydroxyestrone was found to be non- beneficial. High amounts of 16α-hydroxyestrone were found to be the main factor in influencing breast cancer.

This theory of percentages of 2- hydroxyesterone and 16α-hydroxyestrone has been supported over years. According to the theory, all women have these two compounds and what differ are their percentages in the body. If one has high percentage of 2- hydroxyesterone than 16α-hydroxyestrone then she is at a lower risk of suffering from breast cancer. Estrogen Metabolite Ratios – A Naturopathic Perspective Essay. On the other hand, if one has high percentage of 16α-hydroxyestrone compounds in the breast than 2- hydroxyesterone, then she is in a very high risk of suffering from breast cancer.

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In recent years, other researchers have countered the theory. They have found out that the theory was not based on statistical facts rather, very few scientists paid attention on the significance of statistical analysis. A lot of effort has since then been put on the research to determine whether estrogen metabolites are determinants of breast cancer or not. In one of the researches, it was found that the percentage of the two metabolites only applied to women who had not reached their menopause. That is those who feared the risk of breast cancer were those with high percentages of 16α-hydroxyestrone than 2- hydroxyesterone but this was the case only to a limited number of women- those below their menopause age.

In another research, determination of percentages of the two estrogen metabolites, and their effect to breast cancer, it was found that in the selected group of women, no significant difference was ascertained. Women with high ratios of 2- hydroxyesterone and those with 16α-hydroxyestrone had no difference all the same. This research again disapproves the initial theory of estrogen metabolites being key determinants of breast cancer. In a few researches however, there were found a relationship between the estrogen metabolites and breast cancer. Estrogen Metabolite Ratios – A Naturopathic Perspective Essay.

In most cases in the research findings, 2- hydroxyesterone and 16α-hydroxyestrone do not have any influence as to whether one will suffer from breast cancer neither do their percentages in the one’s body control cancer for those already suffering from breast cancer. This again contradicts the theory, which has been embraced and appreciated by many medics over years (Schor 1).

A lot of research and significant data has been done to disapprove the theory of2- hydroxyesterone and 16α-hydroxyestrone in controlling or reducing breast cancer. For instance one researcher, gave breast cancer patients a diet rich in flaxseed which increases 16α-hydroxyestrone which according to the estrogen metabolite theory is harmful but results indicated that the consumption of flaxseed did not influence the risk of cancer. It is therefore evident from recent research that the theory of estrogen metabolite is not true (Schor 1).

Scientist, researchers and medics should therefore embrace and appreciate the facts from recent research findings. There is no type of estrogen compound in the body should be referred to as beneficial or not beneficial and so medics should not recommend dietary restrictions on patients based on whether the foods or supplements are rich in the metabolites.

Opinion from reference

Consumption of flaxseed as a supplement, which increases levels of 16α-hydroxyestrone, has been found not to have any influence in controlling risks of breast cancer in women who have reached their menopause. Medics should not recommend dietary restrictions on patients based on whether the foods or supplements are rich in the estrogen metabolites.

Work Cited

Schor, Jacob. “Estrogen Metabolite Ratios.” Naturopathic Doctor News & Reviews. 2014. Web. http://www.ndnr.com/womens-health/estrogen-metabolite-ratios/

Estrogens are recognized causal factors in breast cancer. Interindividual variation in estrogen metabolism may also influence the risk of breast cancer and could provide clues to mechanisms of breast carcinogenesis.Estrogen Metabolite Ratios – A Naturopathic Perspective Essay. Long-standing hypotheses about how estrogen metabolism might influence breast cancer have not been adequately evaluated in epidemiological studies because of the lack of accurate, reproducible, and high-throughput assays for estrogen metabolites.

Methods
We conducted a prospective case–control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Participants included 277 women who developed invasive breast cancer (case subjects) and 423 matched control subjects; at PLCO baseline, all subjects were aged 55–74 years, postmenopausal and not using hormone therapy, and provided a blood sample. Liquid chromatography–tandem mass spectrometry was used to measure serum concentrations of 15 estrogens and estrogen metabolites, in unconjugated and conjugated forms, including the parent estrogens, estrone and estradiol, and estrogen metabolites in pathways defined by irreversible hydroxylation at the C-2, C-4, or C-16 positions of the steroid ring. We calculated hazard ratios (HRs) approximating risk in highest vs lowest deciles of individual estrogens and estrogen metabolites, estrogens and estrogen metabolites grouped by metabolic pathways, and metabolic pathway ratios using multivariable Cox proportional hazards models. All statistical tests were two-sided.

Results
Nearly all estrogens, estrogen metabolites, and metabolic pathway groups were associated with an increased risk of breast cancer; the serum concentration of unconjugated estradiol was strongly associated with the risk of breast cancer (HR = 2.07, 95% confidence interval [CI] = 1.19 to 3.62). No estrogen, estrogen metabolite, or metabolic pathway group remained statistically significantly associated with the risk of breast cancer after adjusting for unconjugated estradiol.Estrogen Metabolite Ratios – A Naturopathic Perspective Essay. The ratio of the 2-hydroxylation pathway to parent estrogens (HR = 0.66, 95% CI = 0.51 to 0.87) and the ratio of 4-hydroxylation pathway catechols to 4-hydroxylation pathway methylated catechols (HR = 1.34, 95% CI = 1.04 to 1.72) were statistically significantly associated with the risk of breast cancer and remained so after adjustment for unconjugated estradiol.

Conclusions
More extensive 2-hydroxylation of parent estrogens is associated with lower risk, and less extensive methylation of potentially genotoxic 4-hydroxylation pathway catechols is associated with higher risk of postmenopausal breast cancer.

CONTEXT AND CAVEATS
Prior knowledge
Serum estrogen concentration is an established predictor of breast cancer risk in postmenopausal women, but whether biomarkers of estrogen metabolism also predict the risk of breast cancer is unclear because of the lack of accurate, reproducible, sensitive, and high-throughput assays for estrogen metabolites.

Study design
A prospective case–control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial examining associations between the risk of breast cancer in postmenopausal women not currently using menopausal hormone therapy and serum concentrations of 15 estrogens and estrogen metabolites, in unconjugated and conjugated forms, including the parent estrogens and estrogen metabolites in pathways defined by irreversible hydroxylation at the C-2, C-4, or C-16 positions of the steroid ring, as measured by liquid chromatography–tandem mass spectrometry.

Contribution
The serum levels of nearly all estrogens, estrogen metabolites, and metabolic pathway groups were associated with an increased risk of breast cancer, but none of the associations remained statistically significant after adjusting for serum level of unconjugated estradiol. The ratio of 2-hydroxylation pathway estrogen metabolites to parent estrogens and the ratio of 4-hydroxylation pathway catechols to 4-hydroxylation pathway methylated catechols were statistically significantly associated with the risk of breast cancer, even after adjustment for unconjugated estradiol. Estrogen Metabolite Ratios – A Naturopathic Perspective Essay.

Implications
More extensive 2-hydroxylation of parent estrogens is associated with lower risk of postmenopausal breast cancer, and less extensive methylation of potentially genotoxic 4-hydroxylation pathway catechols is associated with higher risk of postmenopausal breast cancer.

Limitations
The study population was restricted to postmenopausal women who were not using menopausal hormone therapy at the time of blood collection, which may limit the generalizability of the findings. There was limited inter-individual variation in serum concentrations of estrogens and estrogen metabolites and high correlations among many of the analytes. There was no adjustment for multiple comparisons and thus, some of the findings could be due to chance.

From the Editors

Prospective studies of postmenopausal women have consistently demonstrated that higher levels of circulating estradiol, estrone, and estrone sulfate are associated with an increased risk of breast cancer (1). These associations have long been thought to result from mitogenic effects mediated by the estrogen receptor.

It has been hypothesized that estrogen metabolism may also play a role in breast cancer etiology. The parent estrogens (ie, estrone and estradiol) can be irreversibly hydroxylated at the C-2, C-4, or C-16 positions of the steroid ring (Figure 1) to produce estrogen metabolites that differ in their bioavailability to breast tissues (3) and activation of estrogen receptors (4). In addition, catechol estrogens, which are formed by 2- and 4-hydroxylation, can be oxidized to form mutagenic quinones (5–7); this process is prevented by methylation of one of the adjacent hydroxyl groups (8). Estrogens and estrogen metabolites can also be conjugated via sulfation or glucuronidation, each of which modifies bioavailability (9). Estrogen Metabolite Ratios – A Naturopathic Perspective Essay.

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Figure 1
Pathways of estrogen metabolism. Adapted from Ziegler et al. (2) and reproduced with permission from Environmental Health Perspectives. The estrogen metabolites are formed by irreversible hydroxylation of the parent estrogens, estrone and estradiol, at the C-2, C-4, or C-16 positions of the steroid ring. The relative size of the chemical structure indicates the relative concentration of the estrogen or estrogen metabolite in serum of postmenopausal women. The structures are for the unconjugated forms of estrogens and estrogen metabolites.

Laboratory and clinical studies have suggested several hypotheses about how estrogen metabolism might influence the risk of breast cancer (10–13). However, robust tests of these hypotheses in population studies have not been possible due to the limitations of the available assays for measuring concentrations of estrogens and estrogen metabolites. The radioimmunoassays and enzyme-linked immunosorbent assays used in recent decades to measure circulating estradiol and other parent estrogens often displayed low specificity due to antibody cross-reactivity and thus limited accuracy, especially at the low estrogen concentrations characteristic of postmenopausal women. Substantial variations in measures of estrogens and estrogen metabolites have been noted across laboratories, assay protocols, and assay kit manufacturing lots (14,15). Moreover, individual assays have not been developed for many estrogen metabolites. However, the recent development (16) of an accurate, reproducible, and relatively high-throughput liquid chromatography–tandem mass spectrometry (LC/MS/MS) assay to measure concurrently 15 estrogens and estrogen metabolites, in conjugated and unconjugated forms, even at the low concentrations characteristic of postmenopausal women, allows for the first time characterization of this phenotype for epidemiological study.

We used the new assay to compare the estrogen and estrogen metabolite profiles in prospectively collected serum from postmenopausal women with and without breast cancer who reported no use of exogenous hormones at the time of blood collection.Estrogen Metabolite Ratios – A Naturopathic Perspective Essay. Because this is the first large nested case–control study of breast cancer, to our knowledge, to measure the 15 estrogens and estrogen metabolites in circulation, five of which are found in both conjugated and unconjugated forms, we have systematically evaluated the association of each with breast cancer. We also evaluated the associations of total estrogens and estrogen metabolites (the sum of all estrogens and estrogen metabolites), seven metabolic pathway groups, and eight metabolic pathway ratios with the risk of breast cancer. The groups and ratios were based on biochemistry, metabolic pathways, and etiologic hypotheses from laboratory and clinical research. We analyzed estrogens and estrogen metabolites, individually and in total, and the metabolic pathway groups and ratios, both alone and in combination with the serum concentration of unconjugated estradiol, which is acknowledged as a strong predictor of breast cancer risk (1), to examine whether any of these measures of estrogen metabolism contributed additional independent information for predicting the risk of breast cancer. We discuss our results with respect to previous hypotheses about mechanisms of estrogen-mediated carcinogenesis, and we consider the use of estrogen and estrogen metabolite profiles for projecting breast cancer risk.

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Methods
Study Design and Population
Incident breast cancer case subjects and control subjects were drawn from the 39 116 female participants, aged 55–74 years, who were randomly assigned from 1993 through 2001 to the screening arm of the multicenter Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) (17). At PLCO baseline, participants completed self-administered questionnaires that covered personal characteristics, medical history, and health-related behaviors. Also at baseline, blood samples were drawn at the screening centers and processed within 2 hours according to a standardized protocol (17). Follow-up was conducted using annual questionnaires that were mailed to the participants. Estrogen Metabolite Ratios – A Naturopathic Perspective Essay. This study was approved by Institutional Review Boards at the US National Cancer Institute and the 10 participating screening centers.

Of the 39 116 female participants randomly assigned to the screening arm of the PLCO, 97% completed a baseline questionnaire and at least one annual follow-up questionnaire. Of these women, 94% were postmenopausal at baseline; of these women, 47% reported no use of menopausal hormone therapy at baseline. Among these postmenopausal non–hormone therapy users, 75% provided blood at baseline and written informed consent for use of the specimen.

In the PLCO, incident breast cancers were ascertained primarily via annual questionnaires; incident breast cancers were also identified through state cancer registries, the National Death Index, physician reports, and next-of-kin reports. Of the breast cancers ascertained by these methods, 96.4% were subsequently confirmed by hospital records (17).

For the initial studies of breast cancer in the PLCO, the cohort of eligible women was defined as all women randomly assigned to the PLCO screening arm who had completed the baseline questionnaire and at least one study update questionnaire, reported no history of breast cancer at baseline, provided blood at baseline or during follow-up, and given written informed consent (16) (Supplementary Table 1, available online). As of June 30, 2005, 1141 incident breast cancers had been ascertained within the study cohort. Control subjects (n = 1141) were randomly selected from women in the study cohort who were alive and free of breast cancer as of June 30, 2005, and were frequency matched to case subjects on age at study entry (55–59, 60–64, 65–69, 70–74 years) and period of blood collection (before vs on or after the median collection date, September 30, 1997). Estrogen Metabolite Ratios – A Naturopathic Perspective Essay.

The subjects for this analysis were drawn from this set of 1141 case subjects and 1141 control subjects, as presented in detail in Supplementary Table 1 (available online). In brief, 440 case subjects and 525 control subjects were postmenopausal and not using hormone therapy at baseline; of these, 424 case subjects and 506 control subjects also had no other cancer, other than nonmelanoma skin cancer, diagnosed during the follow-up period. We excluded an additional 57 case subjects and 70 control subjects who did not have sufficient baseline serum available for the biochemical studies. Also excluded were a total of 13 case subjects and 13 control subjects with extreme values for serum estrogens and estrogen metabolites. Extreme values were defined as those for which the sum of all estrogens and estrogen metabolites was either lower than the 25th percentile of the distribution minus three times the interquartile range, or higher than the 75th percentile of the distribution plus three times the interquartile range.

The remaining 354 case subjects and 423 control subjects represent 80% and 81%, respectively, of the 440 case and 525 control subjects who were postmenopausal and not using hormones at baseline. Of the 354 breast cancer case subjects, 75 had in situ disease and two could not be histologically confirmed and were excluded from the analyses. The final analytic sample included 277 case subjects with histologically confirmed invasive breast cancer and 423 control subjects.

Finally, we considered how these exclusions might affect the characteristics of the study subjects. Of the 1141 potential case subjects and 1141 potential control subjects identified, similar percentages (94%) were postmenopausal at baseline (1076 and 1073, respectively) (Supplementary Table 1, available online). However, of these postmenopausal women, case subjects were more likely (n = 636, or 59%) than control subjects (n = 548, or 51%) to be users of menopausal hormone therapy at baseline, a difference we anticipated given the demonstrated association between menopausal hormone therapy use and risk of breast cancer (18,19). The women not using hormone therapy at baseline, who were included in the analysis, were statistically significantly different with regard to several characteristics from those using hormone therapy, who were excluded; but the differences were similar for case subjects and control subjects (Supplementary Tables 2 and 3, respectively, available online). Estrogen Metabolite Ratios – A Naturopathic Perspective Essay. Specifically, among both the potential case subjects and potential control subjects, those not on hormone therapy tended to be older at baseline, black, more likely to report natural menopause, less likely to have a history of benign breast disease, and more likely to have entered the study early.

By contrast, the subsequent exclusions among non-hormone users (due to insufficient baseline serum for biochemical assays, extreme values for total estrogens and estrogen metabolites, or in situ rather than invasive breast cancer, if a case subject) resulted in only one statistically significant difference between the included and excluded case and control subjects (Supplementary Tables 4 and 5, available online). Only menopausal hormone therapy use before baseline differed statistically significantly between women included and those excluded in this analysis, and only in the control subjects. Thus, the included control subjects were reasonably representative of female participants in the PLCO who were postmenopausal and not using hormone therapy at baseline, and the included case subjects were reasonably representative of the incident cases of invasive breast cancer that occurred among these women.

Laboratory Assays
Serum samples that were stored at −80°C since collection were thawed at 4°C and used for measurement of estrogens and estrogen metabolites. Estrone and estradiol in blood remain stable for years during long-term storage at −70°C or lower (20).

Stable isotope dilution LC/MS/MS was used to measure concurrently 15 serum estrogens and estrogen metabolites, including the parent estrogens (ie, estrone and estradiol); metabolites in the 2-hydroxylation pathway (ie, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestrone-3-methyl ether); metabolites in the 4-hydroxylation pathway (ie, 4-hydroxyestrone, 4-methoxyestrone, and 4-methoxyestradiol); and metabolites in the 16-hydroxylation pathway (ie, 16α-hydroxyestrone, estriol, 17-epiestriol, 16-ketoestradiol, and 16-epiestriol). Details of the method for measuring serum estrogens and estrogen metabolites, including sample preparation and assay conditions, have been published previously (16). For this study, we used six stable isotopically labeled standards to account for losses of the 15 estrogens and estrogen metabolites: deuterated 2-hydroxyestradiol, 2-methoxyestradiol and estriol (C/D/N Isotopes Inc, Pointe-Claire, QC, Canada); deuterated 16-epiestriol (Medical Isotopes Inc, Pelham, NH); and 13C-labeled estrone and estradiol (Cambridge Isotope Laboratories, Andover, MA). Estrogen Metabolite Ratios – A Naturopathic Perspective Essay.

Serum from postmenopausal women contains 15 estrogens and estrogen metabolites. All are found in conjugated forms, attached to sulfate or glucuronide moieties; five (ie, estrone, estradiol, 2-methoxyestrone, 2-methoxyestradiol, and estriol) also exist in unconjugated form. Steps for measurement of unconjugated estrogens and estrogen metabolites in serum included addition of the six stable isotopically labeled standards, extraction with dichloromethane, derivatization with dansyl chloride, and LC/MS/MS. An additional step—enzymatic hydrolysis using a preparation from Helix pomatia with β-glucuronidase and sulfatase activity (Sigma Chemical Co, St Louis, MO)—enables the sum of the unconjugated and conjugated forms of each estrogen or estrogen metabolite to be measured. Subsequently, for each estrogen or estrogen metabolite, the quantity of the conjugated form was calculated as the difference between this sum and the measure of the unconjugated form.

Briefly, to measure estrogens and estrogen metabolites, we added the stable isotopically labeled standards to 1.0-mL serum samples, split the serum samples into two equal aliquots, and hydrolyzed one of the two aliquots with the β-glucuronidase–sulfatase enzyme preparation. We then extracted, derivatized, and performed LC/MS/MS on each aliquot. The single chemical derivatization added a dansyl group (1-dimethyl-amino-naphthalene-5-sulfonyl) to the phenolic hydroxyl present on all estrogens and estrogen metabolites. The bulky charged dansyl group facilitates measuring the neutral lipophilic steroids by mass spectrometry, a technique that separates and detects compounds on the basis of charge and molecular weight.

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For this study, we used serum samples from six postmenopausal women selected to cover a range of circulating estrogen and estrogen metabolite concentrations as quality control samples. Four quality control samples, including two aliquots from the same subject, were randomly inserted in every batch of approximately 40 samples. Estrogen Metabolite Ratios – A Naturopathic Perspective Essay. Laboratory personnel were blinded to both case–control status and quality control samples. Total laboratory coefficients of variation were less than 5% for all individual estrogens and estrogen metabolites measured and less than 3% for estrone, estradiol, and estriol. The published limit of quantitation (ie, the lower limit of absolute concentrations at which reliable precise readings can be obtained) is 8 pg/mL serum (26.5–29.6 pmol/L) for each estrogen and estrogen metabolite (16). However, results from this study suggest that the limit of quantitation is considerably lower. The mean serum concentration of unconjugated estradiol for the six quality control samples was 15.8 pmol/L. The total laboratory coefficient of variation for unconjugated estradiol in the quality control samples was less than 2% and included both within- and between-batch variation over 6 months and all steps of the analytic procedure. Moreover, the total laboratory coefficients of variation for the two least abundant estrogen metabolites—4-methoxyestradiol (mean serum concentration = 2.9 pmol/L) and 17-epiestriol (mean serum concentration = 1.4 pmol/L)—were 4% and 3%, respectively. These low coefficients of variation indicate that estrogen and estrogen metabolite concentrations of 1–2 pmol/L are above the limit of quantitation of our assay. No assays of estrogens or estrogen metabolites in this study resulted in non-detectable readings. Estrogen Metabolite Ratios – A Naturopathic Perspective Essay.