Estrogen Metabolites

Jacob Schor, ND, FABNO

Ten years ago, I commented in this journal about the association between estrogen metabolites and the risk for breast cancer. What I wrote was that the then current science strongly suggested a definitive lack of association between 2-hydroxyestrone to 16α-hydroxyestrone ratio and breast cancer risk. It was clear that risk could not be calculated from lab values of these metabolites and that encouraging patients to test and rely on the results was ethically questionable. I expressed these opinions rather strongly, even when opposed by several eminent practitioners.

It is always difficult to admit that you were wrong about something, but it turns out that I was wrong on this matter. That’s why I’m writing this article now.

1982 is as good a year to begin this story about estrogen metabolism and breast cancer as any. That’s when Fishman and Bradlow used a then newly developed method to track estrogen biotransformations by injecting women with radioactive estrogen and then using serial blood draws, tracking the rate and extent of oxidation measured. Their technique allowed the estimation of the levels of various estrogen metabolites. Fishman and Bradlow compared test results from 33 women with breast cancer with 10 controls and reported that those with breast cancer had 60% higher 16α-hydroxyestrone than healthy women.1 In short order 16α-hydroxyestrone became a marker for elevated breast cancer risk,2 and we started referring to it as the bad estrogen metabolite, in contrast to 2-hydroxyestrone which became the good estrogen. A ratio of the two metabolites became our accepted predictor of risk. It all made good sense.

Except that the data ten years ago failed to support this belief. My initial article was triggered by Mackey et al’s 2012 study.3 It was a prospective case-controlled study nested within the Women’s Health Initiative Hormone Trials (WHI-HT). From that large cohort, confirmed breast cancer cases were matched to controls. Using stored blood serum, biomarkers were measured at baseline and, for those receiving active treatment, at one year. It was a decent-sized study and included 845 women with confirmed breast cancer and 1,690 matched controls. Using stored blood serum, measurements of 16α-hydroxyestrone and 2-hydroxyestrone levels were measured by EIA at baseline and, for those randomized to active treatment (n=1,259), at one year. The women in the WHI-HT study were receiving hormone therapies, estrogen, estrogen plus progesterone, or placebo. The data singled out neither the 16α-hydroxyestrone nor the 2:16 ratio as a reliable predictors of cancer risk.

At that time there had already been eight decent-sized cohort studies published on whether breast cancer was associated with these 2-hydroxyestrone:16α-hydroxyestrone ratios and the results were far from consistent. In those earlier studies, none of the women were using exogenous estrogens. None of the results reached clear statistical significance. Mackey et al was the exception, perhaps because the women were using exogenous hormones. In that trial, which was probably the largest to date, there was a significant 28% increase in risk comparing the highest to lowest quintile of the 2:16 ratio (95% CI=1.00 to 1.63; p-trend=.03). If the ratio had been predictive then we would have thought those in the highest quintile should have had the lowest risk.

A year or so earlier, in October 2011, in the British Journal of Cancer, Zeleniuch-Jacquotte et al reported that this metabolite ratio had no predictive value for endometrial cancer.4

Perhaps the most critical data were those reported by Obi et al. Their February 2011 meta-analysis also asked whether estrogen metabolite ratios changed breast cancer risk. Data from nine prior studies comprising 682 premenopausal cases and 1189 postmenopausal breast cancer cases were combined. In comparing the “… highest compared with the lowest quantile of urinary EMR [estrogen metabolite ratio], nonsignificant associations suggested at best a weak protective effect in premenopausal but not in postmenopausal breast cancer (range of odds ratios: 0.50-0.75 for premenopausal and 0.71-1.31 for postmenopausal)… Circulating serum/plasma EMR was not associated with breast cancer risk. …. Results of the prospective studies do not support the hypothesis that EMR can be used as a predictive marker for breast cancer risk.”5

These were large well-conducted trials that did not support the original small 1982 study’s outcome. I wrote back then something to the effect that we should drop our belief in estrogen ratios.

I was prompted to relook at this issue by a recent email from Dr. Tori Hudson who asked me if there was anything new on this subject because in her observations, the belief that testing and treating the ‘ratio’ was still common in our profession. Dr. Hudson taught many of my classes when I attended naturopathic school at NCNM and I will forever owe her my gratitude for putting up with me. It is unlikely that I was a pleasant student to have in her classroom. Because of her prompting, I’ve been reading through papers that have been published in the intervening years since I first wrote about these estrogen metabolites.

The most important of these papers is likely the one by Regina Ziegler and her colleagues working at the Epidemiology and Biostatistics Program at the National Cancer Institute, that reported results from an epidemiologic study in which they utilized a new and more sensitive technique to measure metabolite levels, that was published in April 2015.6

Ziegler writes that the inconsistency of the study results we had seen previously were due to the inaccuracy of the testing methodology employed. Researchers were measuring estrogen metabolites using enzyme immunoassays (EIA), a detail that I had failed to notice. I’ll spare you the detailed biochemistry here but suffice to say that this methodology lacks the precise discrimination needed to perform assays for this purpose. The eight cohort studies previously mentioned that either lacked significance or in the case of Mackay’s results were the reverse from those predicted, used EIA to measure estrogen metabolites.

Ziegler and colleagues developed a sophisticated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the 15 estrogens/estrogen metabolites that includes a complex purification technique and this has proven to be far more accurate and reliable than the older EIA methodology.7 Their new technology is so accurate that they can measure estradiol levels in post-menopausal breast cancer patients taking aromatase inhibitors.8 This methodology led to the rapid publication of several studies that were made possible by using stored urine samples preserved from prior studies.

All these newer studies used the new LC-MS/MS techniques of Zeigler et al to measure estrogen metabolites. Four were published by 2015, more were in the works at that time.

NHS II – USA: In February 2012 Heather Eliassen and colleagues at the Channing Laboratory at Harvard, using the new methods compared 15 metabolites in urine from 19,521 women. The metabolites of those who eventually were diagnosed with breast cancer (n-247) were matched and compared to two controls each (n-485). Among other significant findings, they reported, “… there was a significant increased risk with higher 16-pathway/parent estrogen metabolite ratio (comparable RR = 1.61; 95% CI, 0.99-2.62; P(trend) = 0.04).”9

PLCO – USA: Fuhrman et al conducted a prospective case-control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) that included postmenopausal women with invasive breast cancer (n=277) and matched controls (n=423). 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). This effect outweighed that of all metabolites, but the ratio of the 2-hydroxylation pathway to parent estrogens (HR = 0.66, 95% CI = 0.51 to 0.87) was significant suggesting that the 2-OH conversion is protective.10

Roni Falk et al studied 215 women with breast cancer along with 215 controls and reported in April 2013 that “more extensive hydroxylation along the 2-pathway may have a reduced risk of postmenopausal breast cancer.”11

B-FIT – USA: In women with breast cancer (n=407), a higher ratio of the 2/16-hydroxylation pathways was associated with reduced risk (HRQ5vsQ1 = 0.60; 95% CI: 0.40-0.90; P trend = 0.002).12

Shanghai Cohort: Moore et al matched postmenopausal invasive breast cancer cases (n=399) with an equal number of matched controls who were participants in the Shanghai Women’s Health Study cohort and again compared estrogens and metabolites. Concentrations of parent estrogens were strongly associated with breast cancer risk; women in the upper quartile compared to those in the lower quartile of estrogen levels had nearly double the risk of getting cancer (ORQ4vsQ1 = 1.94, 95% CI = 1.21 to 3.12, Ptrend = .01). The initial ratios showed significant benefit if the 2-pathway was favored in breaking down initial estrogens, dropping risk by about 40%. Risks also seemed to drop about 25% when these numbers were adjusted for initial estrogen levels and the association sought for 2-pathway effect but lost their statistical significance.

It was postulated that the larger effect in this Shanghai cohort was because their initial estrogen levels are lower than in American women and so as a result the effect of the 2-pathway had greater significance. It may be in the US where initial estrogen levels are higher in post-menopausal women, their impact will overshadow the effect of 2-pathway conversion. “Lower urinary parent estrogen concentrations and more extensive 2-hydroxylation were each associated with reduced postmenopausal breast cancer risk in a low-risk nation. Markedly higher total estrogen/estrogen metabolite concentrations in postmenopausal United States women (Asian Americans) than in Shanghai women may partly explain higher breast cancer rates in the United States.”13 This may be ethnic genetic variations, or lifestyle differences that explain varying risks between Chinese vs US immigrants.14

That last paper was published in 2016, and I find little new information to add since then. The estrogen metabolite ratios play a significant role in breast cancer risk even if it is outweighed by estrogen levels as a predictor.

There have been several clinical trials of natural products, that have been tested in human clinical trials to see whether they improve these metabolite ratios.

A study by Maggie Laidlaw et al, published in 2010 tested a combination of HMR lignan, indole-3-carbinol, calcium glucarate, milk thistle, Schisandra chinesis and stinging nettle. Forty-seven pre-menopausal and forty-nine post-menopausal women were recruited for this study and each woman consumed either treatment or placebo for 28 days. In the pre-menopausal women, treatment resulted in a significant increase (P < 0.05) in urinary 2-OHE concentrations and in the 2:16α-OHE ratio. In post-menopausal women, treatment resulted in a significant increase in urinary 2-OHE concentrations. In pre- and post-menopausal women combined, treatment supplementation produced a significant increase in urinary 2-OHE concentration and a trend (P = 0.074) toward an increased 2:16α-OHE ratio.15

In 2022 a trial of a product called Estrosense was published in the Journal of Complementary and Integrative Medicine. In this randomized, double-blind, cross-over, multicenter, placebo-controlled study in which women (n= 148) were randomized to a treatment sequence that consisted of either EstroSense®, followed by placebo or vice-versa. The product contains calcium D-glucarate, indole-3-carbinol, green tea extract, turmeric extract, rosemary extract, lycopene, sulforaphane, and milk thistle seed extract. After 12 weeks of intervention, the mean urinary 2-OHE1:16α-OHE1 was 4.55 (2.69, 6.42) (p<0.001) higher than placebo adjusted.16

Another trial of interest that looked at the effect of 75 mg/day of Diindolylmethane (DIM) for 30 days was published in 2023 (n=60).17 An earlier 2017 trial had demonstrated a significant improvement in estrogen metabolite ratios in women taking DIM (n=130), but the subjects in this trial were also taking tamoxifen and they took DIM for a full 12 months.18

While these ratios are associated with human breast cancer risk, we are still awaiting information that confirms the link is causal, that altering the ratio will alter risk. The fact that these products are advertised to relieve symptoms of estrogen dominance encourages this writer to think that they provide relief from discomfort and whether it provides long-term benefit might not be as important to patients as if the products work or not, providing moderate-term symptom relief.

And there we have it. I was wrong. My apologies to one and all.

References

  1. Schneider J, Kinne D, Fracchia A, et al. Abnormal oxidative metabolism of estradiol in women with breast cancer. Proc Natl Acad Sci U S A. 1982;79(9):3047-3051.
  2. Bradlow HL, Hershcopf R, Martucci C, Fishman J. 16 alpha-hydroxylation of estradiol: a possible risk marker for breast cancer. Ann N Y Acad Sci. 1986;464:138-151.
  3. Mackey RH, Fanelli TJ, Modugno F, et al. Hormone therapy, estrogen metabolism, and risk of breast cancer in the Women’s Health Initiative Hormone Therapy Trial. Cancer Epidemiol Biomarkers Prev. 2012 Nov;21(11):2022-2032.
  4. Zeleniuch-Jacquotte A, Shore RE, Afanasyeva Y, et al. Postmenopausal circulating levels of 2- and 16α-hydroxyestrone and risk of endometrial cancer. Br J Cancer. 2011;105(9):1458-1464.
  5. Obi N, Vrieling A, Heinz J, Chang-Claude J. Estrogen metabolite ratio: Is the 2-hydroxyestrone to 16α-hydroxyestrone ratio predictive for breast cancer? Int J Womens Health. 2011 Feb 8;3:37-51.
  6. Ziegler RG, Fuhrman BJ, Moore SC, Matthews CE. Epidemiologic studies of estrogen metabolism and breast cancer. Steroids. 2015;99(Pt A):67-75. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722219/pdf/nihms677594.pdf
  7. Xu X, Roman JM, Issaq HJ, Keefer LK, Veenstra TD, Ziegler RG. Quantitative measurement of endogenous estrogens and estrogen metabolites in human serum by liquid chromatography-tandem mass spectrometry. Anal Chem. 2007;79(20):7813-7821.
  8. Lee JS, Ettinger B, Stanczyk FZ, et al. Comparison of methods to measure low serum estradiol levels in postmenopausal women. J Clin Endocrinol Metab. 2006;91(10):3791-3797.
  9. Eliassen AH, Spiegelman D, Xu X, et al. Urinary estrogens and estrogen metabolites and subsequent risk of breast cancer among premenopausal women. Cancer Res. 2012;72(3):696-706.
  10. Fuhrman BJ, Schairer C, Gail MH, et al. Estrogen metabolism and risk of breast cancer in postmenopausal women. J Natl Cancer Inst. 2012;104(4):326-339.
  11. Falk RT, Brinton LA, Dorgan JF, et al. Relationship of serum estrogens and estrogen metabolites to postmenopausal breast cancer risk: a nested case-control study. Breast Cancer Res. 2013;15(2):R34. Published 2013 Apr 22.
  12. Dallal CM, Tice JA, Buist DS, et al. Estrogen metabolism and breast cancer risk among postmenopausal women: a case-cohort study within B~FIT. Carcinogenesis. 2014;35(2):346-355.
  13. Moore SC, Matthews CE, Ou Shu X, et al. Endogenous Estrogens, Estrogen Metabolites, and Breast Cancer Risk in Postmenopausal Chinese Women. J Natl Cancer Inst. 2016;108(10):djw103. Published 2016 May 18.
  14. Visvanathan K, Yager JD. Ethnic Variations in Estrogen and Its Metabolites: Sufficient to Explain Differences in Breast Cancer Incidence Rates? [published correction appears in J Natl Cancer Inst. 2016 Sep;108(9). pii: djw187. doi: 10.1093/jnci/djw187]. J Natl Cancer Inst. 2016;108(11):djw147. Published 2016 Jul 5. https://academic.oup.com/jnci/article/108/11/djw147/2905658?login=false
  15. Laidlaw M, Cockerline CA, Sepkovic DW. Effects of a breast-health herbal formula supplement on estrogen metabolism in pre- and post-menopausal women not taking hormonal contraceptives or supplements: a randomized controlled trial [published correction appears in Breast Cancer (Auckl). 2012;6:101]. Breast Cancer (Auckl). 2010;4:85-95. Published 2010 Dec 16. doi:10.4137/BCBCR.S6505
  16. Green T, See J, Schauch M, et al. A randomized, double-blind, placebo-controlled, cross-over trial to evaluate the effect of EstroSense® on 2-hydroxyestrone:16α-hydroxyestrone ratio in premenopausal women. J Complement Integr Med. 2022;20(1):199-206.
  17. Godínez-Martínez E, Santillán R, Sámano R, Chico-Barba G, Tolentino MC, Hernández-Pineda J. Effectiveness of 3,3′-Diindolylmethane Supplements on Favoring the Benign Estrogen Metabolism Pathway and Decreasing Body Fat in Premenopausal Women. Nutr Cancer. 2023;75(2):510-519
  18. Thomson CA, Chow HHS, Wertheim BC, et al. A randomized, placebo-controlled trial of diindolylmethane for breast cancer biomarker modulation in patients taking tamoxifen. Breast Cancer Res Treat. 2017;165(1):97-107.

Published November 18, 2023

About the Author

Jacob Schor, ND, now retired, had a general practice with a focus on naturopathic oncology in Denver, Colorado. He served as Abstract & Commentary Editor for the Natural Medicine Journal for several years (https://www.naturalmedicinejournal.com/) and posts blog articles on natural therapies, nutrition, and cancer (https://drjacobschor.wordpress.com/). He is a board member of CoAND and past president of OncANP, and someone who is happier outdoors than inside.