Estrogen Metabolism Assessment Test
Topic - Estrogen Metabolism Assessment Test
Essence of the Article:
In a January 2013 article in Townsend Letter Dr. Jacob Schor calls into question the usefulness of looking at estrogen metabolites, specifically the 2/16a-hydroxyestrone ratio, as an insight into vulnerability to related cancers.
While initial studies as early as 1982 supported the link between test findings and estrogen related cancers, more recent and larger studies do not.
"Cauley et al. also failed to report significant association when their study was published in 2003. Estrogen metabolites of 272 women with breast cancer were compared with 291 controls. There was no significant difference in the ratio of between the groups."
There is a rebuttal by Drs. Jonathan Wright and Thomas Klug which is in turn answered by Dr. Schor. A very good exchange worthy of your time to read it all.
Essential Thinking: The most important point here is to make an informed decision on the use and interpretation of this test, and a lot of information is provided in the Townsend piece to do just that.
While the validity of the Estrogen Metabolism Assessment test itself may now be in question, there may be a silver lining. Nutritional substances typically used for a "bad" ratio may indeed provide benefit through alternative routes, including aromatase inhibition, suppression of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, and activation of AMP-activated protein kinase (AMPK) . DIM (diindolymethane) or I3C (indole-3-carbinol), compounds found in cruciferous vegetables, have produced substantial benefits in various tumors.
Article
Estrogen Metabolite Ratios: Time for Us to Let Go
by Jacob Schor, ND, FABNO
http://www.townsendletter.com/Jan2013/estrogen0113.html
For the past 30 years we have relied on a theory that particular estrogen metabolites stimulate hyperproliferation in the hormone sensitive tissues of the breast and uterus and so increase risk of cancer development. Recent studies call this theory into question; it is time to assess whether we should still rely on these ideas for judging patient risk and more importantly for guiding patient treatment. Let us take a few moments to put this story in perspective.The first mention of what we call the "estrogen metabolite ratio" theory was published in May 1982, in a paper by Jill Schneider et al. in the Proceedings of the National Academy of Science. Newly devised radiometric techniques allowed these researchers to accurately measure estrogen metabolites in 33 women with breast cancer and compare their levels with 10 women without cancer. Their data revealed a significantly higher rate of 16-hydroxylation in the cancer patients. These pathways can produce compounds that are potent estrogens, and Schneider et al. were the first to wonder whether this could have a bearing on the etiology of the disease.1Metabolism of estradiol is oxidative and consists of an initial oxidation of a 17-beta-hydroxy group to yield estrone. Estrone is metabolized mainly through two alternate pathways, either hydroxylation at the C-2 or the 16-a position. These two pathways compete for substrate and yield products that, while both biologically active, have different properties. The 16a-hydroxyestrones are tissue stimulating, similar to estradiol. The 2-hydroxyestrogens, in contrast, have almost no estrogenic effect. Thus Schneider et al. theorized that "the relative amounts of specific estrogen metabolites rather than the quantity of the secreted parent substrate increase the risk for the disease [breast cancer] either by prolonging estrogenic activity or by virtue of the unique biological properties of a particular metabolite." They suggested that elevated levels of 16a-hydroxyestrogens could increase risk of breast cancer.Two years later, Fishman along with Schneider and two other colleagues reported in the April issue of the Journal of Steroid Biochemistry that they had measured increased activity of the 16a-hydroxylase activity in women with breast or endometrial cancer. The increase in enzyme activity preceded clinical evidence of disease and so was seen as a significant prognostic or risk factor for estrogen dependent tumors.2Two more years pass, this is 1988 now, and Fishman and Swaneck bring us the next piece to the story. They reported that 16a-hydroxyestrone binds in such a way to the estradiol receptors on breast cancer cells that it sticks, causing long lasting stimulation. As they put it, " … the estrogen bound extensively and irreversibly … "3These early studies did not show up on my radar. The first that I read about the estrogen metabolite theory was in a 2002 article published in Alternative Medicine Review. The article was written by Lord, Bongiovanni, and Bralley, all working for MetaMetrix Clinical Laboratory.By the time of their review, the evidence had grown stronger. Meilahn's prospective study published in 1998 in the British Journal of Cancer had measured estrogen metabolites in 5104 women aged 35 and older with a median follow-up time to cancer diagnosis of 9.5 years. Using the ratio of 2-hydroxyestrone to 16a-hydroxyestrone as a biomarker, they reported that those in the highest tertile compared with those in the lowest tertile showed a 30% lower risk of breast cancer. These differences however did not reach statistical significance (OR = 0.71, 95% CI = 0.29–1.75).4 Even so, they sounded good.What was exciting about the Alternative Medicine Review is that Lord et al. not only brought this theory of how these metabolites could effect cancer risk to our attention, they revealed how we might ameliorate the potential threat that these "bad estrogens" posed through diet and supplements. Apparently all that patients would need do was to consume more cruciferous vegetables. It was all so naturopathic. According to the Lord article, these foods are high in indolymethyl glucosinolates, which in turn release indole-3-carbinol. In 2000 Kishida had reported that high doses of 1-3-carbinol fed to mice reduced 16a-hydroxyestrone.5 Thus, in theory, a woman could increase her 2/16a-hydroxyestrone ratio using diet and supplements, and so lower her risk of cancer. This theory is well summed up on the popular website Natural News:
… the estrogen compounds called 2-hydroxyestrone and 16-a-hydroxyestrone are by far the most important for breast health. 2-hydroxyestrone is considered 'The Good Estrogen' because its presence doesn't seem to increase breast cancer risks, and MAY even be protective against it. However, 16-a-hydroxyestrone is considered 'The Bad Estrogen' because its presence seems to INCREASE breast cancer risk and has even been called a cancer CAUSING agent! All women have both estrogens, but each woman has a different RATIO, and this ratio is more important to health than the total AMOUNTS of the estrogens.6This estrogen metabolite theory quickly became accepted and adopted into alternative clinical practice. It sounded good; it made sense; labs could test hormone metabolites; patients would buy supplements and, if they took them, shift the lab results. There is a problem that we should mention: ongoing research has not supported this theory.There have been at least seven decent clinical studies published over the years that have not found a significant association between these estrogen metabolites and breast cancer. Significance is found for specific subgroups in some trials but these subgroup results lack consistency.Lack of statistical significance in the association between metabolites and breast cancer risk in the Melihan trial from 1998 was already mentioned. A paper by Muti et al. published in November 2000 in Epidemiology also failed to find significant association. Muti conducted a prospective nested case-control study of 10,786 women enrolled in the prospective Italian study "Hormones and Diet in the Etiology of Breast Cancer" (ORDET). After an average of 5.5 years of follow-up, 144 breast cancer cases and 4 matched controls for each case were identified among the participants of the cohort. Among premenopausal women, a higher ratio of 2-hydroxyestrone to 16a-hydroxyestrone at baseline was associated with a nonsignificant reduced risk of breast cancer: women in the highest quintile of the ratio had an adjusted odds ratio (OR) for breast cancer of 0.58 (95% confidence interval [CI] = 0.25–1.34). The odds ratio in postmenopausal women was 1.29 (95% CI = 0.53–3.10).7 Though nonsignificant, the trend could support the theory for premenopausal women but oppose it in postmenopausal women.Cauley et al. also failed to report significant association when their study was published in 2003. Estrogen metabolites of 272 women with breast cancer were compared with 291 controls. There was no significant difference in the ratio of 2- to 16a-hydroxyestrone between the groups. The risk of breast cancer in women with the highest quartile of this ratio compared with those in the lowest quartile was 1.17 (95% confidence interval = 0.73–1.87).8 In this case, though nonsignificant, the trend was opposite of what the theory predicts.Nor did the results of Wellejus et al., published in 2005, support the theory. Data were collected from a cohort of 24,697 postmenopausal Danish women. During follow-up, 426 breast cancer cases were identified. Higher 2-hydroxyestrone levels significantly increased incidence of estrogen receptor-positive breast cancer among current hormone replacement therapy (HRT) users. Higher levels of 16a-hydroxyestrone nonsignificantly lowered risk of estrogen receptor positive breast cancer.9 Again, these trends were opposite of what one would expect based on the theory.Modugno et al. in 2006 did report a significant positive association between 16a-hydroxyestrone in women not taking hormone replacement therapy, especially if they had high BMIs. This was a nested case-control study using data collected from 200 women who developed breast cancer and 200 healthy controls. The 16a-hydroxyestrone levels were modestly yet still significantly higher in HRT users among breast cancer patients. But 2-hydroxyestrone levels were also substantially and significantly higher in HRT users among breast cancer patients. No associations between BMI, estrogen metabolism, and breast cancer risk were found for HRT users. For non-HRT users only, greater BMI and higher 16a-OHE1 levels were individually and jointly associated with increased breast cancer risk (OR for women with high BMI and high 16a-OHE1 compared with those with low BMI and low 16a-hydroxyestrone = 3.51, 95% CI = 1.34–9.16). Estrogen metabolism differs according to both BMI and HRT use, potentially explaining the interaction between BMI and HRT in relation to breast cancer risk.10A 2008 paper is also worth mentioning. Heather Eliassen and colleagues at Harvard's Channing Laboratory reported on their attempt to sort out the impact of these estrogen metabolites. They conducted a prospective case-control study using data and blood samples from the Nurses' Health Study. They tested for 2-hydroxyestrone and 16a-hydroxyestrone in blood samples collected between 1989 and 1990 and compared levels in 340 cases of breast cancer with 677 matched controls. Neither estrogen metabolite appeared to change breast cancer risk. Nor did the ratio between the two metabolites make a significant difference. There was, however, a significant positive association observed for the 2-hydroxyestrone and the 2:16 ratio among women with ER-negative and PR-negative tumors. High numbers for either were associated with triple the relative risk for this small subgroup of breast cancer patients. Again these results are reverse from what the theory predicts. While these results don't support the basic hypothesis that 2-hydroxyestrone lowers breast cancer risk nor that the 2- versus 16-a ratio is predictive of risk, the significant link found with hormone receptor negative breast cancer is worth noting.11 Then there is Arslan et al. 2009: In this case 377 premenopausal breast cancer patients were compared with an equal number of matched controls. Again, " … no significant associations were observed between breast cancer risk and serum levels of 2-hydroxyestrone, 16a-hydroxyestrone, or their ratio."12Examining the results of these various clinical trials led one recent reviewer to conclude, "On the whole, prospective epidemiological data do not support the hypothesis that the 2-hydroxyestrogen pathway is protective, and the 16a-hydroxyestrogen pathway harmful, in hormone-dependent cancers" (Zeleniuch-Jacquotte).Perhaps the most important paper so far is a February 2011 meta-analysis by Obe et al. 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."13 (emphasis added)The data are no more supportive for endometrial cancer. A paper published in October 2011 in the British Journal of Cancer deserves our attention. Zeleniuch-Jacquotte et al. conducted a case-controlled study of 179 endometrial cancer cases and 336 controls. No significant association was observed for the 2- versus 16a ratio. Their results did not support the hypothesis that " … greater metabolism of estrogen through the 2-hydroxy pathway, relative to the 16a-hydroxy pathway, protects against endometrial cancer."14Perhaps the most recent look at this estrogen metabolite theory comes from Mackey et al. published in August 2012. Data that came from 845 women with breast cancer were matched to 1690 control patients. Mackey reports a modest positive association between higher baseline levels of 2-hydroxyestrone and larger 2:16 ratio with breast cancer risk. Again, this is reverse from what the EMR theory suggests. With hormone replacement therapy, breast cancer risk was associated with greater increases in 2-hydroxyestrone and larger 2:16 ratio, but these associations were not significant. Increasing amounts of 16a-hydroxyestrone that resulted from hormone replacement therapy were not associated with breast cancer.15 A modest increase in risk for breast cancer was found in those women with a higher 2-hyrdroxyestrone level at baseline. Increased 16a-hydroxyestrone was not associated with greater risk. Read that over if you need to. This is reverse from what the EMR theory predicts.These multiple large trials have not produced significant or convincing evidence in support of the good vs. bad estrogen theory. At the same time that all this has been going on, there is some evidence that 16a-hydroxyestrone is actually protective in other ways. A 2009 study in the American Journal of Hypertension reported that there is an inverse association between 16a-hydroxyestrone and systolic blood pressure in women; that is, the higher the 16a levels, the lower their blood pressure.16 A July 2011 paper by Patel et al. reported that 16a-hydroxyestrone levels were affected by fiber intake. Soluble fiber has a greater impact than dietary fiber. Dietary fiber is higher in grains and beans while fruits and vegetables provide more soluble fiber. CYP1A2, one of the cytochrome P450 enzymes made by the liver and important in the formation of 16a-hydroxyestrone, is increased by soluble fiber in the diet. Patel hypothesizes that women who eat a lot of fruits and vegetables – because the soluble fiber in these foods increases CYP1A2, which in turn increases 16a-hydoxyestrone – will have lower systolic blood pressures.17 2-hydroxyestrone levels are affected by genetics and lifestyle factors including, weight, smoking, and consumption of hydroxybenzoic acid, anthocyanidins, wine, and caffeine. 18 Exercise generally doesn't increase the 2:16 ratios except in women with very low initial 2:16 ratios; for this subgroup, losing weight and exercising does seem to shift production toward greater 2-hydroxyestrone.19,20 Some would argue though that the "proof is in the pudding." In other words, that supplements which we have associated with "improving the ratio" (increasing the 2:16 ratio) lower breast cancer risk. Even this seems to be unraveling. One example is soy food intake. In a January 2012 study, Morimoto et al. reported that high soy intake (more than 2 servings per day) compared with low soy intake (< 3 servings per week) increased the 2:16a-OH E(1) ratio, a result of a nonsignificant decrease in 16a-OH.21But a more recent paper, published in September 2012 and conducted by the same group on more participants for a longer time, did not find the same effect from high soy consumption: "Contrary to our hypothesis and some previous reports, the results from two well-controlled dietary interventions do not support an effect of a high-soy diet on a panel of urinary estrogen metabolites and the 2-OH/16a-OHE(1) ratio."22Flaxseed consumption would not reduce risk of breast cancer according to this theory as it increases 16a levels, Sturgeon et al. reported in 2010. They fed 43 postmenopausal women 7.5 g/day of ground flaxseed for 6 weeks, and then increased the dose to 15 g/day for an additional 6 weeks. There was no significant change in 2-hydroxyestrone excretion in the urine. The urinary 2:16 ratio was lower at the end of 12 weeks compared with baseline. The authors write, "Based on the current paradigm of the effects of estrogen metabolism on breast cancer risk, the regimen of dietary flaxseed intake used in this study did not appear to favorably alter breast cancer risk through shifts in estrogen metabolism pathways in postmenopausal women."23 In other words, flaxseed meal shouldn't be good for breast cancer risk.As naturopathic physicians, we are often ahead of the curve in translating new theories published in the scientific literature into clinical protocols for use with our patients. This "early adopter" tendency has its merits. We will sometimes find ways to help our patients when "regular" medicine has yet to develop a treatment. Because we limit our interventions to relatively nontoxic, low-risk therapies, we set the bar relatively low for our requirements of proof before experimenting with new ideas. We can justify our experimentation with a "might help and won't hurt" summation of risk analysis. If we were using more dangerous therapies, we would surely raise the bar, asking for stronger evidence before trying a new idea. Thus adopting this estrogen metabolite theory early on before it was well proven did not threaten to hurt anyone if it eventually turned out to be wrong. Except that many people, patients in particular and also some practitioners, forgot that it was theory and considered it proven fact. Being an early adopter does come with responsibility; if a new idea doesn't pan out, we need to abandon it and we need to let others know. It is easier for us to take on new ideas than it is to let go of them. In the case of estrogen metabolites, the theory that the 2-hydroxy form is good for women and that 16a-hydroxyestrone is bad is not holding up. Growing evidence suggests that there is little correlation between these hormones and cancer risk; the situation is more complex than we at first thought. There may be other theories that will make sense of this. My colleagues, after reading the results of these studies, quickly scramble to find alternate theories, "… perhaps there's another metabolite that's the key, maybe the 4 hydroxy?" Maybe, but maybe not.Sometimes you've just got to admit when you were wrong and move on.
It is past time that we let this particular idea go.
Jacob Schor, ND, FABNO
Fellow, American Board of Naturopathic Oncology
President, Oncology Association of Naturopathic Physicians (www.Oncanp.org)
Assistant Editor, Natural Medicine Journal (www.NaturalMedicineJournal.com)
Chair Speaker Selection Committee, Annual Convention of the American Association of Naturopathic Physicians (www.Naturopathic.org)
Notes
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 May;79(9):3047–3051.
2. Fishman J, Schneider J, Hershcope RJ, Bradlow HL. Increased estrogen-16 alpha-hydroxylase activity in women with breast and endometrial cancer. J Steroid Biochem. 1984 Apr;20(4B):1077–1081.
3. Swaneck GE, Fishman J. Covalent binding of the endogenous estrogen 16 alpha-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7831–7835.
4. Meilahn EN, De Stavola B, Allen DS, et al. Do urinary oestrogen metabolites predict breast cancer? Guernsey III cohort follow-up. Br J Cancer. 1998 Nov;78(9):1250–1255.
5. Kishida T, Beppu M, Nashiki K, Izumi T, Ebihara K. Effect of dietary soy isoflavone aglycones on the urinary 16alpha-to-2-hydroxyestrone ratio in C3H/HeJ mice. Nutr Cancer. 2000;38(2):209–214.
6. Knox K. Ratio of good to bad estrogens influences your breast cancer risk [online article]. Natural News.com. http://www.naturalnews.com/027227_cancer_breast_estrogen.html.
7. Muti P, Bradlow HL, Micheli A, et al. Estrogen metabolism and risk of breast cancer: a prospective study of the 2:16alpha-hydroxyestrone ratio in premenopausal and postmenopausal women. Epidemiology. 2000 Nov;11(6):635–640.
8. Cauley JA, Zmuda JM, Danielson ME, et al. Estrogen metabolites and the risk of breast cancer in older women. Epidemiology. 2003 Nov;14(6):740–744.
9. Wellejus A, Olsen A, Tjonneland A, Thomsen BL, Overvad K, Loft S. Urinary hydroxyestrogens and breast cancer risk among postmenopausal women: a prospective study. Cancer Epidemiol Biomarkers Prev. 2005 Sep;14(9):2137–2142.
10. Modugno F, Kip KE, Cochrane B, et al. Obesity, hormone therapy, estrogen metabolism and risk of postmenopausal breast cancer. Int J Cancer. 2006 Mar 1;118(5):1292–1301.
11. Eliassen AH, Missmer SA, Tworoger SS, Hankinson SE. Circulating 2-hydroxy- and 16alpha-hydroxy estrone levels and risk of breast cancer among postmenopausal women. Cancer Epidemiol Biomarkers Prev. 2008 Aug;17(8):2029–2035.
12. Arslan AA, Shore RE, Afanasyeva Y, Koenig KL, Toniolo P, Zeleniuch-Jacquotte A. Circulating estrogen metabolites and risk for breast cancer in premenopausal women. Cancer Epidemiol Biomarkers Prev. 2009 Aug;18(8):2273–2279.
13. Obi N, Vrieling A, Heinz J, Chang-Claude J. Estrogen metabolite ratio: Is the 2-hydroxyestrone to 16a-hydroxyestrone ratio predictive for breast cancer? Int J Womens Health. 2011 Feb 8;3:37–51.
14. 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 Oct 25;105(9):1458–1464. doi: 10.1038/bjc.2011.381. Epub 2011 Sep 27.
15. 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 Aug 29.
16. Masi CM, Hawkley LC, Xu X, Veenstra TD, Cacioppo JT. Serum estrogen metabolites and systolic blood pressure among middle-aged and older women and men. Am J Hypertens. 2009 Nov;22(11):1148–1153.
17. Patel S, Hawkley LC, Cacioppo JT, Masi CM. Dietary fiber and serum 16a-hydroxyestrone, an estrogen metabolite associated with lower systolic blood pressure. Nutrition. 2011 Jul–Aug;27(7–8):778–781.
18. Sowers MR, Crawford S, McConnell DS, et al. Selected diet and lifestyle factors are associated with estrogen metabolites in a multiracial/ethnic population of women. J Nutr. 2006 Jun;136(6):1588–1595.
19. Campbell KL, Westerlind KC, Harber VJ, Bell GJ, Mackey JR, Courneya KS. Effects of aerobic exercise training on estrogen metabolism in premenopausal women: a randomized controlled trial. Cancer Epidemiol Biomarkers Prev. 2007 Apr;16(4):731–739.
20. Westerlind KC, Williams NI. Effect of energy deficiency on estrogen metabolism in premenopausal women. Med Sci Sports Exerc. 2007 Jul;39(7):1090–1097.
21. Morimoto Y, Conroy SM, Pagano IS, Isaki M, Franke AA, Nordt FJ, Maskarinec G. Urinary estrogen metabolites during a randomized soy trial. Nutr Cancer. 2012;64(2):307–14. Epub 2012 Jan 31.
22. Maskarinec G, Morimoto Y, Heak S, et al. Urinary estrogen metabolites in two soy trials with premenopausal women. Eur J Clin Nutr. 2012 Sep;66(9):1044–1049.
23. Sturgeon SR, Volpe SL, Puleo E, et al. Effect of flaxseed consumption on urinary levels of estrogen metabolites in postmenopausal women. Nutr Cancer. 2010;62(2):175–180.
Jacob Schor, ND, FABNO, has practiced as a naturopathic physician in Denver, Colorado, with his wife, Rena Bloom, ND, since they graduated from National College of Naturopathic Medicine in 1991. He was humbled in 2008 when presented with the Vis Award by the American Association of Naturopathic Physicians (AANP). He has had the honor of serving the members of the Oncology Association of Naturopathic Physicians as a board member and currently as president. Dr. Schor began a term on the AANP's board of directors in January 2012. He is a frequent contributor to, and associate editor of, the Natural Medicine Journal.
Rebuttal
by Thomas L. Klug, PhD (April 2013)
Preface
by Jonathan V. Wright, MD
I am greatly honored to write this short preface to the more extensive review of the "2/16 estrogen metabolite ratio" that follows, written by Dr. Thomas L. Klug, one of the most – if not the most – knowledgeable – scientists worldwide about all the myriad details and meaning for health of the ratio of 2-hydroxyestrone (to be technically precise, 2-hydroxyestrone and 2-methoxyestrogens) to 16-alpha-hydroxyestrone (to be technically precise, 16-hydroxyestrogens). As readers of Townsend Letter know, there has recently been concern expressed about the clinical utility of the "2/16" estrogen metabolite test. Based on my clinical observations and very extensive reading, I certainly intend to continue using it in practice. Please excuse these three brief reflections before I "get out of the way" of Dr. Klug's extensive review and analysis of the science of this topic.
1. An Early Clinical Observation involving the '2/16' Estrogen Metabolite Ratio
When the "2/16" estrogen metabolite ratio test first became available commercially at the turn of the century, a coworker at Tahoma Clinic was literally first in line to have it done. She had motivation: all eight of her older sisters had suffered from breast cancer. The test result confirmed her suspicions. Her "2/16" ratio was 0.2. A "minimal" good ratio is 1.0; 2.0 and greater is preferable by many clinicians, including me. (Also for the record, this ratio can be too high and associated with higher osteoporosis risk.) But back to her 0.2 "2/16" estrogen metabolite ratio.First, she tried eating as many cruciferous vegetables as she could, even though she knew that a large quantity of cruciferous vegetables for the "long term" might be goitrogenic. It helped very little. After two months of what her coworkers teasingly called "cruciferizing" herself, her "2/16" ratio was 0.3.She turned to DIM (diindolylmethane) supplementation. She took the largest-dose capsules that she could find at the time, 50 milligrams, taking more and more capsules daily in stepwise fashion, testing her "2/16" ratio at each step. At 3 capsules 3 times daily, her "2/16" ratio rose above 1.0.Even though she was happy that she'd achieved her goal, the expense was unsustainable. She got in touch with the DIM supplement manufacturer, explained her situation, and arranged to buy it at close to cost.Two years later, still cancer free, she left Tahoma Clinic for a higher-paying job – ironically at a cancer treatment facility!It appeared to me that she and her sisters must have a genetic weakness in the estrone ® 2-hydroxyestrone transformation, or – less likely – a very hyperactive estrone ® 16a hydroxyestone pathway, and that (more likely) the weak estrone ® 2-hydroxyestrone transformation was probably contributory to the eight occurrences of breast cancer that affected her eight older sisters.
2. Disappearance of Cervical Intraepithelial Neoplasia Coincident with Improvement in the '2/16' Estrogen Metabolite Ratio
In 1999, along with nearly everyone else attending an ACAM convention, I listened to a breakthrough presentation by Dr. Maria Bell, a gynecologic oncologist at the University of North Dakota (at that time). She reported a 12-week study with 27 women with biopsy-proven stage 2 or stage 3 cervical intraepithelial neoplasia (CIN).1 Ten women took a placebo, 8 women took indole-3-carbinol (I3C; a precursor of DIM) 200 milligrams daily, 9 women took I3C 400 milligrams daily. The 10 women who took the placebo had no regression of the CIN in those 12 weeks. By contrast, 4 of the 8 women who took the 200 milligrams of I3C had complete regression (gone!) of the CIN, as did 4 of the 9 women taking the 400 milligram daily dose. At the same time, the urinary "2/16" ratio declined in the 10 women in the placebo group, but went up in both groups of women taking I3C. All of the women whose cancers completely regressed were checked with colposcopy and biopsy at 12 weeks, as were all the women in the placebo group.To the clinicians listening, the correlation between the improved "2/16" ratio and the previously unheard-of complete regression of CIN in 47% of the women who took CIN was very impressive. It didn't outright prove that the change in estrogen ratios cured the CIN, since the I3C might possibly have had a separate anti-CIN effect of the DIM not related to the change in the "2/16" ratio. However, it's now 2013 – 14 years later – and if there is a nonhormonally related effect of DIM on CIN, it hasn't yet been reported.
3. The '2/16' Ratio and the Severity Of HPV Infection
As readers know, cervical cancer (including CIN) is caused by many different strains of human papilloma virus (HPV). HPV also causes recurrent respiratory papillomatosis (RRP), otherwise known as laryngeal and/or vocal cord polyps. Although frequently "benign," this condition can proceed to actual cancer that can be fatal. RRP had been found to be caused by HPV, specifically HPV-6 and HPV-11, two of the four HPV types that cause cervical cancer. In a 1998 report from Long Island Jewish hospital, researchers noted a close connection between estrogen metabolism and the growth of HPV viruses that cause RRP.2 They wrote: "Our results show an inverse relationship between the ratio of C-2 to C-16 alpha hydroxylated estrogens and the severity of RRP." Translated into English: The lower the "2/16" estrogen ratio, the worse the RRP; the higher the "2/16" ratio, the less severe the RRP. In this early study, the researchers asked RRP sufferers to eat significantly more cruciferous vegetables. Increases in the "2/16" estrogen ratio (caused by compounds in these vegetables) correlated with improvement in RRP. They also noted that in a small group of these research volunteers, the ratio did not change; in this group, the RRP did not improve. But in all research volunteers, the "2/16" ratio correlated inversely with the severity of the RRP.In another study, researchers asked volunteers to take I3C to explore its effects on RRP.3 Thirty-three of these volunteers were "followed" for a mean of 4.8 years. Eleven of the 33 (33%) had complete remission, and another 10 (30%) had a reduction in the growth of RRP, a total of 63%. The other 12 (37%) had no response. As demonstrated by the research described in the last paragraph, one of the components of cruciferous vegetables can favorably influence HPV-associated abnormal growths.
Notes
Although I applaud Dr. Schor for addressing this important topic, I feel strongly that readers of the Townsend Letter deserve a more balanced and comprehensive assessment of the current status of estrogen metabolism and the relevance of the many clinical studies to the general validity and future use of the estrogen metabolite ratio (EMR), specifically, 2-hydroxyestrogen (designated as 2OHE1 for brevity) to 16a-hydroxyestrone ratio. The EMR hypothesis states that if the EMR is found to deviate significantly from normality, then estrogen-dependent pathologies will be found or will develop. A corollary to this theory is that alterations in the EMR will affect estrogen-dependent pathologies. By his own admission, Dr. Schor's exposure to this general concept and clinical studies has been very recent relative to the history of the EMR hypothesis and estrogen metabolism. As developer of the direct enzyme immunoassay (EIA) for 2- and 16a-hydroxyestrone (2OHE1, 16aOHE1) and participant and collaborator in many of original and more recent clinical studies with the commercial EIA kits for the 2OHE1/16aOHE1 ratio (Estramet 2/16), I believe that I am uniquely qualified to respond to Dr. Schor's critique.Rather than address Dr. Schor's critical inferences directly, I think that it would be more informative to briefly review (1) the extensive prior research and clinical studies that have validated the EMR hypothesis, (2) the methods of analysis for estrogen metabolites in physiological fluids and associated problems, and (3) the important distinction to be made between results of small well-designed studies and the more recent large retrospective studies by epidemiologists. Extensive review and citation of references in support of the above points is beyond the scope of this brief communication. A limited number of studies and references will therefore be cited. Over 20 years ago, after we became convinced that estrogen metabolism was very likely an important factor in the etiology of cancer, we approached Drs. Fishman and Bradlow, then at the Rockefeller University, to discuss the possibility of development of immunoassays to detect alterations in estrogen metabolism. At that time, their research had indicated that breast cancer was associated with an increased metabolism of estrogens through the 16a-hydroxylation pathway. Their radiometric methods, however, had not detected a change in 2-hydroxylation in women with breast cancer. After a review of their and other research, we focused on the development of high-affinity murine monoclonal antibodies specific for 2-hydroxy- and 2-methoxyestrogens and 16a-hydroxyestrone and developed a novel metric for assessment of estrogen metabolism, the EMR. The 2/16 EMR is essentially a measure of the balanced competition between the two principal cytochrome p450 (CYP) pathways responsible for oxidative metabolism of endogenous small organic molecules such as estrogens, and exogenous organic molecules like those in foods and drugs. The CYP1A pathway is largely responsible for 2-hydroxylation of estrogens, the CYP3A pathway for 16a-hydroxylation pathway. The 2-hydroxylated estrogens have weak estrogen activity relative to estradiol, whereas 16aOHE1 is as potent as estradiol, but manifests additional carcinogenlike activity. Exogenous organic compounds in foods and drugs that are substrates for these CYPs can induce or inhibit the enzyme activities of CYPs in these two pathways and affect the EMR. Enzyme activity in the CYP3A pathway is largely determined by an individual's genetic makeup, whereas activity of the CYP1A enzymes is determined by both genetic makeup and lifestyle factors such as diet, exercise, lean body mass, and age.After several years of research, we developed an EIA kit for measure ment of these three types of metabolites (2-hydroxyestrogens, 2-methoxyes trogens, and 16a-hydroxy estrone) in urine.1 We under stood that as these metabolites are chemically reactive and unstable, absolute levels would be difficult to measure accurately and routinely. We therefore collaborated with Dr. Herman Adlercreutz, the world's expert in measurement of estrogens, to validate our direct method and together set a "gold standard" for future reference. Adlercreutz's elaborate method utilized isolation of glucuronide and sulfate estrogen metabolite conjugates from urine, derivatization to stabilize estrones, and subsequent enzymatic deconjugation; that is, removal of sugars and sulfates bound to the metabolites. After several ion exchange steps to isolate four fractions, four separate estrogen gas chromatography mass spectroscopy (GCMS) runs were necessary.2 The most important steps are isolation of metabolites from the natural enzyme inhibitors in urine, deconjugation, and removal of contaminants prior to GCMS. We modified our direct EIA procedure until we obtained excellent linear correlation with and identical quantities of metabolites as Adlercreutz's GCMS method.1 The EIA was evaluated independently against this GCMS method externally by researchers at the US National Institutes of Health, and the methods were found to give excellent agreement.3 The only difference between them was that at the lowest concentration of urinary 16aOHE1, as found in some postmenopausal women, the EIA consistently found slightly higher levels. The EIA method has a distinct advantage in the measurement of urinary 16aOHE1 as it recognizes for the majority of 16aOHE1 conjugates directly without deconjugation. The monoclonal antibody to 2OHE1 in the EIA assay recognizes all fully deconjugated 2-hydroxylated estrogens, but is specified as to 2OHE1 for convenience.Contemporaneous to EIA assay development, we developed immunohistologic methods to stain and visualize estrogen metabolites in tissue sections from breast and other tissues.4 Using breast tissues from the NIH Cooperative Human Tissue Network (CHTN), we observed very strong staining for 16aOHE1 in breast cancer ductal cells, and in adjacent parenchymal tissues, whereas staining in normal breast ductal cells and adjacent tissues was very faint. By contrast, staining for 2-hydroxyestrogens (2OHE) in ductal carcinoma cells was very weak, and totally absent from ductal carcinoma cells of abnormal histology. Staining for 2OHE1 in normal well-formed ductal breast cells was strong and associated with the nuclear envelope, although some cytoplasmic staining was occasionally seen. Staining was blocked only by addition of the respective estrogen metabolite in the immunostaining procedure.4 Studies of the EMR comparing that in tissue and urine of 9 women by GCMS found urinary EMR to be a good approximation of the EMR found in breast tumors.5 The key question became whether this shift in estrogen metabolism in cancerous tissues would be reflected in alterations in the amounts of metabolites in urine, serum, or other sources readily available for testing.