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From the Townsend Letter
April 2013

Response to Dr. Jacob Schor's Article 'Estrogen Metabolite Ratios: Time for Us to Let Go'
by Thomas L. Klug, PhD
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Original Article
Estrogen Metabolite Ratios: Time for Us to Let Go
by Jacob Schor, ND, FABNO (January 2013)

Schor's Rebuttal to This Article
Response to Wright and Klug (May 2013)

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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.

1. Bell MC, Crowley-Nowick P, Bradlow HL, et al. Placebo-controlled trial of indole-3-carbinol in the treatment of CIN. Gynecol Oncol. 2000 Aug;78(2):123–129.
2. Auborn K, Abramson A, Bradlow HL, Sepkovic D, Mullooly V. Estrogen metabolism and laryngeal papillomatosis: a pilot study on dietary prevention. Anticancer Res. 1998 Nov-Dec;18(6B):4569-4573.
3. Rosen CA, Bryson PC. Indole-3-carbinol for recurrent respiratory papillomatosis: long-term results. J Voice. 2004 Jun;18(2):248–253.

Response to Dr. Jacob Schor's Article 'Estrogen Metabolite Ratios: Time for Us to Let Go'
by Thomas L. Klug, PhD
President, Immuna Care Corporation

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 measure­ment of these three types of metabolites (2-hydroxyestrogens, 2-methoxyes­trogens, and 16a-hydroxy­estrone) in urine.1 We under­stood 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.

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