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High 4OHE1

4OHE1 is a catechol estrogen formed by aromatic hydroxylation of primary estrogens at either the C-2 or C-4 position.  4OHE1 can be oxidized to catechol estrogen quinones if it is not inactivated by COMT and/or sulfation, glucuronidation, etc. These catechol quinones can react with DNA to produce an excess of estrogen-DNA adducts with subsequent cancer-initiating mutations. This sequence of events is possibly related to uterine fibroids in addition to cancer in the breast, uterus, and prostate.

When a patient is producing too much 4OE1 catechol estrogen, there are 4 primary approaches utilized to decrease the harmful effects and potential increased cancer risk, while still being able to maintain adequate hormone replacement therapy.

1. Decrease the production of 4OHE1 by directing primary estrogen down alternative pathways.
2. Increase the production of the safer estrogen metabolite, 4-MeOE1, by supporting the methylation of 4OHE1 in addition to supporting sulfation and glucuronidation.
3. Protect against the conversion of 4OHE1 estrogens to dangerous quinones that injure DNA.
4. Support glutathione production, because glutathione can detox dangerous quinones.
   

Decrease the production of 4OHE1 by directing primary estrogen down alternative pathways.

Although low levels of 4OHE1 are likely OK, it is very important that your liver metabolizes (breaks down) hormones effectively to avoid the production of too much 4OHE1. The liver has the option of taking estrogen down 3 different pathways, only one of which ends in increased 4OHE1. CYP1B1 is the enzyme pathway in the liver that leads to 4OHE1 production.  There are genetic variants that upregulate the CYP1B1 enzyme, predisposing a person to taking estrogen down the 4OHE1 pathway. There are toxins that turn on the CYP1B1 enzyme, subsequently increasing the production of 4OHE1. There are nutrients found in foods and in supportive supplementation that preferentially turn on the alternative pathways that do not result in 4OHE1 production. Supporting liver metabolism of estrogen down more protective pathways is accomplished with a healthy diet, the avoidance of environmental toxins (including xenoestrogens), and supportive supplementation and hormone optimization.

To inhibit the 4OHE1 pathway and/or turn on the alternative pathways particularly the safer CYP-1A1 enzyme pathway that leads to the production of 2OHE1, a safer estrogen metabolite:

1. Eat a healthy diet
   1. Cruciferous vegetables contain the phytochemical indole-3-carbinol (I3C). In the body, I3C is broken down to 3.3-diindolylmethane – DIM, for short. Cruciferous vegetables include: arugula, broccoli, brussels sprouts, cabbage, cauliflower, collard greens, kale, radishes, rutabaga, turnips and watercress.
   2. Strawberries, blackberries and raspberries (sources of Ellagic acid)
   3. Purple Grapes (a source of Resveratrol)
   4. Green tea (a source of EGCG- epigallocatechin gallate)
   5. Avoidance of Caffeine
   6. Avoidance of simple sugars
   7. Avoidance of excessive omega 6 fatty acids
2. Avoid environmental toxin exposure
   1. Xenoestrogens, a group of either naturally-occurring or artificially-created compounds in the environment, show hormone-like properties: they are both imperfect, potent estrogens and endocrine disruptors. The more efficacious a Xenoestrogen, the more it disrupts the action of physiologic estrogens. In addition, the liver can only do so much. If the liver is busy metabolizing xenoestrogens from the environment, it is not going to do as well metabolizing endogenous estrogens made by the body. Important xenoestrogens to note include: plastics, pesticides, soy isolates, and products from animals raised with inappropriate hormone exposure.
   2. PAHs – (Polycyclic Aromatic Hydrocarbons). PAHs in air are produced by burning wood and fuel for homes. They are also contained in gasoline and diesel exhaust, soot, cola, and smoke from cigars and cigarettes.
   3. PCBs (Polychlorinated Biphenyls). PCBs belong to a broad family of man-made organic chemicals known as chlorinated hydrocarbons. PCBs were domestically manufactured from 1929 until manufacturing was banned in 1979. They have a range of toxicity. Although no longer commercially produced in the United States, PCBs may be present in products and materials produced before the 1979 PCB ban. Products that may contain PCBs include: transformers and capacitors, electrical equipment, oil used in motors and hydraulic systems, old electrical devices or appliances containing PCB capacitors, fluorescent light ballasts, cable insulation, thermal insulation material (including fiberglass, felt, foam, and cork), adhesives and tapes, oil-based paint, caulking, plastics, carbonless copy paper, & floor finish. PCBs can accumulate in the leaves and above-ground parts of plants and food crops. They are also taken up into the bodies of small organisms and fish. As a result, people who ingest fish may be exposed to PCBs that have bioaccumulated in the fish they are ingesting.
3. Optimize hormones
   1. Such as Thyroxine, Progesterone, DHEA
   2. Avoidance of birth control pills
4. Supplementation
   1. EstroPure
   2. Pure Balance
   3. Pure Hormone Support – F
   4. Pure Fish Oil or OmegaAvail Smoothies. Omega -3 found in fish have been shown to promote estrogen using the C-2 pathway. This is particularly true for EPA omega-3 fatty acids.

Increase the production of the safer estrogen metabolite (4-MeOE1) by supporting the methylation of 4OHE1, in addition to supporting sulfation, glucuronidation, and conjugation to glutathione.

If 4OHE1 is overproduced, it is less likely to oxidize to carcinogenic compounds.  That creates DNA adducts if it is neutralized via methylation, sulfation, glucuronidation and/or conjugation to glutathione. Methylation by the enzyme COMT converts the 4OHE1 form of estrogen to 4-MeOE1, a safer form of estrogen. Nutrients to support the COMT enzyme include: Methionine (especially important if low homocysteine), Magnesium, B2, B6, B12, Folate (folinic acid, 5-formyl THF, or 5-methyl THF), TMG (betaine), and choline. Sulfur containing amino acids support sulfation, glucuronidation, and conjugation with glutathione.

1. Supplementation to consider:
   1. Pure Methylation
   2. Homocysteine Supreme
   3. Magnesium Glycinate, Reacted Magnesium.
   4. Caution with excessive calcium supplementation
   5. Sulfur containing compounds/amino acids like: MSM (Methylsulfonlymethane), NAC (N-Acetyl Cysteine), and Methionine
2. Avoid factors that slow or inhibit COMT.
   1. Rule out Mercury toxicity as mercury inhibits the COMT activity through inhibition of S-adenosylmethionine (SAM), a coenzyme for COMT.
   2. Plastics inhibit COMT activity
   3. Chronic Stress – the COMT enzyme is involved in the metabolism of neurotransmitters, using up available nutrients for COMT optimization in the liver.

Protect against conversion of 4OHE1 estrogens to dangerous quinones that injure DNA

1. Avoid trans fats
2. Avoid heavy metal exposure
3. Supplementation:
   1. Melatonin
   2. Theanine
   3. Sulphoraphane

Support glutathione production because glutathione can detox dangerous quinones

1. Diet
   1. Sulfur-rich foods like garlic and onions to provide sulfur containing amino acids
   2. Bioactive Whey Protein, if tolerated – a great source of cysteine
2. Supplementation to boost glutathione production
   1. N-Acetyl Cysteine
   2. Glutamine
   3. Glycine
   4. Selenium
   5. Pure Energy
3. Decrease excessive use of glutathione by: avoiding excessive pharmaceuticals, decreasing heavy metal exposure, decreasing cigarette exposure and air pollution, all of which use up available glutathione.
4. Reactivate glutathione by increasing Vitamin C and Vitamin E.

Total Estrogen and Precision Medicine: Treating Our Patients vs. Treating the Population

Throughout the history of medicine, healthcare trends have evolved toward disease prevention instead of treating disease. Yet, the flood of lifestyle and dietary changes designed to avoid certain diseases seems to be more of a marketing strategy for food distributors, health clubs and supplement companies than realistic and actionable recommendations in practice.  In recent years, a number of studies have discussed the association between genetic mutations (SNPs), estrogen sensitive cancers (breast, uterine and prostate), and estrogen hormone replacement therapies. In these studies, there seemed to be a clear association between orally administered synthetic estrogen and the development of estrogen-sensitive cancer. However, as more and more medical practitioners switched from oral administration of synthetic estrogen to bio-identical creams, patches, gels and pellets, the association between estrogen and breast cancers appeared to decrease. Those prescribing and/or marketing bioidentical hormones made the assumption that breast cancer risk was related to the synthetic make-up in oral administration of estrogen, but although the association to synthetic oral estrogen to cancer is statistically significant, this assumption may be misleading. This article will specifically discuss the pathways of estrogen metabolism, what these pathways represent in clinical practice and how to identify and, subsequently, mitigate the risks associated with developing estrogen-sensitive breast, uterine and prostate cancers in disease prevention.

Estrogen is essential in both men and women for bone health, brain health, cardiovascular health, reproductive health and has even shown positive effects in treating certain cancers. It is clear that maintaining optimal levels of estrogen is very important, because having too much or too little estrogen can present with a myriad of symptoms. Measuring Estrogen before addressing suspected hormone imbalances and monitoring estrogen levels during hormone replacement therapy are crucial in maintaining hormone balance, but the clinical science behind the risks and benefits of Estrogen relating to cancer remained a mystery until estrogen metabolism became more understood. In order to customize therapies for patients while mitigating risks associated with estrogen-sensitive cancers, all primary estrogens (Estrone, Estradiol and Estriol), as well as the downstream estrogen metabolites, must be evaluated.

Most practitioners are accustomed to evaluating serum estradiol by itself when determining if a patient is a candidate for estrogen hormone therapy, but measuring a single estrogen significantly limits practitioners in developing therapies that are precise to each individual patient. As men and women age, aromatase activity takes place at the testosterone precursor, Androstenedione, at a higher rate than the aromatase activity at Testosterone observed in younger populations. The result is a migration toward estrone production over estradiol; so simply measuring estradiol can produce an inaccurate assessment of estrogen effect at the estrogen receptors (ERs).

In addition to assessing Estrone, it is essential to look at the various stages of Estradiol, Estrone and Estrone metabolism collectively. Special attention should be paid to the amount of free hormone that is available to tissues based on the binding affinities of estrone (E1), estradiol (E2), estriol (E3) and 16-alpha-hydroxyestrone at the estrogen receptors.^1,2 The Total Estrogen Effect (TEE) in urinary hormone and hormone metabolite testing is calculated based on the presence of estrone, estradiol, estriol, and their relative binding affinities at the estrogen receptors.^1,2 Estradiol is considered to be the most estrogenic estrogen and is the prevalent primary estrogen produced by menstruating women. Estradiol has a strong and long-binding affinity at the estrogen receptors and is responsible for most cell proliferation at estrogen-sensitive tissues, such as breast and uterine tissues, in menstruating women.^1-6 Based on receptor availability at the tissues, estradiol is converted to estrone and further converted to estriol through the 16-alpha-hydroxyestrone metabolism pathway during Phase I metabolism. Unless a female is pregnant or menopausal, most estrone and estriol is a result of conversion from estradiol. Based on this knowledge, we are able to assume the estrogen effect at the estrogen receptors. The calculation for the TEE assumes certain standard binding affinities at the estrogen receptors. Estriol is the weakest binding primary estrogen at the estrogen receptors and can compete with stronger-binding estrogens.² Due to its competitive nature and its weaker binding affinity, estriol is considered to be a protective estrogen.^1,3,10 Following estriol in binding affinity is estrone (4x more estrogenic than estriol), 16a-hydroxyestrone (9x more estrogenic than estriol), and estradiol (10x more estrogenic than estriol).^2,7 These relative values are added together to establish the Total Estrogen Effect at the ERs.

Table 1: Fictitious Example (If a patient has an estriol of 2, an estrone of 2, a 16a-OHE1 of 2, and an estradiol of 2, then the Total Estrogen Effect (TEE) would be 48 based on their relative binding affinities)

**Although 16a-OHE1 is a Phase I metabolite, it is included in the calculation due to its ability to contribute to estrogen dominance.

Assessing the amount of estrogen effect at the receptor is the best way to decide if a patient is a candidate for estrogen hormone replacement, but this assessment is only the beginning; assessing Phase I and Phase II metabolism of estrogen is where inflammatory responses and cancer risk are assessed and customized therapies are derived.

As shown in Figure 2, there are three distinct pathways of Phase I metabolism of Estrone, resulting in 2-hydroxyestrone (the most favorable pathway of metabolism), 16-alpha-hydroxyestrone (a result of inflammation) and 4-hydroxyestrone (a carcinogenic pathway). These Phase I pathways are directly impacted by lifestyle and dietary choices.

• 4-OHE1 is catalyzed predominantly through CYP1B1
• 2-OHE1 is catalyzed predominantly through CYP1A1
• 16-a-OHE1 is catalyzed predominantly through CYP3a4

Improvements in lifestyle result in a preference of metabolism down the most favorable 2-hydroxyestrone pathway. 2-hydroxyestrone does not bind to the estrogen receptors. However, COMT activity causes the methylation of 2-hydroxyestrone in Phase II metabolism. That results in stable DNA adducts and can slow estrogen-sensitive cell growth and even reverse DNA damage caused by the 4-OHE1 pathway of Phase I metabolism. 2-methoxyestrone has also been shown to reverse inflammatory responses to estrogen dominance and slow or reverse breast, uterine and prostate cancer growth. When estrogen dominance or certain mutations in the CYP1B1 gene are present, Phase I metabolism increases down the 4-hydroxyestrone pathway. 4-Hydroxyestrones are highly reactive and form 3,4 Quinones that can form unstable DNA adducts, resulting in the creation of carcinogenic mutations. The 4-hydroxyestrone pathway is additionally influenced by environmental toxins, so patients who have CYP1B1 SNPs are especially susceptible to estrogen-sensitive cancers. Because 4-OHE1 is a biomarker of CYP1B1 SNPs, patients with increased 4-OHE1 levels should avoid chemical toxins and improve methylation to drive Phase II detoxification of 4-OHE1. When 4-OHE1 is methylated in Phase II metabolism, the carcinogenic effects of 4-OHE1 are completely neutralized.  Finally, the 16-alpha-hydroxyestrone pathway increases in the presence of inflammation. While most of this inflammation stems from the gut, estrogen dominance and the presence of estrogen-sensitive cancers can increase 16-a-OHE1 during Phase I metabolism as well. The best way to assess 16-a-OHE1 levels is the relative rate of metabolism of 2-OHE1 to 16-a-OHE1 via the 2:16 ratio. When the 2:16 is low, supporting Phase I metabolism and reducing gut inflammation are the primary ways to redirect Phase I metabolism down the 2-OHE1 pathway. Additionally, driving the 16-a-OHE1 pathway through 16-a-OHE1 to Estriol (via 16-hydroxylase activity) is another way to protect the tissues from estrogen dominance through weaker competitive binding of estriol at the ERs. 16-a-OHE1 covalently binds to ERs, resulting in long-standing action at the target tissues. This covalently-binding estrogen metabolite has positive effects in cell proliferation and has even been used to treat certain cancers, but it can cause an existing cancerous tumor or damaged estrogen-sensitive tissue to grow aggressively as well. This unique action of increased 16-a-OHE1 in the presence of inflammation, followed by increase cell proliferation, makes 16-a-OHE1 advantageous in the absence of free-radicals and un-repaired DNA damage. On the other hand, this action makes 16-a-OHE1detrimentally aggressive in the presence of cancer or certain genetic predispositions to increases in 4-OHE1. This is why assessing estrogen metabolism, modulating Phase I metabolism through lifestyle, and supporting Phase II metabolism through methylation and glutathione activity are essential in customizing therapies for patients and optimizing clinical outcomes.

Provided there is sufficient glutathione activity, the removal of DNA-damaging free-radicals is a regular event inside of cells. In the event of DNA mutations, insufficient DNA repair and the initiation of cancerous tissue, these mechanisms of cellular defense are likely compromised significantly. Un-repaired DNA damage is a major cause of cancer initiation. Again, the 2-hydroxy ➝ 2-methyoxyestrone pathway is the most advantageous pathway for repairing DNA damage and reversing the effect of free-radicals, as well as glutathione activity.

A number of nutrients, botanicals and nutrient compounds have been identified as having varying effects on the estrogen-metabolizing and detoxifying pathways. Implementation of many of these compounds through an individualized process affords great potential for those affected by the potentially deleterious effects of aberrant estrogen metabolism.

• DIM (diindolylmethane) – DIM is used to stimulate 2 hydroxylation (neutral estrogen pathway) via CYP1A1 and reduce expression of 16 hydroxylation (potential harmful estrogen pathway) through inhibiting CYP3a4. There is far more potential therapeutic action to DIM, however. DIM has been shown to reduce DNA hypermethylation of CpG islands (hallmark feature of cancer activity), reduce intestinal inflammation, function to mildly inhibit aromatase, and enhance DNA repair mechanisms.
• Flax seeds – Flax seeds are a promoter of CYP1A1 and an inhibitor of CYP1B1. Thus, flax seeds are promoters of 2 hydroxylation (neutral estrogen) and inhibitors of 4 hydroxylation (potentially undesirable). Flax also has shown to inhibit CYP3a4 and reduce the excretion of 16OHE1, another potentially-problematic estrogen.
• Berries – Numerous types of berries (blackberries, raspberries, grapes, blueberries) are a rich source of polyphenolic compounds, including ellagic acid. Ellagic acid is a promoter of glutathione transferase (GSTM), as well as NQO1 (quinone reductase). These 2 enzymes are important in the detoxification of 3,4 semi-quinones. Additionally, ellagic acid has been shown to increase DNA repair genes, as well as reduce DNA adducts that have been formed by carcinogens.
• Grapefruit & Citrus peel – Are sources of hesperidin. Hesperidin, at high doses, inhibits CYP1B1 and also CYP3a4. Grapefruit is notorious for inhibiting CYP3a4. Citrus peel contains a considerable amount of hesperidin; that is especially true of dried tangerine peel. An assortment of studies done on hesperidin have found an overall increase in blood flow and circulation, reduction in blood pressure, and reduction in symptoms of cell adhesion factors, which may disrupt cancer activities.
• Calcium D-glucarate – Is a form of calcium that promotes phase 2 glucuronidation. This phase 2 reaction makes molecules more water-soluble. Additionally, it is believed that calcium d-glucarate is a beta glucuronidase inhibitor, which acts to prevent the reabsorption of detoxified estrogens through 2nd pass metabolism.
• Glutathione promoters and/or cofactors: NAC, lipoic acid, selenium, B-2, B-6, zincⁱ

REFERENCES

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We Are Officially in the New Lab

Back in 2015, we started designing and developing our new laboratory. After repeated trial and error in the old lab, we knew EXACTLY what we were looking for. There was plenty of space, but the layout and instrumentation needed to be strategically planned. We wanted to expand our base hormone panel but needed a more sensitive LCMS/MS, so we set out to find the best one money could buy, using data instead of emotion to make our final decision. We then took these instruments for a test drive, using real patient samples and our own preparation method. First, we split urine samples into several plates, performed our own sample preparation method, and sent them out to all the big vendors. Then, we sat back and let the results compete with each other. We then purchased two Sciex 6500+ LCMS/MS instruments, an additional Thermo Scientific Vantage LCMS/MS, three liquid handling Tecans, and more. We even installed an in-house nitrogen generator system that supplies enough nitrogen for every instrument and process required in the lab, with additional built-in expansion (and we even have a backup tank for that!). We now have built-in automation and redundancy in every area of the new lab. Literally, every piece of equipment and every process in this laboratory has a back-up, which is used for clinical operations only. In fact, we have an entire lab dedicated to Research and Development that could serve as a back-up!

Unfortunately, we had to learn the hard way that Clinical Operations and Research and Development couldn’t coexist without dedicated instrumentation and staff. When it came down to making a decision on whether to use the instruments for R&D or clinical ops, clinical would win 100% of the time. R&D would make a little progress, but then Operations would need the instruments. R&D would then have to throw their work away and start over when they became available again. This vicious cycle went on for too long before we finally realized that we needed to split the two up, so we built the new lab with the intent of keeping everything in the old lab and turning it into Research and Development. We also decided to keep the CLIA license there and apply for another CLIA license in the new building, creating the added benefit of a fully equipped, licensed back-up lab should our current lab’s license ever become compromised. With the rapidly-evolving LCMS industry, regulatory compliance became very difficult to keep up with. For this reason, we have contracted a team of consultants to come in and perform mock inspections on a quarterly basis. These are former inspectors who specialize in this area and keep us informed of any changes that we may not be aware of. This also keeps us on our toes and ensures that we are always “inspection ready”. Having two complete, CLIA-licensed labs made the process of moving the operation a lot easier. The idea of shutting down the lab for a week to move simply wasn’t realistic, so we kept running in the old lab while building out, testing, validating, and correlating the instruments in the new lab. Finally, in February, we passed regulatory inspection and obtained our new CLIA license. We then started moving one process at a time, with the option of reverting back to the old lab if need be.

As of October 1st, we are fully operational in the new lab. Not only are we excited to be utilizing our new, state-of-the-art equipment, but we’re happy to be reunited with the rest of our Physicians Lab family! Beyond that, we’re already prepared for further expansion!

Jessica M. Faupel-Badger, Barbara J. Fuhrman, Xia Xu, Roni T. Falk, Larry K. Keefer, Timothy D. Veenstra, Robert N. Hoover, and Regina G. Ziegler

Source: Cancer Epidemiol Biomarkers Prev. 2010 January ; 19(1): 292–300. doi:10.1158/1055-9965.EPI-09-0643.

Abstract

Absolute and relative concentrations of estrogens and estrogen metabolites (EM) are important for clinical decisions, as well as for epidemiologic, experimental, and clinical research on hormonal carcinogenesis. Radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) are routinely used for measuring EM in blood and urine due to efficiency and low cost. Here we compare absolute and ranked concentrations of estrone, estradiol, and estriol measured by indirect RIA and of 2-hydroxyestrone and 16α-hydroxyestrone measured by ELISA to the concentrations obtained using a novel liquid-chromatography-tandem mass spectrometry (LC-MS/MS) method which measures 15 EM concurrently.

We used overnight urine samples collected from control women (362 premenopausal, 168 postmenopausal) participating in a population-based case-control study of breast cancer among Asian-American women ages 20–55 years. When comparing RIA or ELISA levels to LC-MS/MS, absolute concentrations for the five EM ranged from 1.6–2.9 and 1.4–11.8 times higher in premenopausal and postmenopausal women, respectively, (all p<0.0001). However, LC-MS/MS measurements were highly correlated [Spearman r (rs) =0.8–0.9] with RIA and ELISA measurements in premenopausal women, and moderately correlated (rs=0.4–0.8) in postmenopausal women. Measurements of the 2-hydroxyestrone:16α-hydroxyestrone ratio, a putative biomarker of breast cancer risk, were moderately correlated in premenopausal women (rs=0.6–0.7) but only weakly correlated in postmenopausal women (rs=0.2). LC-MS/MS had higher intraclass correlation coefficients (≥99.6%) and lower coefficients of variation (≤9.4%) than ELISA (≥97.2% and ≤14.2%) and RIA (≥95.2% and ≤17.8%).

Comparison with the LC-MS/MS method suggests that the widely used RIA and ELISA EM measures may be problematic, especially at low EM levels characteristic of postmenopausal women.

Link to Study

J Am Pharm Assoc. 2003;43:724–6. – Jolena Hagen, Nicolette Gott, and Donald R. Miller

Recent reports concerning the safety of hormone replacement therapy (HRT) have led many women to seek alternative ways to treat menopausal symptoms. A new alternative that has become very popular is bioidentical HRT.  This therapy is often based on the results of saliva hormone tests, which may be purchased from pharmacies and physician offices or over the Internet. Compounding pharmacies individualize HRT based on the results of these tests. Although saliva hormone testing is easy and convenient for a patient to perform at home, it has potential problems.2–4 Given the importance of these tests to compounding pharmacists, we decided to perform a simple check on the tests’ reliability.

Conclusion: Our findings suggest that laboratory values for saliva hormone samples collected with at-home test kits are not reliable. The individualization of HRT for patients is impossible without a reliable analysis. We suggest that compounding pharmacists periodically send in replicates of their own samples to test the reliability of the laboratories that they use.

Link to Study