4-Hydroxyestradiol

4-Hydroxyestradiol (4-OH-E2) is a metabolite of estradiol, the primary form of estrogen in the body.  It is produced through the action of cytochrome P450 enzymes, specifically CYP1A1 and CYP1B1. 

As a catechol estrogen, 4-OH-E2 features a distinct dihydroxyphenyl structure, which plays a significant role in its biological activity.  

This metabolite may undergo further conversion by catechol-O-methyltransferase (COMT) into the less reactive 4-methoxyestradiol (4-MeOE2), but if not methylated, it can form reactive estradiol-3,4-quinones. 

These quinones are capable of causing DNA damage, which has been linked to increased cancer risks, particularly breast cancer. 

The production and metabolism of 4-OH-E2, therefore, have significant implications for estrogen-related pathologies, highlighting its importance in studies of estrogen metabolism and cancer risk assessment.

What is 4-OH-E2?  [3., 5., 8., 14.] 

4-OH-E2, or 4-hydroxyestradiol, is a metabolite of estradiol, a primary bioactive form of estrogen.  It is formed through the 4-hydroxylation pathway of estradiol (E2) metabolism. 

4-OH-E2 is a catechol estrogen, meaning It has a catechol (dihydroxyphenyl) structure with two hydroxyl groups at the 3 and 4 positions of the steroid nucleus.  [5.]

4-OH-E2 is produced from E2 by the cytochrome P450 enzymes CYP1A1 and CYP1B1 in the Phase I metabolism of estrogens.  CYP1A1 is expressed in many tissues including the liver, breast, and adipose tissue.  CYP1B1 is also expressed in various tissues including breast, prostate, and liver, suggesting 4-OH-E2 can be produced in these tissues.

In Phase II metabolism, 4-OH-E2 can be further metabolized by the enzyme catechol-O-methyltransferase (COMT) to form 4-methoxyestradiol (4-MeOE2), a less reactive and potentially less harmful metabolite. 

However, if not methylated, 4-OH-E2 can undergo oxidation to form reactive estradiol-3,4-quinones (E2-3,4-Q), which can cause DNA damage.  4-OH-E2 has been associated with increased cell proliferation, malignant transformation of breast cells, and tumor formation.   [3., 8.]

Catechol Estrogens  [5.] 

Catechol estrogens, a type of estrogen metabolite, undergo processes known as redox cycling, producing reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals. 

These metabolites, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone, and 2-hydroxyestriol, are implicated in the development of breast cancer due to their mutagenic properties and ability to cause DNA damage. 

Unlike their parent compounds (estradiol, estrone, estriol), these catechol estrogens actively participate in redox cycling, significantly contributing to oxidative stress within breast epithelial cells. 

4-OH-E2 And Breast Cancer Risk  [3., 5., 8., 9., 10.] 

4-OH-E2 has been strongly linked to breast cancer due to its production of reactive oxygen species including hydrogen peroxide, H2O2, and hydroxyl radicals.  These stimulate the production of reactive quinone species which ultimately cause DNA damage which leads to carcinogenesis.  [5.]

The reactive quinones derived from 4-OH-E2 can form DNA adducts such as 4-OH-E2-1-N3 adenine and 4-OH-E2-1-N7 guanine, which have carcinogenic potential.  

Human breast cancer tissue has been found to produce higher levels of 4-OH-E2 compared to normal breast tissue.   [9.]  Women with certain polymorphisms in CYP1B1 may have an increased risk of developing breast cancer, potentially due to higher 4-OH-E2 levels.  [9.]

The ratio of the 4-hydroxylation pathway metabolites 4-OH-E1 and 4-OH-E2 to parent estrogens (4-OH-E1 + 4-OH-E2):(E1 + E2) has been positively associated with breast cancer risk in premenopausal women.  [8.]

Laboratory Testing for 4-OH-E2

Overview of Testing, Sample Collection and Preparation

Urine samples are commonly used for 4-OH-E2 testing.  

Estrogen metabolites can be excreted in the urine, making it a reliable method for testing estrogen detoxification and comparing ratios of estrogen metabolites.  Urine testing specifically assesses phase I estrogen detoxification, and it can also be used to assess phase II methylation detoxification.    

Urine collection can be easier and less stressful for patients compared to blood draws, as samples can be collected at home without the need for a clinical setting.  Additionally, urinary levels can reflect longer-term hormone exposure rather than the transient levels often seen in blood, as it reflects detoxification patterns (rather than providing snapshots of levels in the bloodstream).

Interpretation of 4-OH-E2 Test Results

Reference Range for 4-OH-E2

It is important to consult with the lab company providing testing for 4-OH-E2 levels.  For reference, one lab provides the following reference range for urine 4-OH-E2 levels:  [11.]

For cycling women in the luteal phase: 0-0.5 ng/mg

For postmenopausal women not supplementing with hormones: 0-0.1 ng/mg

Optimal Levels of 4-OH-E2

Hormones never act alone, and their effects are nuanced.  Optimal levels of 4-PH-E2 in urine tests may vary depending on individual health conditions, gender, and age, although lower levels are considered optimal.

One recommendation is that 60-80% of a woman's circulating estrogen utilize the 2-OH pathway; that 13-30% utilizes the 16-OH pathway; and that the remaining 7.5-11% utilizes the 4-OH pathway.  [11.]

Health professionals often recommend that women remain within the reference range of 0-0.5 ng/mg in urine samples for cycling women, or 0-0.1 ng/mg in urine samples for postmenopausal women.  

However, a professional's recommendation will be affected by many factors including the patient’s overall health, detoxification capacity, personal and family health history, time of life, diet and lifestyle, medications, and other factors.  

Regular monitoring through urinary tests is essential to ensure that the metabolite levels are within a safe range, thereby reducing the potential for DNA damage and promoting better hormonal balance and overall health.

Clinical Significance of High 4-OH-E2 Levels  [5., 8.]

The 4-hydroxylation pathway is considered the most genotoxic, as its metabolites can create reactive quinone species that can damage DNA and potentially lead to carcinogenesis.

If 4-OH-E2 is not properly methylated to the less harmful 4-methoxyestradiol (4-MeOE2), it can accumulate and convert to carcinogenic 3,4-quinones.

In postmenopausal women receiving hormone replacement therapy, elevated levels of 4-OH-E2 may indicate an excessive amount of hormone supplementation, which is then increasing production of 4-OH-E2.  

These women should have their dosages assessed by a healthcare professional, and estrogen detoxification support may be considered.   

Premenopausal women with elevated levels of 4-OH-E2 should discuss these findings with a healthcare professional; a comprehensive assessment of male and female sex hormones, adrenal and thyroid hormone levels, as well as estrogen detoxification support may be considered.  

Premenopausal women, or women supplementing with estrogen who complain of estrogen excess symptoms, should be assessed for estrogen and estrogen metabolite levels.  

See below for more information on natural methods to promote hormone balance.   

Clinical Significance of Low 4-OH-E2 Levels

Typically, declining levels of estrogen and its metabolites are seen postmenopausally.  Testing of estrogen metabolites may be recommended for women complaining of menopausal symptoms.  

Additionally, postmenopausal women with a family or personal history of osteoporosis or low bone mineral density should consider testing their estrogen and estrogen metabolite levels. 

4-OH-E2 Related Biomarkers to Consider

Several other biomarkers are associated with estrogen metabolism and activity.  Other estrogen metabolites including 2-hydroxyestrone (2-OH-E1), 16-hydroxyestrone (16-OH-E1), 4-hydroxyestrone (4-hydroxyestrone) and methoxyestrogens; estrone (E1), estradiol (E2), and estriol (E3) levels should also be considered.  

2-Hydroxyestrone (2-OH Estrone)

2-OH estrone is a direct precursor in the metabolic pathway that leads to 2-Methoxyestrone, another methylated metabolite of estrone. 

Evaluating both metabolites provides a more comprehensive view of estrogen metabolism, particularly in the 2-OH pathway, and helps in understanding the balance between estrogen metabolites that may influence cancer risk and other hormone-related conditions. 

16-Hydroxyestrone (16-OH estrone)

16-Hydroxyestrone (16-OH E1) is a metabolite of estrone, one of the three main naturally occurring estrogens in the human body.  

Unlike some other estrogen metabolites, 16-OH E1 exhibits relatively strong estrogenic activities. It binds to estrogen receptors, potentially influencing estrogen-responsive gene expression and cellular functions.

 This metabolite has been associated with various physiological effects and is implicated in different health conditions, including increased risks for certain types of cancers due to its potent estrogenic properties.

4-Hydroxyestrone (4-OH estrone)

4-OH estrone is another metabolite of estrone with strong estrogenic properties and potential carcinogenic effects. 

 Specifically, 4-OH estrone is known for its potential to form quinones that can directly damage DNA and generate reactive oxygen species, increasing the risk of mutagenesis.

Measuring 4-OH estrone alongside 16-OH estrone and 4-Methoxyestrone can help assess the overall estrogenic and carcinogenic potential within the body.

Methoxyestrogens

Testing for methoxy-estrogens like 4-methoxyestradiol (4-MeOE2) and 2-methoxyestradiol (2-MeOE2) along with 4-hydroxyestradiol (4-OH-E2) provides valuable information about the body's ability to detoxify and eliminate potentially harmful estrogen metabolites.  

4-OH-E2 can be further metabolized by the enzyme COMT to form the less reactive and less DNA-damaging 4-MeOE2.  Measuring the ratio of 4-OH-E2 to 4-MeOE2 indicates how efficiently the methylation detoxification pathway is working to neutralize 4-OH-E2.

High levels of 4-OH-E2 combined with low levels of 4-MeOE2 could signal poor methylation capacity, allowing 4-OH-E2 to accumulate and potentially increase cancer risk through oxidative DNA damage.

Assessing both metabolites together gives a more complete picture of estrogen metabolism and detoxification status compared to just measuring 4-OH-E2 alone.

Estrone (E1)

Estrone is a weaker estrogen compared to estradiol but is prevalent in postmenopausal women and can be converted back to estradiol. 

Testing for estrone is important for understanding the overall estrogenic activity, especially in postmenopausal women who are at increased risk for estrogen-sensitive cancers.

Estradiol (E2)

Estradiol is the most potent estrogen and has significant implications for bone density, reproductive health, and cardiovascular health.  Monitoring estradiol levels is essential for assessing reproductive health and menopausal status, and for managing hormone replacement therapy effectively.

Estriol (E3)

Estriol is a weak estrogen predominantly produced during pregnancy. Outside of pregnancy, its levels are very low, but it has been suggested to have protective effects against breast cancer. 

Testing for estriol, especially in non-pregnant states, might provide additional insights into estrogenic activity and potential protective mechanisms against estrogen-related pathologies.

Natural Ways to Promote Hormone Balance

It is always essential to work with a qualified healthcare professional in any case of hormone imbalance.  The following diet and lifestyle measures have been shown to naturally promote healthy hormone balance:

Dietary Fiber Increase: consuming more fiber helps bind estrogen in the digestive tract, promoting its excretion and reducing reabsorption.  [6.]  

Interestingly, one study of 240 women also showed a correlation between increased fiber intake and anovulation, possibly due to lower estrogen levels.  [6.]

Cruciferous Vegetables: foods like broccoli, cauliflower, and Brussels sprouts contain indole-3-carbinol, which aids in detoxifying excessive estrogen and optimizing hormone balance.  [2.] 

Regular Exercise: physical activity can help balance hormones by improving metabolism and reducing fat, which is significant since body fat can produce and store estrogen.  [13.]

Probiotics and Gut Health: a healthy gut flora supports proper digestion and detoxification processes, including the breakdown, elimination and balance of hormones like estrogen.  [7.]

‍Limit Alcohol and Caffeine: reducing intake of substances that can impair liver function helps ensure the liver effectively processes and removes excess hormones.  [4., 12.]

Stress Management: stress may have an impact on estrogen levels and metabolism; techniques such as yoga, meditation, or even simple breathing exercises can reduce cortisol levels and help maintain a healthy hormonal balance.  [1.]

Order 4-OH-E2 Testing

Click here to explore testing options and order testing for 4-OH-E2 levels.