2-OH (E1 + E2)/16a-OH-E1 Ratio

The 2-OH (E1 + E2)/16a-OH-E1 ratio reflects the balance between the following estrogen metabolites: the 2-hydroxylated estrogens (estrone and estradiol) to 16α-hydroxyestrone.  This ratio is used to assess the balance between these metabolites, which can influence disease risk and progression.

The 2-OH (E1 + E2)/16α-OH-E1 ratio is useful in evaluating the metabolic pathways of estrogen metabolism, where higher ratios may indicate a greater conversion of estrogens into less estrogenic and potentially anti-estrogenic metabolites, compared to the more potent 16α-hydroxyestrone. 

This ratio can help identify individuals at a lower risk of estrogen-related diseases such as certain cancers, by indicating a predominance of less aggressive estrogen metabolites.

Understanding the 2-OH (E1 + E2)/16a-OH-E1 Ratio is vital not only for grasping a person's hormonal balance but also for predicting and managing health risks related to estrogen metabolism.

What is 2-OH (E1 + E2)/16a-OH-E1 Ratio?

Definition and Significance

The 2-OH (E1 + E2)/16a-OH-E1 Ratio is a measure comparing the levels of 2-OH estrogens to 16-OH estrogen in the body.  Hydroxylation, or adding an -OH group to the estrogen molecule, occurs in phase I estrogen detoxification via the CYP family of enzymes.  An -OH group may be added at the 2-C, 4-C, or 16-C position, indicating the estrogen metabolite produced.  

2-OH metabolites are known for their less potent estrogenic activity, while 16a-OH E1 is a more potent estrogen metabolite. The balance between these metabolites illustrates an individual’s estrogen balance within the body, and may inform one’s disease risk.   

Note that this ratio compares the metabolites produced through 2-hydroxylation (2-OH E1 and E2) and those produced through 16-alpha-hydroxylation (16a-OH E1).

2-OH estrogens are also considered catechol estrogens, which have clinical implications. 

2-OH (E1 + E2) 

2-OH-E1 (2-OH Estrone)  [5., 12., 20.]

2-hydroxyestrone (2-OH-E1) engages estrogen receptors with weaker potency compared to other estrogens, making it relatively less carcinogenic. 

It plays a distinct role in cell physiology by inhibiting the proliferation of certain breast cancer cells, suggesting anti-estrogenic properties. 2-OH-E1 influences critical signaling pathways like mTOR and Akt, reducing cancer cell growth and promoting anti-inflammatory and antioxidative responses. 

While traditionally considered to offer a protective effect against breast cancer, especially in premenopausal women, recent studies indicate that the protective capacity of 2-OH-E1, particularly its ratio to 16α-hydroxyestrone, might be minimal and varies with hormone receptor status of tumors. 

Further research is needed to clarify its impact on different types of breast cancer and its overall role in cancer risk assessment.

2-OH-E2 (2-OH Estradiol)

2-Hydroxyestradiol (2-OH-E2), considered a safer estrogen metabolite than other estrogen metabolites, plays multiple protective roles in the body. It is particularly effective in reducing lipid peroxidation and protecting against oxidative stress in neural cells, indicating significant neuroprotective properties.  [18.]

This metabolite also exhibits potential anti-cancer effects in breast tissue by binding weakly to estrogen receptors, inhibiting rapid cell proliferation, and possibly inducing apoptosis in malignant cells.  Research suggests 2-OH-E2 might serve as a predictive biomarker for breast cancer, especially in premenopausal women.  [9.]

In cardiovascular health, 2-OH-E2 promotes endothelial function and vasodilation through increased nitric oxide release and acts as an antioxidant, reducing the risk of atherosclerosis and mitigating vascular oxidative stress.  [11.]

Additionally, it has complex roles in endometrial cancer, involving both pro-carcinogenic and anticarcinogenic effects related to oxidative DNA damage and nitro-oxidative stress responses.  [3.]

Catechol Estrogens

2-OH and 4-OH estrogens are considered catechol estrogens.  Typically, those estrogens hydroxylated via the 2-OH pathway are considered safer than those hydroxylated via the 4-OH pathway.  

However, catechol estrogens can undergo processes known as redox cycling, producing reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals.  [7.] 

Catechol estrogens 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.  [7., 19.] 

Additionally, catechol estrogens exhibit varied binding affinities to estrogen receptors, which can influence their roles as neurotransmitters in neuronal cells and as hormones in other tissues.  [19.]

Relative to estradiol-17β (considered as a baseline with a relative binding affinity of 100), 2-hydroxyestradiol and 4-hydroxyestradiol show high affinities (100–150) when assessed using human estrogen receptors in cell-free setups.  However, their affinity varies significantly with the receptor source.

Conversely, methoxylated derivatives like 4-methoxyestradiol and 2-methoxyestradiol demonstrate minimal affinity (less than 0.1 to 1).  

Notably, catechol estrogens also interact with membrane-associated receptors, showing potential competitive inhibition against catecholaminergic agonists and antagonists in the brain, and bind with sex-hormone binding globulin with varying efficacies, suggesting a broad spectrum of physiological roles.

16-OH E1  [4., 5., 17.]

The estrone metabolite 16-hydroxyestrone (16-OH-E1) displays estrogenic activity, which although less potent than estradiol, can still significantly influence estrogen-receptor-positive (ER+) cancer progression. 

Elevated levels of 16-OH-E1 are linked to a higher risk of hormone-dependent cancers, such as breast and endometrial cancers, and may also increase the risk of cardiovascular disease and metabolic disorders. 

On the contrary, higher levels might offer protective benefits against osteoporosis by supporting bone health. 

Further research is essential to fully understand 16-OH-E1's diverse roles in health and disease, including its implications in cardiovascular health and its potential as a biomarker for cancer risk in postmenopausal women.

Physiological Relevance of the 2-OH (E1 + E2)/16a-OH-E1 Ratio

The 2-OH (E1 + E2)/16a-OH-E1 Ratio describes estrogen metabolism in the body.

Relative to the more biologically active 16-a-OH-E1 metabolite, the 2-hydroxy estrogens are generally considered protective, associated with a lower risk of estrogen-related cancers and other health issues. 

Conversely, the 16-alpha-hydroxy estrone is linked with an increased risk of certain cancers and other estrogen-related conditions. The ratio between these metabolites provides insight into the body's estrogenic environment, which can be critical for assessing disease risk and tailoring medical interventions. 

Lab Testing for 2-OH (E1 + E2)/16a-OH-E1 Ratio

Overview of Testing, Sample Collection and Preparation

Urine samples are commonly used for testing 2-OH (E1 + E2)/16a-OH-E1 ratio.  

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 2-OH (E1 + E2)/16a-OH-E1 Ratio Test Results

Reference Range for 2-OH (E1 + E2)/16a-OH-E1 Ratio 

It is important to consult with the lab company providing testing for 2-OH (E1 + E2)/16a-OH-E1 ratio.  

For reference, one lab provides the following reference range for urine 2-OH (E1 + E2)/16a-OH-E1 ratio for premenopausal women, or women on estrogen replacement therapy: 1.29-5.49  [14.]

Optimal Levels of 2-OH (E1 + E2)/16a-OH-E1 Ratio

Hormones never act alone, and their effects are nuanced.  An optimal 2-OH (E1 + E2)/16a-OH-E1 ratio in urine tests varies depending on individual health conditions, gender, and age, although a higher ratio is typically preferred.   

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.  [13.]

Health professionals often recommend that women remain within the reference range of 1.29-5.49 in urine samples.  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 a High 2-OH (E1 + E2)/16a-OH-E1 Ratio 

A higher 2-OH (E1 + E2)/16a-OH-E1 ratio is preferred because it indicates a higher proportion of the 2-OH estrogen metabolites present, relative to the 16-OH estrogen metabolites.  

While both are considered safer than the 4-OH metabolites of estrogen, 16-OH-E1 is still recognized as having a high degree of estrogenic activity, which may promote estrogen positive cancers. 

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

Clinical Significance of Low 2-OH (E1 + E2)/16a-OH-E1 Ratio

A lower 2-OH (E1 + E2)/16a-OH-E1 ratio indicates a relatively higher amount of 16-OH-E1, which can signify excessive estrogenic action.  This may increase an individual’s risk of estrogen-positive cancers, although results should be interpreted in the context of an individual’s overall health history, family history, and other important factors.  

A low 2-OH (E1 + E2)/16a-OH-E1 Ratio may warrant additional assessment of estrogen levels and other estrogen metabolites for a better understanding.   

2-OH (E1 + E2)/16a-OH-E1 Ratio-Related Biomarkers to Consider

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

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 2-OH estrone can help assess the overall estrogenic and carcinogenic potential within the body.

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.  [8.]  

Interestingly, one study of 240 women also showed a correlation between increased fiber intake and anovulation, possibly due to lower estrogen levels.  [8.]

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.  [16.]

Probiotics and Gut Health: a healthy gut flora supports proper digestion and detoxification processes, including the breakdown, elimination and balance of hormones like estrogen.  [10.]

Limit Alcohol and Caffeine: reducing intake of substances that can impair liver function helps ensure the liver effectively processes and removes excess hormones.  [6., 15.]

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.]

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