EndoAxis Clinical Team
In functional medicine, urinary hormone metabolites—especially ratios such as 2-OHE₁ : 16-OHE₁, or the value of 4-OH E1 and E2—are sometimes criticized for lacking strong causal evidence in disease. While direct causation has not been firmly established in human cohorts, research consistently finds correlations between certain ratios and hormone-sensitive conditions, including breast cancer. More importantly, these metabolites can help uncover that there is oxidative stress and altered redox homeostasis at play that can alter health and influence hormone symptoms.
It’s important to remember that sex hormones are cellular messengers. From synthesis to utilization to clearance, their impact on DNA expression can be shaped by numerous influences,including gut health, immune activity, insulin sensitivity, thyroid status, environmental exposures, and nutritional factors.
By understanding the enzymes that drive steroid synthesis and metabolism, and the factors influencing their regulation, we can uncover a far richer picture than absolute metabolite values alone can provide. Each metabolite represents the end point of multiple enzymatic steps—steps that can be traced back to reveal why a pathway may be up- or downregulated.
The Limits of “Single-Metabolite” Thinking
Studies have examined individual metabolites (e.g., 4-OHE₁) or ratios (e.g., 2-OH : 16-OH E₁) in relation to cancer risk. These are not strong causational studies, but they do reveal patterns—especially when combined with an understanding of the metabolic drivers that influence these metabolites. When interpreted alongside patient history, other risk factors, and markers of oxidative stress or detoxification capacity, these patterns can guide targeted antioxidant strategies and lifestyle interventions that support cellular health.
Disease is never the result of a single metabolite. Clinical studies, by necessity, ask simplified questions like “Does 4-OHE₁ cause cancer?” The answer is: possibly—but the real question is why is that metabolite elevated in the first place.
If we think about where 4-OH E1 is derived from, it is the hydroxylation of estrone through CYP1B1. CYP450 enzymes are a family of enzymes responsible for the conversion of toxic substances and steroid/fat-based molecules (like sex hormones and cortisol) into more water-soluble intermediates. Although this occurs heavily in the liver, knowing where these enzymes express holds valuable importance when assessing metabolite concerns. CYP1B1 expression, for example, is not heavily expressed in the liver – it is an extrahepatic isoform of the CYP1 family, expressed in breast, prostate, ovaries, pancreas and testes (1).
CYP1B1 is responsible for hydroxylation of polyaromatic hydrocarbons (PAH’s) as well as estrone, and estradiol. Expression is regulated by estradiol, xenobiotics exposure and PAH (2).
Greater expression increases 4-OH E1, which will result in the tissues where it is expressed (breast, prostate, ovary, pancreas, testes).
4-OH E1 can either methylate or oxidize into estrone/estradiol-3,4-quinone. This process is regulated by peroxidase. Quinones cause damage to cellular DNA, as they create unstable adducts that act like speedbumps that alter DNA replication, leading to aberrant mutation altering cell replication (3).
If 4-OH E1 is elevated, and expression occurs in hormone-sensitive tissues, then the risk increases that 4-OH E1 will become an estrone-3,4-quinone and damage the DNA of the cells in which it forms (breast, prostate, ovaries, pancreas, testes).
But we cannot easily measure these quinones. So instead, we can use the immediate upstream metabolite, 4-OH E1. With the knowledge that higher levels increase risk for oxidative potential, and infer risk, when reviewed in tandem with methyl clearance, pyroglutamate status and 8OHdG activity.
So the question is not: “Does 4-OH E1 cause cancer?”
The question should be: “Why is 4-OH E1 favored, and what would cause enzyme expression to increase, and do those substances harm human health and warrant further support or targeted care, and is there evidence that intervention helps?”
For example:
- Are PAHs genotoxic and mutagenic? Strong association, including early onset breast cancer (4,5,6, 7)
- Do PAHs increase CYP1B1 expression? Yes (8).
- Does CYP1B1 drive 4-hydroxylation of Estrogens? Yes (9).
- Does 4-OHE₁ cause cancer? Possibly. Specifically, estrone-quinones. As summarized from a study by Cavalari and Rogan, 2021: Higher CYP1B1 activity increases the formation of estrone/estradiol-3,4-quinones, leading to greater DNA adduct formation and a higher likelihood of cellular transformation. In contrast, increased catechol-O-methyltransferase (COMT) activity reduces E1(E2)-3,4-Q production, lowers adduct burden and decreases transformation risk. Notably, MCF-10F cells—an ER-negative breast epithelial cell line—can still undergo transformation by estrogens even in the presence of antiestrogens such as tamoxifen or ICI-182,780, indicating that the estrogen receptor is not required for this process. These findings underscore a key carcinogenic mechanism: estrogens can initiate cancer through metabolic conversion to E1(E2)-3,4-Q, which subsequently reacts with DNA to produce characteristic mutations (10).
- Can we reduce quinone activity? Yes. Both NAC and resveratrol show potent support in the reduction of quinones, while NRF2-NQO1 promoters like sulforaphane, pomegranate and green tea show strong evidence in reducing quinone concentration and reducing oxidative burden that can alter DNA activity (9,11)
If 4-OHE₁ is high, it informs providers that there is an abnormal preference for CYP1B1 activity—a pattern that should not be favored in healthy physiology. Whether the metabolite itself is the culprit or merely a byproduct of carcinogenic exposure, it reveals metabolic dysfunction that warrants attention.
The Fate of 4-OHE₁: Detoxification or DNA Damage
4-OHE₁ can follow two major fates:
- Methylation via COMT → 4-methoxy E₁ (water-soluble, stable, readily eliminated)
- Conversion to 3,4-estrone quinones → DNA-reactive, potentially carcinogenic
Protective systems can mitigate quinone damage:
- Glutathione conjugation (high glutathione production and activity, supported by adequate pyroglutamate)
- NRF2 and NQO1 activation (enhancing antioxidant enzyme expression and DNA repair)
Unfortunately, most studies on 4-OHE₁ do not assess these protective factors. They focus on correlation with disease rather than the upstream and downstream metabolic context.
If a patient has:
- Poor methylation capacity
- Low glutathione production
- High oxidative DNA damage (e.g., elevated 8-OHdG)
- And elevated 4-OHE₁
…this combination represents a metabolic “perfect storm”—one that becomes strongly suggestive of high oxidative burden and clinical intervention, regardless of whether the metabolite itself is the primary cause of disease.
Unfolding the Enzymatic Story
Urinary hormone metabolites are not isolated biomarkers—they are windows into the enzymatic dynamics of steroid metabolism.
Phase I metabolism
CYP1B1 → 4-hydroxylation
CYP3A4 → 16-hydroxylation
CYP1A1 → 2-hydroxylation
Phase II clearance
- COMT: methylates catechol estrogens (2-OH often protective; 4-OH potentially genotoxic)
- UGT and SULT: glucuronidation and sulfation to inactivate estrogens for elimination
Abnormal patterns may indicate:
- Overactive Phase I pathways (e.g., elevated 16- or 4-hydroxylation)
- Impaired Phase II clearance (e.g., low COMT function; low SAMe for COMT; overwhelmed conjugation systems)
These are signs of metabolic imbalance, not merely “toxic metabolites.”
Toward a Systems-Based Interpretation
Urinary metabolites, more than serum levels, provide a dynamic, time-integrated view of hormonal balance. They can help reveal:
- Detox capacity (methylation, glucuronidation, sulfation efficiency)
- Stress and inflammation effects on enzyme regulation
- Adipose-driven hormone modulation (e.g., altered T/E₂ ratio, slower cortisol clearance)
- Phase I vs. Phase II imbalances
High-risk patterns like elevated 4-OHE₁ gain true clinical meaning only when considered with:
- Oxidative stress markers (8-OHdG)
- Methylation capacity (SAMe status)
- Antioxidant defenses (glutathione levels, NRF2/NQO1 activity)
The Secret Behind Our Formulations
It is through this biochemical lens that we have always crafted our supportive blends. Each formula is designed with a clear purpose: to influence the enzymes driving abnormal hormone metabolite patterns. Our focus is not on chasing symptoms or absolute hormone levels, but on uncovering and addressing the why. Why is 4-OH E1 elevated, and how can we reduce the oxidative stress contributing to impaired estrogen clearance? Why is metabolized cortisol persistently high, and what interventions can rebalance that pathway?
By asking the right questions and pairing the answers with targeted minerals, amino acids, and botanicals, we can influence the key enzymatic steps within critical biochemical pathways—supporting the root drivers of hormone imbalance and the symptoms they produce.
Of course, truly effective care must also integrate the environmental, nutritional, mental, emotional, and psycho-spiritual dimensions of health—elements we explore in depth within the EndoAxis report. But by combining that broader context with precise nutritive interventions, we aim to provide more targeted support, ultimately enhancing clinical response and improving patient outcomes.
Key Takeaways
The 2/16 ratio shows consistent correlation—but not causation—with breast cancer risk.
Urinary metabolites should be seen as sentinels—pointing to upstream enzymatic regulation or dysregulation.
Interpretation must integrate metabolites, detox pathways, oxidative stress burden, and environmental/lifestyle influences.
Clinical insight lies in contextual pattern analysis, not isolated thresholds.
By embracing the metabolic narrative, clinicians can move past “high vs. low” interpretations, toward root-cause-informed care that supports detoxification capacity, optimizes enzymatic function, and strengthens systemic resilience.
