Ensure Effective Bio-Identical Hormone Replacement: Select the Right Hormone Test for Your Patient

by Lara Pizzorno, MDiv, MA, LMT, with Pushpa Larsen, ND

Part I: The Advantages and Disadvantages of Saliva, Serum and Urine Tests

Treating the sequelae of the age- and stress-related decline in adult hormones with bio-identical hormone replacement (BHRT) can restore more youthful hormone levels and significantly alleviate symptoms associated with “normal” aging, optimizing health, happiness and quality of life.  Successful and safe BHRT, however, necessitates laboratory testing to assess the patient’s current hormonal status, monitor treatment, and ensure that hormones are being metabolized in ways that reduce risks for cancer, cardiovascular disease, osteoporosis, other age-related diseases and declines in cognitive and sexual function.

Hormones can be assayed using saliva, blood (serum), and urine. Each testing method has advantages and disadvantages.  Which of the three hormone test methods, or which combination of tests, you will wish to utilize will depend upon what information you need in a given clinical situation.

Salivary Hormone Testing

Advantages

Saliva testing may be helpful for monitoring:

• Cortisol circadian rhythm
• Cyclical pattern of estradiol and progesterone throughout the menstrual cycle in a premenopausal woman

For the four-point (6am, 12pm, 6pm, and 12am) cortisol assay required to evaluate cortisol circadian rhythm, salivary testing is the only practical option.

Saliva testing may also be useful, and is surely a more practical option than blood draws, to assess the cyclical estrogen and progesterone pattern throughout the month in a cycling or peri-menopausal woman. Saliva samples have been demonstrated to enable differentiation between the follicular and luteal phase for both estradiol and progesterone. Saliva collected daily throughout the menstrual cycle shows a specific pattern, with a mid-cycle rise and a peak in the early luteal phase. Mean salivary progesterone concentrations in the follicular phase range from 20 to 100 pmol/L, whereas peak concentrations during the peri-ovulatory period may attain 300 pmol/L. This significant difference is consistently reported and allows for an assessment of ovarian function.¹

The clinician can see if the patient is cycling, where hormone levels are off during the cycle, and can get an overview of the cycle’s pattern.

 Disadvantages

Saliva test readings may be unreliable. Evaluation of saliva samples obtained from the same subject at the same time and sent to two different labs has been shown to produce results with rates of variation from 35 to 75% for estradiol, 8 to 103% for progesterone, and 13 to 40% for testosterone.²

Salivary testing has repeatedly been shown to be highly unreliable for testosterone. Salivary testing of postmenopausal women receiving transdermal testosterone supplementation found no correlation with salivary testosterone levels for any of the serum testosterone subtypes (total testosterone, bioavailable testosterone [free and albumin-bound testosterone], and free testosterone.)³

It has been well documented that salivary testosterone measurements, more so than other commonly measured salivary analytes (i.e., cortisol), can be substantially influenced during the process of sample collection.  Materials commonly used to absorb the saliva sample (cotton and polyester swabs) or stimulate saliva flow (powdered drink-mix crystals and/or chewing gum) can artificially inflate testosterone results as much as two- to three-fold.⁴

Salivary testosterone levels are also susceptible to distortion resulting from the leakage of blood (plasma) into saliva as a result of micro-injury to the oral mucosa. In one study confirming this, the micro-injury group (n=35) donated a baseline saliva sample, then manually brushed their teeth for 2 minutes using an ADA recommended medium bristle brush without toothpaste. Saliva samples were collected immediately after brushing and then every 15 minutes for one hour. A control group (n=10) provided saliva samples without brushing teeth. In the tooth-brushing group, the resulting micro-injury elevated the presence of blood and its components in saliva within minutes, and correspondingly, testosterone levels in saliva increased and remained elevated over baseline well after micro-injury, even in samples that did not appear visually contaminated with blood. The effect of micro-injury was specific for testosterone; neither salivary cortisol nor dehydroepiandrosterone (DHEA) levels differed from baseline after brushing.⁴

In addition to possible blood contamination, the presence of both corticosteroid-binding globulin and sex hormone binding globulin (SHBG) in uncontaminated saliva renders questionable whether salivary steroids can accurately reflect circulating free steroid levels ¹(particularly in the older patient since, as noted earlier, SHBG levels increase with age).

Another potential confounder is the presence of the enzyme that converts cortisol to cortisone, 11β-hydroxysteroid dehydrogenase II (11β-HSD II), in salivary glands. Significant conversion of cortisol to cortisone (the inactive / storage form) may occur in the salivary glands via the activity of 11β-HSD II. This enzymatic conversion is often underappreciated in salivary cortisol measurements with the result that the data reflect not cortisol, but falsely increased measurements of both glucocorticoids together.¹  Furthermore, ascertaining the ratio of cortisol: cortisone (the ratio of active hormone: hormone reserves) provides significant insight into the patient’s adrenal health. (The cortisol: cortisone ratio is easily determined using the 24-Hour Urine test, discussed below.)

Storage temperatures can also contribute to error in the values estimated for salivary testosterone. One group of researchers evaluating the reliability of salivary testosterone found a striking linear increase in testosterone levels across four weeks for samples stored at 4 °C. On average, after one week of storage at 4 °C, measured testosterone levels increased 20.59% (9.1 pg/ml); by four weeks, testosterone levels had increased 330.77% (150.5 pg/ml) over baseline!⁴

Artificially elevated results have been reported not only for testosterone, but DHEA, progesterone and estradiol using cotton absorbent materials. On the other hand, salivary cortisol values are reduced by more than 50% when saliva is not retrieved immediately from cotton buds.⁵⁶

In an effort to mitigate the numerous problems with saliva sample reliability, a recent review recommends collection of unstimulated saliva into small plain tubes and storage at −20° C as the best method to avoid known pitfalls. If long term storage is anticipated, storage at −80° C is advised.⁶

However, there are additional problems for which no recourse is currently available. As we age, we produce less saliva, fewer hormones, and more sex hormone binding globulin (SHBG). Since saliva tests reflect only the metabolic process of salivary glands and a limited passive diffusion of the hormones from the bloodstream, levels may be below detection limits in elderly patients.

On the other hand, saliva testing has repeatedly been found to indicate significantly higher than physiological levels of hormones assayed in samples from patients using transdermal creams to deliver bio-identical hormone replacement therapy. This gives a false impression of overdosing.⁶ A possible explanation may be that red blood cells passing through capillaries rapidly uptake steroid hormones, which are lipophilic, and quickly transport them to salivary glands and other tissues. This results in elevated hormone concentrations in saliva, while serum and urine levels remain low.⁷⁸⁹

Saliva tests cannot capture hormone metabolites, so they do not provide the clinician with information essential to safe hormone replacement therapy: e.g., what percentage of estrogens are being converted into carcinogenic rather than protective compounds; the extent of adrenal fatigue; the activity level of  5α-reductase (the enzyme that converts testosterone to the more potent [and potentially prostate-carcinogenic] DHT; DHT also promotes benign prostatic hypertrophy and male pattern baldness in men, and hirsutism and polycystic ovarian syndrome [PCOS] in women.) The 24-Hour Urine test reports all of these variables.

Like serum testing, saliva tests also offer only a ‘snapshot’ look at hormones that ebb and flow throughout a 24-hour period. Due to this fact, the conclusion drawn in a 2009 review entitled, “Salivary steroid assays – research or routine?” was:  “The diagnostic value of salivary estradiol, progesterone, testosterone, dehydroepiandrosterone and aldosterone testing is compromised by rapid fluctuations in salivary concentrations of these steroids. Multiple samples are required to obtain reliable information, and at present the introduction of these assays into routine laboratory testing is not justified.”¹⁰

In sum, salivary steroid testing may be useful for assessing circadian patterns for cortisol and for evaluating estrogen and progesterone patterns (ovarian function) in cycling and peri-menopausal women. For these indications, collection of unstimulated saliva into small plain tubes and storage at −20° C is recommended to avoid known pitfalls. Saliva testing is not a reliable option for even a snapshot evaluation of androgen levels or for monitoring the absorption of sex hormones from transdermal creams, which are likely to show elevated salivary hormone levels, while plasma and urinary levels remain low.¹

Blood (Serum) Hormone Tests

Advantages

Serum testing offers a reliable and is the preferred option for testing a number of hormones including:

• Follicle Stimulating Hormone (FSH)
• Luteinizing Hormone (LH)
• Prolactin
• Thyroid Stimulating Hormone (TSH)
• Reverse T3
• Insulin
• Dihydrotestosterone (DHT)

Measurement of these compounds in serum is the best choice for two reasons. Most of these hormones are proteins and therefore do not show up in urine in significant quantities if the kidneys are functioning normally. Others, e.g., reverse T3, are extremely small, so are more easily measured in serum than urine.

Additionally, while most testosterone metabolites are easily measured in urine, dihydrotestosterone (DHT) is not. DHT is very difficult to measure in any medium, but most accurately measured in serum using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Disadvantages

Blood (serum) hormone tests directly assess the amount of hormones in the circulation; however, significant limitations must be noted.

Most important is that measurement of sex hormones in serum is necessarily a ‘snapshot’ peek at hormones that fluctuate significantly during the day both premenopause/andropause and in individuals using any form of hormone replacement therapy. In premenopausal women, the ovaries secrete hormones in pulses. In men, the testes, in which Leydig cells produce 95% of the body’s testosterone, perform similarly. Postmenopausal women using BHRT (or HRT) typically take their replacement hormones once or twice daily, as do men using BHRT (fortunately for 21st century men, the formerly patented, carciogenic testosterone analogue, methyltestosterone—widely and enthusiastically prescribed for men in the 1940s and early 1950s, as was Premarin® for women from the 1980s until 2002—is hardly every prescribed at present).

Adding to the “snapshot” limitation is the fact that most serum tests report only “total” hormone values, i.e., the sum of free, conjugated and bound forms of the hormones—again, with the exception of testosterone for which both free and total values are typically reported. Free estrone, free estradiol, and free progesterone are rarely measured in serum. Serum hormone tests typically include measurements only of total estradiol (E2), although total estrone (E1, the most potent and potentially oncogenic estrogen) may be available. Since bound estradiol and estrone are inactive, serum tests do not provide feedback regarding levels of active hormone.

Another distinction not taken into consideration in serum tests is that among free, conjugated and bound forms. Unlike bound forms, which have been completely inactivated, conjugated steroids, which have combined with simple molecules—glucuronic acid or sulfates in Phase II liver conjugation—can be activated by glucuronidases and sulfatases. Sulfatases are not only highly prevalent in breast tissue, but are also found in numerous other tissues including the endometrium, ovaries, bone, brain, and prostate. Quantitative data show that the sulfatase pathway, which transforms estrogen sulfates into bioactive unconjugated estradiol, is actually 100–500 times more active than the aromatase pathway, which converts androgens into estrogens. Thus, the distinction between conjugated and bound forms of estradiol is clinically important since the sulfatase pathway is a significant contributor to body load of bioactive estradiol. Conjugated estradiol cannot be discounted as a factor in estrogen-related oncogenesis.¹¹

In addition, conjugated estrogens can be re-activated in the intestines and returned to circulation via the action of beta-glucuronidase enzymes, which are produced by E. coli, Clostridum and Bacteroides¹², thus intestinal dysbiosis may increase circulating levels of active estrogens). For these reasons, hormone replacement therapy assessment (whether BHRT or HRT) should incorporate both free and conjugated estrogens. The 24-hour Urine Test does so; serum tests do not.

Levels of unconjugated estriol (E3, considered protective and the weakest form of estrogen) are sometimes available in serum tests.  While measurement of unconjugated estriol may be helpful in terms of evaluating a woman’s risk of cancer (an “Estrogen Quotient” [estriol divided by the sum of estrone and estradiol] of ≤ 1.0 suggests lower cancer risk), its usefulness is significantly hampered because 90% of estriol is conjugated.

As a consequence of all of the above, serum testing does not provide the clinician with adequate information to safely prescribe and evaluate the effects of hormone replacement therapy (BHRT or HRT). For example, a menopausal woman whose serum test results indicate total estradiol at normal levels may still be experiencing hot flashes and other common climacteric symptoms if most of her estrogen is bound, which it likely is, as levels of sex hormone binding globulin (SHBG) increase with age.¹³

In addition, prescribing estradiol to this patient to alleviate her symptoms may result in increasing her levels of estrone since estradiol readily converts to estrone. This may not be advisable since estrone may be metabolized to either protective 2-hydroxyestrone (2-OH), 2-methoxyestrone (2-CH3O) or Estriol (E3) OR into pro-carcinogenic 4-hydroxyestrone (4-OH estrone) and 16α-OH estrone. Serum testing will not indicate which pathways are capturing her estrogens, so will not provide the clinician with the insight necessary to safely alleviate this woman’s symptoms. Furthermore, BHRT typically involves using a compounded bi-est containing a combination of estriol and estradiol (often 80% and 20%, respectively).  Yet serum testing does not usually measure estriol.

To be able to safely and effectively prescribe hormone replacement therapy (BHRT or HRT) will necessitate knowing not only how much estrone, estradiol and estriol are in circulation, but how much is active (free plus conjugated [potentially active]), and into what forms each estrogen is being metabolized: Is she predominantly producing protective or carcinogenic estrogen metabolites? This data is provided by the 24-Hour Urine Test (see below).

Urine Hormone Testing

Advantages

The 24-Hour Urine sample may be relied upon to accurately evaluate and monitor:

• Total daily production of hormones
• Overall hormonal balance (sex hormones, glucocorticoids, mineralocorticoids)
• Levels of active (free plus conjugated) estrogens (estrone, estradiol, & estriol)
• Progesterone
• Female and male sex hormone balance and metabolites
• Free androgens (DHEA & testosterone)
• Androgen balance
• Androgen metabolites (however, new serum tests provide information on additional analytes not reported in urine testing)
• Activity of the 5α-reductase and aromatase enzymes
• Breast and prostate cancer hormonal risk factors (e.g., in women the 2:16α ratio, 4-OH estrone, the Estrogen Quotient; in men, the 2:16α ratio, the Testosterone:Estrogen ratio [low T:E suggests insulin resistance which correlates with increased risk for prostate cancer])
• Liver function as indicated by assessment of Phase I and II metabolites
• Human growth hormone
• Adrenal health / adrenal reserves
• Response to and safety of  hormone replacement therapy (BHRT or patent medicine, e.g., Premarin® and Provera®)

Urine hormone testing is well-established in medical literature as a reliable method of assessing levels of active (free & conjugated) hormones and their metabolites, and has been shown in clinical settings to correlate well with patient symptoms and reflect the beneficial impact (or lack of benefit and potentially oncogenic effect) of therapeutic interventions.¹⁴¹⁵

Measuring steroid hormones via urine testing using both gas chromatography-mass spectrometry (GS-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been shown to produce highly accurate results, but radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA, also known as enzyme immunoassay [EIA]) analysis may not be accurate. Comparisons of RIA and ELISA (methods routinely used to measure estrogen metabolites in blood and urine due to efficiency and low cost) with GS-MS and LC-MS/MS have shown RIA and ELISA estrogen metabolite measures to be much less accurate, especially at the low estrogen metabolite levels characteristic of postmenopausal women. The clinical takeaway: ask the lab you use what procedures they are using.¹⁶¹⁷¹⁸

A 24-hour urine sample is practical (collection is non-invasive and easy for the patient) and provides a more accurate indication of hormonal output since it averages out the hour-to-hour fluctuations seen in both serum and salivary measurements.

In addition, a 24-Hour Urine test captures numerous metabolites that are not measurable in saliva and cannot be reliably measured by a single, or even multiple, blood draws. Estrogen metabolites that have been shown to impact estrogen-related cancers (more detail provided in Part II of this article) offer a prime example:

(1) The 2/16α ratio: a decrease in this ratio of the estrone metabolites, 2-hydroxyestrone and 16α-hydroxyestrone, is associated with an increased risk of breast and cervical cancer. Optimal ratio is 2.0 – 4.0.

(2) The “Estrogen Quotient” (EQ): also available only via the 24-hour urine test since it requires assessment of the levels of free estrone, free estradiol and free estriol. Another indicator of breast cancer risk, the EQ, is calculated by dividing the amount of estriol by the sum of that of estrone and estradiol. An EQ <1.0 is suggestive of increased breast cancer risk.

Measurement of urinary estrogens also provides insight into liver and gut function. Urinary levels of Phase I and Phase II estrogen metabolites serve as a functional indication of liver detoxification capability. Abnormal levels may indicate exposure to compounds that stress liver function, such as heavy metals, environmental chemicals, or pharmaceuticals including conjugated equine estrogens and progestins.

Urinary estrogen levels outside the reference range may also be suggestive of intestinal dysbiosis since beta-glucuronidase enzymes, which are produced by E. coli, Clostridum and Bacteroides in the intestines, can de-conjugate conjugated estrogens, enabling their return to the circulation.¹²  For these reasons, interventions that improve liver and gut function may assist in gradual normalization of estrogen levels.

A number of glucocorticoid and mineralocorticoid metabolites measured in a 24-hour urine hormone profile, but not in a serum or saliva assay, provide greater insight into long-term adrenal health, short term stress response, the cortisol/cortisone balance, and other measures of adrenal health and function than assessment of cortisol alone. These include cortisone, tetrahydracortisone, allo-tetrahydrocortisol, tetrahydrocortisol, aldosterone, allo-tetrahydrocorticosterone, tetrahydocorticosterone and 11-dehydrotetrahydrocorticosterone, and are discussed in Part II of this review.

Disadvantages

Circadian fluctuation of cortisol cannot be measured using 24-hour urine collection, nor can it easily show the monthly cyclical pattern in estrogen and progesterone production in a menstruating or peri-menopausal woman. You could, technically, do 24-hour urine collections for each of the days (as many as 11) tested in a saliva panel for this.  But consider the logistics and expense! A single 24-hour urine test cannot provide the clinician with insight into where hormone levels are off during the cycle or whether the patient is ovulating; however, very low progesterone levels in a 24-hour urine sample collected during the mid-luteal phase (days 19-21) would suggest anovulation, and the timing and type of symptoms can give further clues to hormonal imbalance.

The physician should confirm that patients have a clear understanding of collection instructions since sample collection should occur during the mid-luteal phase (days 19-21) if the patient is premenopausal, or postmenopausal and on hormone replacement therapy; if postmenopausal and not on hormone replacement therapy, sample collection may be taken on any day.

To prevent sample contamination, patients applying transdermal cream to the vaginal labia should be instructed to apply to the perianal area or inner thigh on the day of collection.

Part II: The 24-Hour Urine Test: Analyte Interpretation and Evaluation for Women

Following is an explanatory listing of the steroids evaluated by the most comprehensive of the 24-hour urine tests, the Comprehensive PLUS Hormone Profile with hGH.  Levels of all steroids reported indicate free plus conjugated forms.

This article discusses 24-Hour Urine Test results primarily in relation to female patients. Part III of this review will focus on BHRT serum and 24 Hour Urine test results in relation to male patients.

For a schematic overview of the metabolism of these steroids, please see Figure 1, “Metabolism of Select Steroids.”

This chart is reproduced with permission from Meridian Valley Lab, www.meridianvalleylab.com.

Click Here to View Larger View of Metabolism of Select Steroids.

24 Hour Urine Test analytes and adult reference ranges for women are summarized in Figure 2 Below.

24 Hour Urine Test Analytes

Creatinine: a break-down product of creatine phosphate in muscle, which is produced by the body at a fairly constant rate and filtered out of the blood by the kidneys. Creatinine concentration provides a marker for a full – neither under nor over – urine collection.

The adult reference range is 0.5 – 2.0 gm/24hr. Because it is a muscle breakdown product, creatinine will typically be higher in men than women (especially if the patient exercised on the day of collection).

Total urine volume: typically ranges from 1,200 – 3,000 mL.

SEX HORMONES: ESTROGENS

Estrone (E1): The most potent estrogen, estrone is synthesized from androstenedione by aromatase in the ovaries and adipose tissue in premenopausal women, and in adipose tissue in postmenopausal women. Estrone, which constitutes ~33% of circulating estrogens in cycling women (compared to 44% for estradiol and 10% for estriol)¹⁹,  is the most abundant estrogen in postmenopausal women, especially those with a high percentage of adipose tissue (including apparently “lean” women with sarcopenia).

In its conjugated form as estrone sulfate, estrone is a major source of local bioactive estrogen formation in bone and plays an important role in bone maturation and homeostasis, especially in elders.  Additionally, estrone sulfate is a long-lived derivative that serves as a reservoir for the more active estradiol (E2), into which estrone is easily converted via 17β-hydroxysteroid dehydrogenase (17β-HSD).

Estrone’s metabolites are known to play both oncogenic and anti-oncogenic roles.  Estrone’s oncogenic metabolites, 4-hydroxyestrone (4-OH estrone, considered the most carcinogenic estrogen metabolite) and 16α-OH estrone (which is needed in small amounts because of its bone-building actions), are produced by Phase I metabolism. Estrone’s protective metabolites: 2-hydroxyestrone (2-OH estrone), 2-methoxyestrone (2-CH3O-estrone) and estriol (E3), are produced in Phase II metabolism.

The Luteal (days 17-26 of the menstrual cycle) Reference Range for estrone is 3.3 – 44.6 μg/24hr; levels in postmenopausal women not on BHRT range from 1.0-7.0.

High levels of estrone may result from oral supplementation with DHEA. Switching the delivery form of DHEA to a transdermal or transmucosal cream will often lower EI to within normal range.²⁰²¹

Estradiol (E2): The most active estrogen, estradiol is the primary “estrogen” used in conventional hormone replacement (HRT) whether the delivery form is oral or patch. In humans, it is produced both from the conversion of estrone by 17β-HSD and from testosterone via aromatase. Estradiol is also readily converted to estrone via 17β-HSD, which is reversible. Small amounts are also produced in the adrenal cortex, adipose tissue, brain, arterial wall and, in males, in the testes. Unless converted to estrone, estradiol is metabolized into protective 2-OH estradiol (Phase I) and 2-methoxyestradiol ([2-CH₃0 estradiol], Phase II).

The Luteal (days 17-26 of the menstrual cycle) Reference Range for estradiol is 1.4 – 12.2 μg/24hr; levels in postmenopausal women not on BHRT range from 0 – 4 μg/24hr.

Estriol (E3): Derived from estrone through the intermediate (bone-building but potentially oncogenic) 16α-OH estrone, estriol has 20-30% less affinity for estrogen receptors and is the weakest estrogen, with anti-proliferative effects in estrogen sensitive tissues (e.g., breast and endometrium). Most receptors for estriol are found in the vagina.

Estriol has been found to have beneficial immune-modulating effects in patients with multiple sclerosis, increasing protective immune responses and decreasing the number and volume of lesions seen in cerebral MRIs.²²  Estriol is the primary estrogen produced during pregnancy, when it is made by placenta from 16α-OH DHEA sulfate (DHEA-S), an androgen made in the fetal liver and in the adrenal glands.²³

The Luteal Reference Range for estriol is 6.1 – 32.4 μg/24hr; in postmenopausal women not on BHRT, the reference range runs from 0 – 30 μg/24hr.

Estrogen Quotient: EQ = Estriol (E3) divided by the sum of Estrone (E1) + Estradiol (E2). Originated 1970s by Dr. Henry Lemon, who tested estrogen levels in 24 hour urine samples and found that an EQ >1 strongly correlated with a higher survival rate after breast cancer.²⁴  Further research conducted by Lemon, Heidel, et al., a meta-analysis of published fractional estrogen excretion collected from 2,846 healthy women worldwide aged 15 to 59 years, with a risk of breast cancer varying five-fold, found that an EQ <1 reflects increased rates of oxidation of estrone or estradiol to 4-OH catechols (also referred to in the literature as the 3,4-catechol estrogen quinones), which have been identified as the principal proximal human mammary carcinogens after menarche, while an EQ >1 reflects conversion to protective 2-OH estrogen metabolites.²⁵²⁶

In relation to breast cancer prevention, the amount of estriol relative to estradiol and estrone (the EQ, which should be >1), appears to be more important than the absolute amount of estriol.

If the EQ is <1, supplementation with 6-15 drops of Lugol’s solution QD (or 6-10 drops of SSKI or equivalent amounts of Iodoral QD [1 tablet of Iodoral = 12.5 mg iodine]) has clinically been shown to be helpful in promoting conversion of 16α-OH estrone to estriol.²⁷²⁸   Some women initially require high doses of iodine to jump start the conversion of 16α-OH estrone to estriol, after which iodine supplementation can be cut back to lower amounts.  The conservative approach is to start at the lower end, and if you do not get results, to then increase the dose; however, a significant percentage of women will need higher doses.  And, of course, monitoring thyroid function is especially important the higher the dose of iodine.

2/16α ratio: The ratio of 2-hydroxyestrone (2-OH E1) to 16α-hydroxyestrone (16α-OH E1) is an indicator of an individual’s risk of developing breast or prostate cancer. 2-OH E1, a Phase I metabolite of estrone, has weak estrogenic activity and is associated with protection against breast and prostate cancer. 16α-OH E1, also a Phase I estrone metabolite, is an estrogen agonist, stimulates cell mitosis and proliferation, and is associated with increased risk of breast, endometrial and prostate cancer. However, 16α-OH estrone is also involved in bone building, so very low levels indicate increased risk for osteopenia/osteoporosis in men as well as women.^{29303132333435}   Confirming this, a positive family history of osteoporosis has been found to be associated with preferential metabolism of estrogen through the inactive 2-OH pathway.³⁶

The Luteal Reference Range for 2-OH estrone is 3.8 – 38.1 μg/24hr ; in postmenopausal women not on BHRT, the range is 0.2 – 5.4 μg/24hr.

The Luteal Reference Range for 16α-OH estrone is 2.1 – 7.9 μg/24hr; in postmenopausal women not on BHRT, the range is 0.15 – 3.5 μg/24hr.

The ideal 2/16α Ratio range is 2 to 4. A 2/16α Ratio of >2 indicates increased risk for positive and negative estrogen receptor breast cancer, prostate cancer, cervical cancer, ovarian cancer, and laryngeal cancer. A 2/16α Ratio of <4 indicates increased risk for osteopenia, osteoporosis.

Fortunately, the 2/16α ratio is highly modifiable with natural interventions. Estrone’s conversion to (pro-carcinogenic) 16α-OH E1 is promoted by obesity, hypothyroidism, pesticides, alcohol and cimetidine. Conversion to (protective) 2-OH E1 is promoted by indole-3-carbinol (I3C), 3.3- diindoylmethane (DIM), exercise flax, soy and omega-3 fatty acids. (I3C and DIM are phytochemicals in Brassica vegetables that induce CYP1A1, a Phase I enzyme that stimulates formation of 2-OH E1 while downregulating formation of 16α-OH E1. DIM forms spontaneously from dimerization of I3C in the presence of stomach acid and is thought to be the primary active agent in I3C.)^{37383940414243}

4-OH estrone (4-OH E1): An estrone metabolite that can be enzymatically oxidized to catechol estrogen quinones, which can damage DNA by forming predominantly depurinating adducts. This results in the accumulation of mutations and promotes transformation into malignant cells.⁴⁴

Obviously, low end of the Reference Range, (Luteal is 0.8 – 5.9 μg/24hr; postmenopausal not on BHRT is 0.6 – 5.0 μg/24hr) is desired.

2-methoxyestradiol (2-CH₃0E2) and 2-methoxyestrone (2-CH₃0 E1) are Phase II metabolites of estradiol and estrone. 2-CH₃0 E2 has generated a lot of research excitement lately since it not only has anti-angiogenic and anti-tumor effects, but has been shown to inhibit bone resorption, both by inhibiting osteoclast differentiation and by being cytotoxic to osteoclasts, while harmless to most normal cells.^45464748

Recent papers have even suggested that the cardioprotective protective effects of estrogen may be largely mediated by 2-CH₃0 E2, which inhibits vascular smooth muscle cell growth in arteries. Of note, in systemic and pulmonary vascular endothelial cells, smooth muscle cells, and fibroblasts, 2-CH₃0 E2 exerts stronger anti-mitotic effects than estradiol itself. Estradiol and 2-CH₃0 E2, despite having similar effects on other cardiovascular cells, have opposing effects on endothelial cells; in endothelial cells, estradiol is pro-mitogenic, pro-angiogenic and anti-apoptotic, whereas 2-CH₃0 E2 is anti-mitogenic, anti-angiogenic and pro-apoptotic. 2-CH₃0 E1 appears to be a metabolite that itself lacks biologic activity but may be converted to (and serves as a reservoir for) 2-CH₃0 E2.

Levels of 2-CH₃0 E2 and 2-CH₃0 E1 also provide an indirect assessment of liver function since these metabolites are formed from their respective estrogen precursors via methylation in the liver. If levels are below, or at the low end of the reference range, supplementation with methylation cofactors can help optimize Phase II metabolism of estrogen to these methylated derivatives. Methylation cofactors include SAMe (whose production requires magnesium as well as methionine), B6, B12, folate, zinc, and betaine (aka trimethylglycine).

The Reference Ranges are 2-CH₃0 E2: luteal 0.1 – 2.2 μg/24hr; postmenopausal 0.03 – 0.54 2 μg/24hr, and 2-CH3O E1: luteal 2.2 -14.4 μg/24hr; postmenopausal 0.3 -4.1 μg/24hr.

Practical Application: Normalizing Estrogen Levels

High overall estrogen levels (> mid-luteal range) may indicate insulin resistance and/or a need to improve estrogen detoxification.

As insulin levels increase, this signals the ovaries to decrease sex hormone binding globulin (SHBG), thus increasing circulating levels of free estrogens.¹³⁴⁹⁵⁰

Lessening intake of refined carbohydrates while increasing fiber intake may be sufficient to increase SHBG. If metabolic syndrome or type 2 diabetes has been verified, a low glycemic index, high-fiber diet is recommended.

Calcium D-glucarate supports proper estrogen excretion

The major route of estrogen elimination in humans is through Phase II glucuronidation (conjugation with glucuronic acid) in the liver. Glucose and inositol are oxidized in the liver to glucuronic acid and then bound into uridine diphosphate glucuronate (UDG), which is used by the enzyme uridine diphosphate transglucuronylase to convert 2-OH estrone, 4-OH estrone, 16α-OH estrone, estradiol or estriol into water-soluble glucuronides, which can be excreted through the urine as well as bile.

Calcium-D-glucarate is the calcium salt of D-glucaric acid. Oral supplementation with calcium-D-glucarate has been shown to inhibit beta-glucuronidase, an enzyme produced by colonic microflora (e.g., E.coli) that is capable of hydrolyzing glucuronide conjugates, thus returning estrogen readied for excretion back into circulation. Elevated beta-glucuronidase activity is associated with an increased risk for various cancers, particularly hormone-dependent cancers such as breast, prostate, and colon cancers.⁵¹⁵²

Normalizing estrogen hyperexcretion

If despite carefully increasing doses of bio-identical estrogens, your patient is still experiencing hot flushes and other menopausal symptoms suggestive of insufficient estrogen (e.g., night sweats, insomnia, memory and concentration difficulties, anxiety), she may be hyperexcreting estrogen due to upregulated Phase I (the cytochrome P450 family of enzymes) activity. A 24-Hour Urine test will typically reveal high levels of excreted estrogens, while a serum test will show low estrogen levels.

Cytochrome P450 (CYP) enzymes well known for their involvement in the metabolism of tobacco carcinogens are also involved in estrogen metabolism, and many are regulated by estrogens (e.g. CYP 1A1, CYP 1B1, CYP 17)⁵³⁵⁴, CYP 1B1, in particular, is upregulated by equilin and equilinin (hormones from pregnant mares found in Premarin®) which converts these estrogens to 4-OH (pro-carcinogenic ) metabolites. In contrast, human estradiol is catalyzed by CYP1A1, producing 2-OH (protective) metabolites.⁵⁵

Not infrequently, Phase I is upregulated in women who have taken conventional HRT (e.g., Premarin®). Microdoses of cobalt chloride (300 – 700 mcg QD) will inhibit heme synthesis, normalizing Phase I activity and decreasing the rate of estrogen biotransformation and excretion. Obviously, switching to BHRT is advised.⁵⁶  Doses of cobalt this small are quite safe; this amount is present in the daily diet in some areas of Europe and North America.⁵⁷  Cobalt supplementation usually restores this abnormality of estrogen metabolism to normal within 10 to 20 weeks, after which time, supplementation with cobalt can be discontinued.²⁰²⁷

SEX HORMONES: PROGESTERONES

Progesterone: The major naturally occurring member of a class of structurally very similar hormones, progesterone is produced by the ovaries, brain, and during pregnancy, the placenta. Progesterone opposes, modulates and balances estrogen’s effects, not only in the uterus but in numerous other tissues including the breast, brain, bones, urinary tract, and skin. Unopposed estrogen (“estrogen dominance”) can excessively stimulate cell growth in the breast, uterine lining and endometrium, leading to hyperplasia and cancer.⁵⁸  Levels of progesterone are low during the pre-ovulatory (follicular) phase of the menstrual cycle, rise at ovulation and are elevated during the luteal phase.

The body’s innate awareness of the importance of balance between the estrogens and progesterone is illustrated by the fact that estrogen increases the expression of progesterone receptors.⁵⁹⁶⁰⁶¹

Among its numerous other beneficial functions, bio-identical progesterone also acts as a natural anti-depressant⁶²⁶³ and diuretic agent,⁶⁴⁶⁵ activates osteoblasts to build bone⁶⁶⁶⁷⁶⁸ normalizes blood clotting,⁶⁹  prevents blood brain barrier breakdown after stroke by reducing expression of metalloproteinases and inflammation,⁷⁰  and plays a significant role among factors contributing to thyroid stimulating hormone (TSH) and free thyroxine (T4) regulation.⁷¹

Symptoms of progesterone deficiency include:  agitation, irritability, anxiety, anger, weight gain, water retention, headaches, swollen or tender breasts, fibrocystic breasts, breakthrough bleeding, spotting, prolonged cycles, mood swings, insomnia, achy joints, excessive bleeding, and endometriosis. Since progesterone is the precursor of the glucocorticoids (e.g., cortisol), low levels can significantly contribute to adrenal fatigue.

Progesterone is an aromatase inhibitor,⁷² so may also help improve libido via increasing testosterone levels by preventing its aromatization to estradiol in women. (In men, progesterone’s inhibition of aromatase may be useful for treating benign prostatic hyperplasia [BPH]. In men, progesterone levels should be similar to women’s levels during the follicular phase.)

Pregnanediol, an inactive progesterone metabolite, is used to provide an indirect (but accurate) measure of progesterone levels in the body since progesterone’s structure prevents it from being eliminated in urine in significant quantities.⁷³⁷⁴

The Reference Ranges for pregnanediol are: Luteal 1450 -1640 μg/24hr; Follicular 0-2500 μg/24hr in women, 70-1050 μg/24hr in men; Postmenopausal 200 -1000 μg/24hr. In women on BHRT, the 24-Hour Urine sample should be collected during the Luteal phase (days 19-21 of the hormone replacement cycle).

SEX HORMONES: ANDROGENS

DHEA: produced in the adrenals, DHEA is the most abundant steroid in the human body. Levels peak between 25 to 30 years of age and then decline. After conversion into androstenedione, DHEA may be used to produce testosterone and its metabolites (for which reason DHEA replacement may boost libido in women) or estrone, and thus other estrogens.⁷⁵

DHEA helps build new bone tissue, primarily through its indirect elevation of serum levels of estradiol, and has been shown to significantly improve bone mineral density in older adults.⁷⁵⁷⁷⁷⁸  Low levels are associated with increased risk of fracture and osteoporosis.⁷⁹

DHEA and its metabolite etiocholanolone (also reported by the 24-Hour Urine Test, see below), inhibit glucose-6-phosphate dehydrogenase (G6PD), an enzyme that plays a key role in anerobic glycolysis, the major route of energy production for cancer cells.⁸⁰⁸¹

DHEA has significant immune-modulatory functions—both immune-stimulatory and anti-glucocorticoid effects. Evidence is accumulating that DHEA may be effective as a treatment for the immunological abnormalities that arise in subjects with low circulating levels of this hormone, including the impaired immune response of older individuals and immune dysregulation in patients with chronic autoimmune disease.⁸²

The Reference Range for DHEA is 100 – 2000 μg/24hr.

Etiocholanolone: One of two DHEA metabolites reported on the 24-Hour Urine Test, etiocholanolone, as noted above, has cancer-preventive anti-proliferative effects via its inhibition of G6PD. Etiocholanolone is produced from androstenedione by the enzyme 5β-reductase followed by 3α-hydroxysteroid dehydrogenase (3α-HSD). Excessive DHEA supplementation (>25 mg/day in females; > 50 mg/day in males) may be the cause of high etiocholanolone levels.

The Reference Range for etiocholanolone is 500 – 5000 μg/24hr.

Androsterone: A DHEA metabolite derived from androstenedione via the activity of 5α-reductase followed by 3α-hydroxysteroid dehydrogenase (3α-HSD), and therefore useful for monitoring 5α-reductase activity. If androsterone is high in relation to etiocholanolone, 5α-reductase activity may be elevated, resulting in increased conversion of testosterone to dihydrotestosterone (5α-DHT).

5α-DHT is 10 times more powerful than testosterone and unlike testosterone, cannot be aromatized to estradiol. Thus, in women, elevated 5α-reductase may contribute to PCOS. (In men, excess 5α-reductase is associated with male pattern baldness.) Excessive DHEA supplementation (>15 mg/day in females; >50 mg/day in males) is a possible cause of high androsterone levels.  However, if androsterone is low in relation to etiocholanolone in men, then 5α-reductase activity may be low as well, leaving more testosterone to be aromatized to estradiol.

The Reference Range for androsterone is 500 – 3200μg/24hr in women; 2000 to 5000μg/24 hr in men.

Testosterone: Primarily secreted by the testes in men and the ovaries in women, but also produced in the adrenals, liver, skin and brain, testosterone is important not only for its effects on the female as well as male libido,⁸³⁸⁴  but on bone mineral density. In a yearlong double-blind study, postmenopausal women were given either sublingual micronized estradiol + micronized progesterone alone or with the addition of micronized testosterone. Bone mineral density in the lumbar spine increased by +2.2 +/- 0.5% the HRT alone group and by + 1.8 +/- 0.6% in the HRT + T group. Total hip bone mineral density was maintained in the HRT alone group (+0.4 +/- 0.4%) and increased in the HRT + T group (+ 1.8 +/- 0.5%).⁸⁵  (That said, it would be much safer to use a topical delivery form for these hormones; there are serious problems with taking either estrogen or testosterone orally. Unlike topical estrogens, oral estradiol increases risk of heart attack, stroke and deep vein thrombosis.^{86878889909192939495969798}  Transmucosal delivery of both these hormones is preferred.)

In addition to its positive effects on BMD, testosterone also significantly lessens the age-related decline in, and preserves, lean body mass and muscle strength in postmenopausal women as well as in men.⁹⁹

Since women as well as men metabolize DHEA to testosterone, DHEA replacement in a female patient may render testosterone replacement unnecessary; however, if the 24-Hour Urine test shows insufficient conversion of DHEA to testosterone, replacement (at a fraction of the male dose [An adult male produces 40-60x more testosterone than an adult female] may be indicated if flagging libido or excessive bone loss is an issue.

For women, the Reference Range for testosterone is 5 – 35μg/24hr. A typical female BHRT dose is 1 mg QD. In women, testosterone may be metabolized to estrogens (estradiol), so follow-up testing to monitor hormone balance is essential.

5α-Androstanediol: a testosterone/5α-DHT  metabolite produced via the activity of 5α-reductase. High levels indicate testosterone is primarily being metabolized through 5α-DHT, especially if levels of 5β-androstanediol are low. As noted above, 5α-DHT is highly potent—10 times more powerful than testosterone. In women, elevated 5α-reductase activity is linked to acne, hirsutism, hair loss, hypothyroidism, PCOS, insulin resistance, and obesity.

The Reference Range for 5α-Androstanediol in women is 3.0 – 35.0μg/24hr.

5β-Androstanediol: a testosterone/5β-DHT metabolite produced via the activity of 5β-reductase. High levels indicate testosterone is being metabolized through 5β-DHT, especially if levels of 5α-androstanediol are low. 5β-DHT is weak testosterone metabolite.

The Reference Range for 5β-Androstanediol in women is 13.0 – 180.0μg/24hr.

GLUCOCORTICOIDS

Pregnanetriol: A progesterone metabolite and indicator of sufficient substrate for the cortisol pathway. Progesterone supplementation may increase pregnanetriol levels, but very high levels—while rare–are suggestive of 21-hydroxylase deficiency, which is the proximate underlying cause of adrenal hyperplasia. When 21-hydroxylase is dysfunctional, conversion of progesterone to both glucocorticoids and mineralocorticoids is blocked. Disorders resulting from glucocorticoid and mineralocorticoid deficiency as well as androgen excess result, including PCOS, infertility and depression. DHEA may also be elevated.¹⁰⁰¹⁰¹

The Reference Range for pregnanetriol is quite wide (100-1500). Mid-range or higher is desirable since pregnanetriol is an indicator of substrate availability for the cortisol pathway.

Cortisol: After DHEA, cortisol is the second most plentiful steroid in a healthy person. Cortisol increases gluconeogenesis, affects protein and fat metabolism, and thyroid metabolism. (Both excess and insufficient cortisol inhibit, while normal levels promote the conversion of T4 to T3.)^{102 103}  Cortisol has potent immunosuppressive and anti-inflammatory activity.

In excess, however, cortisol promotes insulin resistance and many features of the metabolic syndrome (e.g., glucose intolerance, hypertension, dyslipidemia). High levels of cortisol also decrease the ability of osteoblasts to synthesize new bone and interfere with absorption of Ca2+ from the gastrointestinal tract.

In 21st century life, cortisol is often elevated due to unremitting stress and sleep deprivation, and in “Catch 22” fashion, elevated cortisol promotes both. Elevated cortisol (or cortisone, see below) is associated with Cushing disease, unipolar depression, sleep deprivation, anxiety, panic disorder, PTSD in its early stages, exogenous cortisol supplementation, high-dose licorice root supplementation, intense physical exercise, and acute ingestion of alcohol.

Clinical signs of adrenal excess include insomnia, anxiety, insulin resistance, obesity (especially truncal adiposity), hyperglycemia, hypertension, easy bruising in the extremities due to loss of subcutaneous adipose and connective tissue, bone loss, muscle weakness and sarcopenia. If causes are not addressed, adrenal fatigue and cortisol insufficiency is the likely outcome.

The Reference Range for cortisol is 30 – 170μg/24hr, which is quite wide and does not represent optimal levels. Optimal range for Cortisol is ~90μg/24hr.

Cortisone is the inactive metabolite of cortisone and serves as a “cortisol reserve,” in the body. Cortisone is produced by the action of 11β-hydroxysteroid dehydrogenase (11β-HSD), an enzyme with two isoforms, the first of which, 11β-hydroxysteroid dehydrogenase I (11β-HSD I) catalyzes cortisone into cortisol, enabling rapid supply of the active hormone as needed. The second isoform, 11β-HSD II, inactivates cortisol to cortisone (an action that is reversible via the activity of 11β-HSD I).

Decreased cortisol or cortisone seen with adrenal insufficiency is associated with Chronic Fatigue Syndrome, fibromyalgia, rheumatoid arthritis, and late stage Panic Disorder. Clinical signs of adrenal insufficiency include fatigue, exercise intolerance, hypoglycemia, salt craving, insomnia, depression, irritability, positive Hippus test (greater light exposure should result in pupil contraction), and low blood pressure.

The Reference Range for cortisone is 31 – 209μg/24 hr, which is quite wide and does not represent optimal levels. Optimal range for Cortisone is ~120-130μg/24hr.

Cortisol:Cortisone Ratio:  The ideal cortisol:cortisone ratio is 0.7 (i.e., cortisone should be ~30% higher than cortisol), as this indicates slightly more storage (cortisone) than active (cortisol) hormone. Sleep problems are common when this ratio gets to ≥1.

A ratio greater than 1.4 is considered possibly suspicious for the hypertensive syndrome “Apparent Mineralocorticoid Excess Type 2” (AME Type 2 is a much milder version of AME Type 1, a severe and lethal congenital deficiency of 11β HSD II).

Tetrahydrocortisone, Tetrahydrocortisol, and Allo-tetrahydocortisol: are metabolites of cortisone (tetrahydrocortisone), and cortisol (tetrahydrocortisol, and allo-tetrahydocortisol), and can be used to determine daily cortisol output.

When their 24 Hour Urine Test values are added together, these three metabolites account for approximately half of daily cortisol output. Taking the sum of the three, doubling it and moving the decimal 3 points to the left will give, in milligrams, about how much cortisol is being made each day. For a woman, these three metabolites should add up to 5,000 to 7,000, which translates to a cortisol output of 10-14 mg/day. (For a man, the three metabolites should add up to between 8,000 and 10,000, which corresponds to a cortisol output of 16-20 mg/day.)

Low levels are a very strong indication of weak adrenal function. (If allo-tetrahydrocorticotsterone (5α-THB), tetrahydrocorticosterone (THB) and 11-dehydrotetrahydrocorticosterone (THA) levels are also low, this is a very strong indication of long term adrenal insufficiency. These analytes are discussed below.)

11β-OH Androsterone and 11β-OH Etiocholanolone: terminal metabolites of cortisol. Their values will confirm if cortisol production is excessive or insufficient. Often, as patients become insufficient, cortisol levels may still appear within normal range, but downstream metabolites will be low.

MINERALOCORTICOIDS

Aldosterone: The major mineralocorticoid, aldosterone is part of the renin-angiotensin system and acts on the distal tubules and collecting ducts of the nephron (the functional unit of the kidney) to cause conservation of sodium, secretion of potassium, increased water retention, and increased blood pressure. Aldosterone levels are usually a reliable indication of whether a person is on a normal, low or high salt diet.

Aldosterone reverses certain types of hearing loss inn experimental animals^{104 105 106 107} and levels of serum aldosterone have been inversely correlated with degree of hearing in humans.¹⁰⁸  Case studies at Tahoma Clinic, Renton, Washington, have found that a significant percentage of individuals with hearing loss and low levels of aldosterone recover some of their hearing when exogenous aldosterone is taken.¹⁰⁹

(For further discussion and possible mitigating circumstances, see “Activity of 11β-Hydroxysteriod dehydrogenase I and II” below.) Reference ranges are: Normal Diet = 6.0 -25.0 μg/24hr; Low Salt = 17.0 -44.0μg/24hr; High Salt = 0.0 – 6.0μg/24hr.

Allo-tetrahydrocorticostersone (5α-THB), Tetrahydrocorticosterone (THB) and 11-dehydrotetrahyrdocorticosterone (THA): metabolites of aldosterone that serve as sensitive markers for monitoring acute adrenal stress. These metabolites are the first to rise in the ACTH stimulation test; high levels suggest acute stress at the time of collection. Low levels are a good indication of chronic adrenal fatigue. References ranges: 5α-THB = 130 – 600μg/24hr; THB = 30 – 240μg/24hr;   THA = 62 – 293μg/24hr.

The most comprehensive 24-Hour Urine Test also reports levels of human Growth Hormone, free T3 and T4 (the thyroid hormones).

Human Growth Hormone: Produced in the anterior pituitary and regulated from hypothalamus by growth hormone releasing hormone and growth hormone inhibiting hormone (aka somatostatin), human growth hormone (hGH, aka somatotrophin) enters the circulation and is delivered to the liver where it is converted to growth factors that initiate muscle, bone, and cartilage production; improve kidney function, skin elasticity, and cell repair and regeneration. One thing growth hormone does not increase is body fat; hGH decreases adipose tissue.

IGF1, used to evaluate hGH levels in serum tests, is not considered a reliable marker^{110 111}; urine hGH is a better indicator.^{112 113 114 115 116}

Growth hormone levels are increased by deep sleep,¹¹⁷ arginine (more effective in younger people, but useful in elders as well),^{118 119} glutamine (helpful for older people, 2 grams at bedtime),¹²⁰ and ornithine alpha-ketoglutarate (may boost the use of glutamine in arginine metabolism, 0.25 mg/kg),¹²¹ resistance training, short intense bursts of exercise, and vigorous aerobic exercise, ^{122 123} (although the effect of exercise is more pronounced in younger subjects),¹²⁴ and adequate protein.¹²⁵

A number of hormones increase hGH secretion including testosterone (the most potent secretagogue for hGH), estrogen, progesterone, thyroid, melatonin, and growth hormone releasing hormone (GHRH).^{126 127 128 129}  hGH is decreased by a sedentary lifestyle, inadequate protein, poor sleep, and insufficient endogenous hormones.

The adult Reference Range for hGH is 1065 – 4722μg/24hr. Mid-range is optimal.

Thyroid Hormones

Upon stimulation by thyroid-stimulating hormone (TSH), the thyroid produces two main hormones: thyroxine (T4), the major form of thyroid hormone in the blood, and triiodothyronine (T3), the active hormone (three to four times more potent than T4), which primarily regulates the metabolic machinery inside cells. (The ratio of T4 to T3 released into the blood is roughly 20 to 1.) Thyroid hormones’ effects include controlling the speed of protein synthesis, energy use, and sensitivity to other hormones.

Both thyroid hormones combine tyrosine with iodine (T4 with 4 iodine molecules, and T3 with 3 iodine molecules), thus iodine insufficiency prevents adequate thyroid hormone formation.

Even if TSH is effectively signaling the thyroid gland, and iodine is present in sufficient amounts for adequate production of T4 and T3 (blood test levels thus appearing normal), intra-cellular conversion to T3 may not occur for several reasons. T4 is converted to the T3 within cells by deiodinases (5′-iodinase), enzymes for which selenium is the required cofactor. Cortisol is also required for the conversion of T4 to T3; long-term stress, which depletes adrenal reserves of cortisone, will therefore cause inhibition of the T4 to T3 conversion. Inflammatory cytokines (notably interleukin-2) can promote formation of autoantibodies to the thyroid, again inhibiting the T4 to T3 conversion.¹³⁰   Thus, a patient can have hypothyroid symptoms despite normal serum levels of thyroid hormones. The 24-Hour Urine test gives a better indication of what is happening inside the cell.¹³¹

Ideally, one wants to see higher levels of free T3 than free T4.  The richest food source of selenium, Brazil nuts may help improve conversion of T4 to T3, and may also help lessen inflammation since selenium is a cofactor for reduction of glutathione peroxidases. One caveat here: no more than 2 Brazil nuts daily, 5 days each week. One Brazil nut provides ~ 100 mcg of selenium; 200 mcg is the recommended dosage. Excessive selenium can interfere with enzyme systems related to sulfur metabolism and can be toxic in amounts greater than 900 mcg/day.^{132 133 134 135}

Urinary Minerals

Sodium: Reference range 40 – 220 mmol/24hr

Potassium: Reference range 25 – 150 mmol/24hr

Urinary levels of sodium and potassium clearly reflect dietary intake. The ideal ratio of sodium: potassium is 1.5. Due to the typical Western diet, which contains a disproportionate amount of high-sodium processed foods and few servings of potassium-rich green leafy and other vegetables, the ratio of sodium: potassium is typically elevated.

Bringing this ratio into ideal range is well recognized to be of vital importance in the prevention of hypertension, myocardial infarction, stroke and kidney failure.^{136 137 138 139}

When salt intake is high, even a modest reduction for a duration of 4 or more weeks has a significant and important beneficial effect on blood pressure in individuals with normal as well as elevated blood pressure. A modest and long-term reduction in population salt intake could significantly reduce strokes, heart attacks and heart failure.¹⁴⁰   Decrease sodium by avoiding processed foods; increase potassium by increasing green leafy vegetable intake.

On the other hand, a 24 Hour Urine sodium on the low end of the reference range is not uncommon in people with low adrenal function.  Many people have heard that it is beneficial to reduce salt and are over-zealous about it, and thus are not getting the sodium they require for good adrenal function.

Enzyme Activity

5α-Reductase is the enzyme that converts testosterone to the more potent (and potentially prostate-carcinogenic) 5α-DHT. In men, excessive 5α-DHT activity promotes BPH and male pattern baldness. In women, excessive 5α-DHT activity is associated with hirsutism, acne and polycystic ovarian syndrome (PCOS).  In both sexes, upregulated 5α-reductase is associated with insulin resistance and obesity.

5α-reductase activity is reflected by two ratios on the 24-Hour Urine test:

(1) Androsterone/Etiocholanolone Ratio and (2) Allo-tetrahydrocortisol/tetrahydrocortisol Ratio.

If excessive, 5α-Reductase can be inhibited by zinc, GLA, saw palmetto, progesterone, green tea extract, finasteride, and dutasteride. (However, dutasteride, and presumably finasteride, can over-inhibit 5α-reductase.

In the Prostate Cancer Prevention Trial, a study involving more than eighteen thousand men, subjects taking dutasteride had a lower rate of prostate cancer, but a significantly higher rate of aggressive prostate cancers than the placebo group.  This resulted in a higher absolute number of aggressive prostate cancers in the dutasteride group than in the placebo group – even though the placebo group had a higher rate of prostate cancer.¹⁴¹ Thus, as we have seen elsewhere, e.g., rofecoxib (Vioxx®), celecoxib (Celebrex®), balance in biological processes is more beneficial than absolute interruption.)

11β-Hydroxysteriod dehydrogenase I and II are the enzymes that convert cortisone to cortisol and visa versa.

11β-Hydroxysteriod dehydrogenase I converts cortisone to cortisol. 11β Hydroysteroid dehydrogenase II converts cortisol to cortisone. 11β HSD I is inhibited by estradiol and HGH. 11β HSD II is inhibited by licorice and cadmium; this latter inhibition explains some of the hypertensive effects of these two substances.

11β-HSD I is highly expressed in key metabolic tissues including the liver, adipose tissue, and the central nervous system, where it reduces cortisone into the active hormone,  cortisol.

11β-hydroxysteroid dehydrogenase II (11β-HSD II) is found in salivary glands and aldosterone-selective tissues, e.g. kidneys, where it oxidizes cortisol to cortisone to prevent activation of the mineralocorticoid receptor. As noted in Part I on this review, the presence of 11β-HSD II in salivary glands can invalidate salivary cortisol measurements.¹

Activity of 11β-Hydroxysteriod dehydrogenase I and II is reflected by two ratios provided by the 24-Hour Urine Test:

(1) Cortisone: Cortisol Ratio

This ratio of hormone reserve (cortisone) to active hormone (cortisol),  shows enzyme activity in adipose tissue and kidneys and provides significant insight into the patient’s adrenal health. The ideal ratio is 0.7. Licorice, which contains glycyrrhetinic acid, can inhibit 11 β-HSD II, causing increased conversion of (storage) cortisone to (active) cortisol. (See Cortisol: Cortisone Ratio above for brief discussion of Apparent Mineralocorticoid Excess syndromes)

(2) Tetrahydrocortisol + allo-tetrathydrocortisol/Tetrahydrocortisone Ratio

Low ratios for these enzymes are associated with obesity and insulin resistance, while elevated ratios are associated with low-renin hypertension, high dose licorice, and exogenous cortisol. Ideal ratio is 0.9.

In patients with essential hypertension, elevated ratios may also be a sign of primary aldosteronism (PA), for which recent reports suggest incidence may be as high as 10-15% in hypertensive patients. PA may be missed in these patients because it can exist for many years before hypokalemia is demonstrable. Such patients are often mis-placed on anti-hypertensive medications, which do not prevent progression of the hypertensive vascular complications induced by hyperaldosteronemia (i.e., heart attack, stroke, kidney failure). If 11β-HSD ratios are elevated, hyperproduction of aldosterone will be detectable by ACTH-stimulated venous sampling.¹⁴²

CONCLUSION

24-Hour Urine Hormone Testing—Sine Qua Non for Optimal BHRT

Despite the brevity of this introductory overview of a very complex subject—how to safely and effectively monitor BHRT in your aging patients–the necessity of distinguishing between levels of bound, free and conjugated hormones, and of evaluating hormone metabolites has, hopefully, been demonstrated. The top labs offer clinicians free consults with physicians who specialize in BHRT, utilize the most comprehensive 24-Hour Urine tests, and are happy to mentor other physicians and assist them in learning how to interpret test results.

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