Bio-Identical Hormone Replacement: Selecting the Right Hormone Test(s) for Your Male Patient

by Lara Pizzorno, MDiv, MA, LMT with Barry Wheeler, ND

Treating the sequelae of andropause, the age- and stress-related decline in hormones in the adult male, 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 he is metabolizing hormones in ways that reduce his risks for prostate cancer, cardiovascular disease, metabolic syndrome, osteoporosis, and the “low grade” depression often seen in older men, as well as declines in cognitive and sexual function.

As discussed in detail in Part I of this review and recapped briefly here in relation to the male patient, hormones can be assayed using saliva, blood (serum), and urine. Which of the three hormone test methods, or which combination of tests, you will wish to utilize will depend upon what information you require in a given clinical situation.

Salivary Hormone Testing

Advantages

In males, saliva testing may be helpful for monitoring cortisol circadian rhythm. For the four-point (6am, 12pm, 6pm, and 12am) cortisol assay required to evaluate cortisol circadian rhythm, salivary testing is the only practical option.

Disadvantages

While saliva testing can help determine the circadian rhythm of cortisol, it cannot provide information on tetrahydrocortisol (THE), tetrahydrocortisone (THF), and allo-tetrahydrocortsiol (5a-THF)  levels, which is needed to determine adrenal reserve, 1 ,2   and thus, whether prescribing herbs, such as licorice, withania somiferum (ashwaganda), Panax ginseng or others, will be potentially beneficial or if replacement with hydrocortisone is indicated. Furthermore, it is difficult to estimate total production of cortisol since a night time spike, which is common in those with sleep disturbances, may be missed.

As discussed in detail in Part I  of this review, salivary testing has repeatedly been shown to be highly unreliable for testosterone.

Like serum testing, saliva tests offer only a ‘snapshot’ look at hormones that ebb and flow throughout a 24-hour period, for which reason its diagnostic value for testosterone, estradiol, progesterone, dehydroepiandrosterone (DHEA) and aldosterone is compromised by rapid fluctuations in salivary concentrations of these steroids. Samples would need to be taken multiple times throughout the day and night to obtain reliable information. 

More importantly, saliva tests cannot capture hormone metabolites, so cannot provide the clinician with information essential to safe hormone replacement therapy, e.g.:

  1. What percentage of testosterone is being converted into 5α-DHT, and from there through 5α-androstanediol into 5α-androstane-3α,17β-diol (aka 3α-adiol, a storage form of 5α-DHT) and 5β-androstane-3α,17β-diol (aka 3β-adiol, a cancer-preventive metabolite.)  A newly available serum Testosterone Metabolites test, discussed below, assesses these metabolites.
  2. The activity level of 5α-reductase (5AR), the enzyme that converts testosterone to 5α-DHT. Excessive 5AR activity promotes benign prostatic hypertrophy and male pattern baldness; however, under activity of this enzyme can promote prostate cancer since inhibition of 5α-DHT production will also inhibit production of its protective metabolite, 3β-adiol. 
  3. Levels of metabolites that indicate the extent of adrenal reserve or fatigue, e.g., tetrahydrocortisone (THE), allotetrahydrocortisol (5α-THF), tetrahydrocortisol (THF), allotetrahydrocorticosterone (5α-THB), tetrehydrocorticosterone (THB), 11-dehydrotetrahydrocorticosterone (THA). These metabolites are reported by the 24-Hour Urine test (discussed with a focus on women in Part I of this review and summarized in relation to men below).

In sum, in the male patient, salivary steroid testing may be useful for assessing circadian patterns for cortisol. For this indication, collection of unstimulated saliva into small plain tubes and storage at −20° C is recommended to avoid known pitfalls (See Part I). Saliva testing is not a reliable option for even a snapshot evaluation of androgen levels.

Blood (Serum) Hormone Tests

Advantages

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

Measurement of these compounds in serum is the best choice because most are proteins and thus do not show up in urine in significant quantities if the kidneys are functioning normally. Others, e.g., reverse T3, are extremely small, and thus more easily measured in serum than urine. Additionally, DHT, which is very difficult to measure in any medium, is most accurately measured in serum using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).56 

Disadvantages

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 pre-andropause and in men using any form of hormone replacement therapy (as indicated by Figure 1 below).  The testes, in which Leydig cells produce 95% of the body’s testosterone, secrete hormones in pulses throughout the day.  In men given testosterone injections, levels increase dramatically, peak within two to four days, then drop rapidly.  Men supplemented with natural testosterone may be applying transdermal creams or gels once or twice daily or may be using a patch or sublingual tablet; regardless of the delivery form chosen, testosterone levels will fluctuate throughout the day.

Figure 1: Serum Testosterone Concentration: Differences Among 3 Delivery Forms

Adding to the “snapshot” limitation is the fact that -- with the exception of testosterone for which both free and total values are typically reported -- most serum tests report only “total” hormone values, i.e., the sum of free, conjugated and bound forms of the hormones. Free estrone, free estradiol, and free progesterone are rarely measured in serum. In men, the ratio of free testosterone : free estrogens should be ≥4. If <4, this indicates excessive aromatization and possible insulin resistance.

Another distinction not taken into consideration in serum tests is that among free, conjugated and bound forms. Bound forms have been completely inactivated, but conjugated steroids (which have combined with simple molecules, i.e., 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 in men as well as women, but are also found in numerous other tissues including prostate bone, and brain. 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 in men since the sulfatase pathway is a significant contributor to body load of bioactive estradiol. Conjugated estradiol is a factor in estrogen-related prostate cancer (via its potential for metabolism to 16α-OH estrone).12 

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 Bacteroides 13 ; thus intestinal dysbiosis may increase a man’s circulating levels of active estrogens). For these reasons, evaluation of hormone replacement therapy in men should assess not only testosterone, but both free and conjugated estrogens. The 24-hour Urine Test does so; serum tests do not.  

As a consequence of all of the above, serum testing does not provide the clinician with adequate information to safely prescribe or evaluate the effects of androgen replacement therapy. For example, an andropausal man whose serum test results indicate total testosterone at normal levels may still be experiencing loss of muscle and libido, prostate enlargement, and erectile dysfunction, along with increased risk of cardiovascular disease, osteoporosis and depression, if most of his testosterone is bound, which becomes increasingly likely in men >50, the age at which testosterone levels typically begin to decline, while levels of sex hormone binding globulin (SHBG) increase with age.14  

Prescribing testosterone to this patient to alleviate his symptoms may result in increasing his levels of estradiol if his aromatase enzyme, which readily converts testosterone to estradiol, is overactive.  Excessive aromatization, in addition to its “feminizing” effects, may increase prostate cancer risk since estradiol can be metabolized to estrone, which can then be converted to either protective 2-hydroxyestrone (2-OH) or Estriol (E3) OR into potentially pro-carcinogenic 16α-OH estrone. On the other hand, insufficient aromatization not only promotes bone loss in men since some 16α-OH is required for bone remodeling, but has recently been shown to promote prostate cancer by inhibiting the production of 3β-adiol (discussed next).  Serum testing will not indicate which estrogen metabolites a man is producing.

When testing male hormones in serum, a morning blood draw is recommended since that is when they are typically at their highest levels. More typically, serum free testosterone will be low in an adropausal man experiencing the symptoms noted above. Nevertheless, to reduce risks, more information is needed than typical serum testing can provide.

Testosterone Metabolites Serum Testing

A recently developed serum test, the Testosterone Metabolites Profile, does provide significant insight into prostate cancer risk, however. This test captures levels of not only 5α-DHT, but its metabolites: 5α-Androstane-3α-17β-diol (aka 3α-adiol) and 5α-Androstane-3β-17β-diol (aka 3β-adiol).

Low levels of 5α-DHT are indicative of over-inhibition of 5α-reductase (5AR), the enzyme that converts testosterone to 5α-DHT, which is ~2-3 times as potent as testosterone due to 5α-DHT’s greater affinity for androgen receptors.15 

Drugs designed to inhibit 5AR (e.g., finasteride [Propecia, Proscar], dutasteride [Avodart]) and botanical supplements (e.g., Serenoa repens [Saw palmetto]) are taken by hundreds of thousands of men around the world to combat benign prostatic hypertrophy and androgenic alopecia (male pattern baldness). It has also been assumed that 5α-DHT is a key contributor to prostate cancer initiation and progression, so 5AR inhibitors are commonly used to treat prostate cancer. However, recent studies show a correlation between low levels of DHT and decreased survival in prostate cancer patients, rendering this assumption highly questionable.16 ,171819  According to one recent paper, “Low dihydrotestosterone in the prostate is probably sufficient to propagate the growth of aggressive prostate cancer.” 20 

The American Society of Clinical Oncology/American Urological Association 2008 Clinical Practice Guideline states: “Men who are taking 5-ARIs for benign conditions such as lower urinary tract symptoms (LUTS) may benefit from…understanding that the improvement of LUTS relief should be weighed with the potential risks of high-grade prostate cancer from 5-ARIs.” 21

Mechanisms through which 5α-reductase inhibitors promote prostate cancer

Over-inhibition of 5AR results in increased availability of testosterone, which may be shunted, via aromatase, to estradiol, causing “feminization,” e.g., gynecomastia, one of the side effects of 5AR inhibitor drugs; and decreased production of not only 5α-DHT, but its metabolites, 5α-Androstane-3α-17β-diol (aka 3α-adiol, a storage form of 5α-DHT), and 5α-Androstane-3β-17β-diol (aka 3β-adiol), an estrogen receptor beta (ERβ) ligand that promotes normal cellular differentiation, thus lessening risk of benign prostatic hypertrophy and prostate cancer.22 ,2324

ERβ is involved in several biological functions in the prostate, including regulation of cell proliferation, apoptosis and differentiation. 3β-adiol, as an alternative ligand to estradiol of ERβ (the concentration of 3β-adiol in prostate is 100-fold higher than that of estradiol), mediates the majority of these effects. ERβ expression has been revealed to be greatly decreased in malignant prostate tissue, reaching nearly undetectable levels with tumor progression in more than 75% of cases. Reintroduction of ERβ triggers apoptosis, and decreases proliferation and invasiveness of malignant cells. In addition, prostate hyperplasia has also been described in mice lacking ERβ, and 3β-adiol has been shown to more potently induce expression of ERβ in the rat prostate than estradiol or 5α-DHT.23

3β-adiol has lately also been revealed to be a key means through which the neuroendocrine response to stress is modulated in males. 3β-adiol suppresses excessive hypothalamic-pituitary-adrenal (HPA) axis activation, which would otherwise promote depression and neurodegeneration.2526

Balancing Men’s Hormones Summary:

Issues to Consider

        Under activity of 5α-Reductase (5AR): 

Over activity of 5α-Reductase:

Excessive aromatization: 

Dysbiosis:

  1. Remove toxins:  i.e., allergic foods, parasites, pathogenic  bacteria or yeast overgrowth). This might involve using an allergy “elimination diet” or prescribing drugs or herbs to eradicate a particular bug. It might also involve making healthier dietary choices: a whole foods, Mediterranean style, lower carb diet;  less processed, high glycemic index foods.
  2. Replace: replace digestive enzymes, hydrochloric acid and bile acids whose secretion may have been compromised by diet, drugs, diseases, aging.
  3. Reinoculate:  with probiotic foods (e.g., yogurt with active cultures), supplements that contain bifidobacteria and lactobacillus species, and consumption of the high soluble fiber foods that contain fructooligosaccharides (FOS) and other prebiotics.
  4. Repair:  supplement with key nutrients utilized by GI tract cells for repair, e.g., zinc; vitamins A, C, and E; fish oil; glutamine.
  5. Rebalance:  lifestyle choices – sleep, exercise and stress -- can all affect the GI tract.

Imbalanced 2/16α ratio:

Promoting 3β-adiol Production

Monitor Impact of All 5α-Reductase Inhibitors

Given the importance of 3β-adiol for prostate cancer prevention, stress reduction and prevention of age-related cognitive decline, men using any type of 5AR inhibitor -- natural compounds as well as patent medicines -- should be monitored for over-inhibition. If over-inhibition is found: (1) use a lower dose of, or a less potent, 5AR inhibitor (2) replace 3β-adiol by asking your compounding pharmacy to include it in your patient’s testosterone cream.27

5α-Reductase Inhibitors 

5α-Reductase Agonists

DHEA as well as free testosterone upregulates 5AR activity in the prostate3435; levels of both are assessed on a 24-Hour Urine Hormone Profile.
Exercise, even a single session on a treadmill, significantly increases 5AR expression—at least in male and female mice. 36  

Indirect Assessment of 5α-Reductase Activity

A 24-Hour Urine test provides two ratios that assess 5AR activity: Andosterone:Etiocholanone and Allo-tetrahydro-cortisol:Tetrahydro-cortisol. These are discussed in Part II of this review.
Very low ratios of the above hormones in urine indicate low 5AR activity and are suggestive of very low 5α-DHT production. Follow-up testing with a serum Testosterone Metabolites Profile can confirm since serum testing, which allows assessment of two other important ratios: the ratio of Testosterone:5α-DHT and that of 5α-DHT:3β-adiol, is a better indicator of elevated, low or normal levels of DHT.

The serum Testosterone Metabolites Profile also allows assessment of two other important ratios: the ratio of Testosterone:5α-DHT and that of 5α-DHT:3β-adiol:

Testosterone:5α-DHT: In men, (average age 64 +/- 9 years), a higher testosterone to 5α-DHT ratio has been associated with a 42% decreased risk of BPH.37  A significant inverse association has also been shown between Testosterone:5α-DHT and lower urinary tract symptoms (LUTS). Additionally, men with higher concentrations of bioavailable testosterone had a 56% decreased risk of LUTS compared with those with hypogonadal concentrations.38  

As early as 1966, a study found an inverse relationship between tumor volume and the testosterone : DHT ratio in men screened for prostate cancer. Lower DHT values were seen in more advanced tumors. Testosterone levels were also lower in patients with cancer than in the control group. An inverse relationship was also found between tumor volume and 5α-DHT level; 5α-DHT levels tended to be lower among cases and in those with more advanced tumors, and the testosterone : 5α-DHT ratio was higher in patients with more advanced tumors.39  

More recently, a 2008 study found that plasma 5α-DHT levels were reduced when testosterone was given to elderly men with below normal levels of testosterone and elevated 5α-DHT levels at baseline. 40

Several possible mechanisms could explain why: (1) Since 5α-DHT is ~3x as potent as testosterone, 5α-reductase activity may be increased to compensate for the low testosterone levels pre-treatment. When testosterone is given, the need to compensate via increased production of 5α-DHT is removed; the androgen receptors are now also being activated by testosterone, resulting in signaling for reduced synthesis of 5α-reductase and/or reduced 5α-reductase activity.41  In sum, the research suggests that testosterone replacement promotes normalization of 5α-reductase activity, 5α-DHT levels and its metabolism to 3β-adiol.

5α-DHT : 3β-adiol : While 5α-DHT has been seen only as a potential promoter of proliferation and growth in the prostate, its metabolite 3β-adiol is a differentiating agent that activates ERβ and may help prevent cancer. A growing body of evidence suggests that the ratio of 5α-DHT to 3β-adiol may much more important than their absolute values.22,23,24,42 However, the ratio of 3β-adiol : (5a DHT + 3α-adiol) may be even more useful since 3α-adiol serves as a “reservoir” which re-cycles back to 5α-DHT.23

Urine Hormone Testing

Advantages

As discussed in Part II of this review, urine hormone testing using both gas chromatography-mass spectrometry (GS-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well-established in the medical literature as a reliable method of assessing levels of active (free & conjugated) hormones and their metabolites, correlates well with patient symptoms, and reflects the beneficial impact (or lack of benefit and potentially oncogenic effect) of therapeutic interventions.43, 444 Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA, also known as enzyme immunoassay [EIA]) analysis may not be accurate. The clinical takeaway: ask the lab you use what procedures they are using.45, 46, 47

A 24-hour urine sample is non-invasive, 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 metabolites that are not measurable in saliva and cannot be reliably measured by a single, or even multiple, blood draws, e.g., 16α-OH estrone, an estrogen metabolite shown to impact bone loss in men, and a number of glucocorticoid and mineralocorticoid metabolites (discussed in Part II of this review)  that 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.

Disadvantages

Circadian fluctuation of cortisol cannot be measured using 24-hour urine collection. To prevent sample contamination, patients applying transdermal cream to the scrotum or testicles should be instructed to apply to the perianal mucosal area on the day of collection. 

The 24-Hour Urine Test: Analyte Interpretation and Evaluation for Men

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.

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

24 Hour Urine Test analytes and adult reference ranges for men 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. Since it is a muscle breakdown product, creatinine is typically higher in men than women (especially if the patient exercised on the day of collection).

Total urine volume: ranges from 1,200 – 3,000 mL; most likely to be ≥1,500 in men.

SEX HORMONES: ESTROGENS

Estrone (E1): The most potent estrogen, estrone is derived from testosterone via prior aromatization to estradiol and estradiol’s conversion by 17β-hydroxysteroid dehydrogenase (17β-HSD) to estrone.  Because of its bone-building actions, small amounts of the estrone metabolite, 16α-OH estrone (which is produced by Phase I metabolism) are required by men. The adult male reference range is 3 – 11.4 μg/24 hr.

Estradiol (E2): Estradiol is produced 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-CH3O estradiol], Phase II). The adult male reference range is 0.8 – 4.6 μg/24 hr.

Estriol (E3): Derived from estrone through the intermediate (bone-building but potentially oncogenic) 16α-OH estrone, estriol is the weakest estrogen with 20-30% less affinity for estrogen receptors. Also estriol stimulates ERβ and has anti-proliferative effects in estrogen sensitive prostatic tissues. The adult male reference range is 3.5 – 13.7 μg/24 hr.

Elevated total estrogens in men may be due to excessive testosterone supplementation (≥75 mg/day) or excessive aromatase activity, which is nearly always associated with insulin resistance.

If excessive aromatase activity is indicated, check for insulin resistance. The most accurate and definitive test for insulin resistance is the procedure published by Kraft.48

In addition to diet and exercise, supplementation with vitamin D,49, 50;  chromium51,52,  (most likely to be effective in insulin-resistant vs. insulin-sensitive patients and when combined with biotin53,54); and especially the natural plant extract berberine  are very likely to be helpful.55, 56, 57, 58, 59, 60, 61, 62  

Supplementation with Myomin (a combination of Chinese botanicals, 2 to 3 capsules BID63  and Chrysin (liposomal drops, 6 drops sublingually BID64,65 will also lessen aromatase activity; however, retest with 24-Hour Urine after 3 months to ensure sufficient 16α-OH estrone is present for bone health; run Serum Testosterone Metabolites to check 3β-adiol levels for protection against prostate cancer.

2/16α ratio: The ratio of 2-hydroxyestrone (2-OH E1) to 16α-hydroxyestrone (16α-OH E1) is one indicator of a man’s risk of developing prostate cancer. 2-OH E1, a Phase I metabolite of estrone, has weak estrogenic activity and is associated with protection against 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 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.6667, 6869, 70, 7172  

The ideal 2/16α Ratio range is 2 to 4. A 2/16α Ratio of <2 indicates increased risk for prostate 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), flax, soy, omega-3 fatty acids and exercise. (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.)73, 74, 7576, 7778, 79

Levels of 16a-OH E1 can also be lowered by increasing its conversion to estriol. This conversion is stimulated by iodine and iodide.80

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 prostate, and colon cancers.81,  82

SEX HORMONES: PROGESTERONES

Progesterone: The major naturally occurring member of a class of structurally very similar hormones; in males, progesterone is produced by the brain.

Progesterone is an aromatase inhibitor,83 so may be useful for treating benign prostatic hyperplasia (BPH). Follow-up testing with the Serum Testosterone Metabolites assay is recommended to ensure over-inhibition of 5AR is not occurring.

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.8485 
In men, the Reference Range for pregnanediol is70-1050 μg/24hr.

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 help boost testosterone levels) or estrone, and thus 16α-OH estrone86

For this reason, DHEA helps build new bone tissue and has been shown to significantly improve bone mineral density in older adults.8788, 89  Low levels are associated with increased risk of fracture and osteoporosis.90 

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.9192  

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.93  

In men, as noted above, DHEA is a 5AR agonist, thus promoting conversion of testosterone to DHT, and has also been shown to exert anti-atherogenic effects; higher serum levels of DHEA-S correlate inversely with carotid artery intima-media thickness, plaque and blood flow volume.94  

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 (>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 5AR followed by 3α-hydroxysteroid dehydrogenase (3α-HSD), and therefore useful for monitoring 5AR. If androsterone is high in relation to etiocholanolone, 5AR activity may be elevated, resulting in increased conversion of testosterone to dihydrotestosterone (5α-DHT).

5α-DHT is ~3 times more powerful than testosterone and unlike testosterone, cannot be aromatized to estradiol. Thus, excess 5AR is associated with male pattern baldness. Excessive DHEA supplementation (>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 5AR activity may be low as well, resulting in insufficient production of 3β-adiol (thus increasing prostate cancer risk) and leaving more testosterone to be aromatized to estradiol.

The Reference Range for androsterone is 2000 to 5000μg/24 hr in men.

Testosterone: Primarily secreted by the testes in men, but also produced in the adrenals, liver, skin and brain, testosterone is important not only for its effects on the male libido95,96, but on lean body mass and muscle strength, cardiovascular health, cognitive function and bone mineral density.97  

The Reference Range for testosterone is 20 – 200μg/24 hr in men.

The range is wide because of the many different body types included in its calculation, which is based on 30+ years of 24 Hour Urine tests on thousands of men. Some men may produce less testosterone than other men and may have more active 5AR to compensate. For anti-aging, we generally recommend that, with treatment, men be between 80 - 150μg/24 hr. While some clinicians prefer to see testosterone levels in the upper end of the reference range to help with anti-aging, even though the patient could likely be symptom free with less HRT, we believe the key question is: “Is the testosterone dose sufficient to render the patient symptom-free?” In addition, hormones should always be evaluated comprehensively because of they constitute an interactive web.  We can often get more benefit with less because when they are all balanced, they “cooperate" with each other. In terms of testosterone, if the patient is in the upper end of the reference range, it is essential to also run the Serum Testosterone Metabolite profile to check DHT and its metabolites.

5α-Androstanediol: On the 24 Hour Urine test, the 5α-androstanediol analyte contains both the 3α-adiol (aka 5α-Androstane-3α-17β-diol) and 3β-adiol (aka 5α-Androstane-3β-17β-diol) subcomponents, and does not differentiate between these final downstream metabolites of 5α-DHT. The 3α-adiol and 3β-adiol metabolites are able to be further differentiated and are reported on the Serum Testosterone Metabolite profile. 5α-Androstanediol is a testosterone/5α-DHT metabolite produced via the activity of 5AR. 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: ~3 times more powerful than testosterone. Elevated 5AR activity may be linked to increased risk for male pattern baldness and benign prostatic hypertrophy. Over-inhibition of 5AR, however, has been linked to increased risk of aggressive prostate cancer. (As explained above, a Serum Testosterone Metabolite test will give you more insight into testosterone’s metabolization and potential over-inhibition.)

The Reference Range for 5α-Androstanediol in men is 22.0 – 131.0μg/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 a weak testosterone metabolite.

The Reference Range for 5β-Androstanediol in men is 40.0 – 401.0μg/24hr.

GLUCOCORTICOIDS

Pregnanetriol: A progesterone metabolite and indicator of sufficient substrate for the cortisol pathway. The Reference Range for pregnanetriol is quite wide (200 – 1,500 μg/24hr). Mid-range is desirable.

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.)98, 99   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 cortisol 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. In men, the three metabolites should add up to between 5,000 and 10,000, which corresponds to a cortisol output of 10-20 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 animals100, 101102103,  and levels of serum aldosterone have been inversely correlated with degree of hearing in humans.104  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.105 

(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 marker106106; urine hGH is a better indicator.108, {ref109, 110111112  

Growth hormone levels are increased by deep sleep,113  arginine (more effective in younger men, but useful in elders as well)114, 115, glutamine (helpful for older people, 2 grams at bedtime),116  and ornithine alpha-ketoglutarate (may boost the use of glutamine in arginine metabolism, 0.25 mg/kg)117,  resistance training, short intense bursts of exercise, and vigorous aerobic exercise118, 4119, (although the effect of exercise is more pronounced in younger subjects)120,   and adequate protein.121 

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).122, 123, 124125   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.126  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.127 

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 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.128, 129130131

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.132133134, 135   

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.136  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 5α-DHT. Upregulated 5α-DHT activity promotes BPH and male pattern baldness, and is associated with insulin resistance and obesity.

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

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

If excessive, 5AR can be down-regulated by zinc137, 138,   GLA,139 EPA,139 vitamin D3,140  saw palmetto141,  progesterone142, 143,   green tea extract144,  finasteride, and dutasteride.

Over-inhibition of 5AR, however, can promote aggressive prostate cancer.  In the Prostate Cancer Prevention Trial, a study involving more than 18,000 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.145  (See above “Mechanisms through which 5α-reductase inhibitors promote prostate cancer”) (Neal, please make this a link to this section above) 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 vice 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.146

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.

(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.147

CONCLUSION

Serum and 24-Hour Urine Hormone Testing—Sine Qua Non for Optimal Men’s BHRT

Despite the brevity of this introductory overview of a very complex subject—how to safely and effectively monitor BHRT in your aging male patients—the necessity of assessing hormone metabolites has, hopefully, been demonstrated.  By utilizing the latest serum Testosterone Metabolites tests in combination with the most comprehensive 24-Hour Urine tests, physicians can gain unprecedented insight into their male patients’ health, ensuring safe and effective BHRT. 

 Figure 2

24-Hour Urine Comprehensive Profile:  Analytes & Reference Ranges for Men

Analyte

Adult Reference Range*

Creatinine

0.5 – 2.0 gm/24hr

Total urine volume

1,200 – 3,000 mL

Steroid

Amount excreted in μg/24 hr

Sex Hormones

 

 

Estrone (E1)

3.0 – 11.4

Estradiol (E2)

0.8 -4.6

Estriol (E3)

3.5 – 13.7

Total Estrogens

7.3 -29.7

2-OH Estrone  (Phase I metabolite)

1.9 – 15.8

16α-OH Estrone (Phase I metabolite)

0.2 – 5.9

2/16α Ratio (Ideal = 2 - 4)

1.2 – 4.9

Pregnanediol (progesterone metabolite)

70 – 1,050

DHEA

100 – 2,000

Androsterone (DHEA metabolite)

2,000 – 5,000

Etiocholanolone (DHEA metabolite)

1,400 – 5,000

(Androsterone:etiocholanolone ratio, if low, suggestive of low 5AR activity, low 5α-DHT, low 3β-adiol)

Testosterone

20 - 200

5α-Androstanediol

22 - 131

5β-Androstanediol

40 - 401

Glucocorticoids

Pregnanetriol  ( want mid-range, indicator of substrate availability for cortisol pathway )

200 – 1,500

Cortisone (optimal range ~120-130μg/24hr)

31 - 209

Cortisol  (optimal range ~90μg/24hr)

30 - 170

Cortisone: Cortisol Ratio (always interconverting)

Ideal = 0.7

Tetrahydrocortisone (THE, cortisone metabolite)

2,100 – 7,400

Allo-tetrahydrocortisol (5α-THF, cortisol metabolite)

700 – 3,800

Tetrahydrocortisol (THF, cortisol  metabolite)

1,200 – 4,500

(THE + 5α-THF + THF) x2 = daily cortisol output

8,000 – 10,000

11β-OH Androsterone (terminal metabolite of cortisol; low value confirms  insufficiency)

613 – 2,298

11β-OH Etiocholanolone (terminal metabolite of cortisol, low value confirms insufficiency)

153 - 950

Mineralocorticoids

Aldosterone(indicator of salt level in diet, low may indicate adrenal fatigue)

Normal Diet = 6.0 – 25.0

Low Salt: 17.0 – 44.0

High Salt: 0.0 – 6.0

Allo-tetrahydrocorticosterone (5α-THB, low = long term adrenal insufficiency, high = acute stress at collection)

130 – 1600

Tetrahydrocorticosterone (THB, low = long term adrenal insufficiency, high = acute stress at collection)

30 -240

11-dehydrotetrahydrocorticosterone (THA, low = long term adrenal insufficiency, high = acute stress at collection)

76 - 456

Human Growth Hormone

1,065 – 4,722

Thyroid Hormones

Free T3

470 – 1,750

Free T4

430 -3,200

T3 + T4 (if <1000 adrenal support may be indicated)

~1000

Urinary Sodium and Potassium

Sodium

40 - 220

Potassium

25 – 150

Sodium:Potassium Ratio (ideal 1.5, if low consider malabsorption)

1.2 – 4.8

Enzyme Activity

5α-Reductase enzyme (converts testosterone to more potent 5α-DHT) activity as shown by:

Androsterone/Etiocholanolone Ratio

Ideal = Mid-range 0.7

Allo-tetrahydrocortisol /Tetrahydrocortisol Ratio

Ideal = Mid-range 0.6

Elevated 5AR activity associated BPH, premature baldness, obesity, insulin resistance in men

Low 5AR activity suggestive of reduced conversion of testosterone to DHT, undervirilization, low 3β-adiol production, increased prostate cancer risk

 

11β-Hydroxysteriod dehydrogenase I and II (isoforms of enzyme that converts cortisol to cortisone) activity shown by:

Cortisol: Cortisone Ratio

Ideal 0.7

Tetrahydrocortisol + allo-tetrahydrocortisol: Tetrahyrdocortisone Ratio

Ideal 0.9

Low ratios associated w/obesity & insulin resistance. Elevated ratios w/low-renin hypertension, high dose licorice, exogenous cortisol.

*Reference ranges vary by lab and can may  be expressed in different units of measure

 

 

 


 

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