Urine Steroid Hormones Accession Number:

105-32 Royal Vista Dr NW Calgary, AB T3R 0H9 www.rmalab.com Phone: 403-241-4513 Fax: 403-241-4516 Date of Collection: 23-Oct-2013 Sample Received: 24-Oct-2013 Reported Date: 28-Nov-2013 Sample Type: Urine Urine Steroid Hormones Accession Number: 333333 Healthcare Professional: Patient: Gender : M Jane Doe Date of Birth : 01-Nov-1967 Age: 45 Phone Fax Hormone Therapies Last Used Biometrics Height (in) : 72 Weight (lb) : 210 Waist (in) : 40 Hip (in) : 41 N/A: not available All units µg/24hrs Cortisol/ Cortisol Metabolites Analyte Result 0% 20% 40% 60% 80% 100% %ile Cortisol 47 40-98 24% Male Tetrahydrocortisol (THF) 1,300 480-1,200 89% Male allo-tetrahydrocortisol (athf) 780 200-620 93% Male Cortisone 110 62-150 55% Male Tetrahydrocortisone (THE) 3,500 1,400-3,200 90% Male 17 Hydroxyprogesterone Metabolites Analyte Result 0% 20% 40% 60% 80% 100% %ile Tetrahydrodeoxycortisol (THS) 35 33-98 18% Male Pregnanetriol (P3) 2,000 560-1,400 95% Male Total 17-Hydroxysteroids = athf + P3 + THE + THF + THS Analyte Result 0% 20% 40% 60% 80% 100% %ile Total 17-hydroxysteroids (Total 17HS) 7,700 3,000-6,100 0.95 95% Male a division of LifeLabs LP Page 1 of 9 CPSA Accreditation # L0154200

Urine Steroid Hormones Accession Number: 395634 Mineralocorticoids Analyte Result 0% 20% 40% 60% 80% 100% %ile Aldosterone N/A 1.5-4.4 100% Male THA 210 32-110 99% Male allo-tetrahydrocorticosterone (athb) 250 50-190 93% Male Progesterone/ Progesterone Metabolites Analyte Result 0% 20% 40% 60% 80% 100% %ile Progesterone (Pg) 0.30 0.23-0.50 35% Male Pregnanediol (P2) 960 150-560 95% Male Cortisol Metabolite Ratios Analyte Result 0% 20% 40% 60% 80% 100% %ile athf / THF 0.59 0.29-0.75 68% Male (athf + THF) / THE 0.60 0.39-0.77 64% Male 17 Ketosteroids Analyte Result 0% 20% 40% 60% 80% 100% %ile Dehydroepiandrosterone (DHEA) 100 30-1,000 26% Male Androstenedione (ANDN) 3.0 1.9-6.7 37% Male Testosterone 33 19-69 38% Male Dihydrotestosterone (DHT) 6.6 2.8-8.5 71% Male 3-alpha androstanediol 57 19-61 81% Male Etiocholanolone (ECHL) 2,600 1,200-3,200 71% Male 11-ketoetiocholanolone (11KE) 250 75-340 68% Male 11-hydroxyetiocholanolone (11HE) 570 100-500 89% Male Androsterone (ANDS) 3,500 1,400-3,200 88% Male 11-ketoandrosterone (11KA) 34 17-53 54% Male 11-hydroxyandrosterone (11HA) 650 230-660 83% Male Page 2 of 9

Urine Steroid Hormones Accession Number: 395634 Total 17-Ketosteroids = DHEA + ECHL + ANDS + 11HA + 11HE + 11KA + 11KE Analyte Result 0% 20% 40% 60% 80% 100% %ile Total 17-ketosteroids (Total 17KS) 7,700 3,400-8,700 78% Male Anabolic Catabolic Ratio Analyte Result 0% 20% 40% 60% 80% 100% %ile Anabolic 17KS / Catabolic 17HS 1.0 0.90-1.7 23% Male Androgen Ratios Analyte Result 0% 20% 40% 60% 80% 100% %ile DHT / Testosterone 0.20 0.083-0.25 76% Male Androsterone / Etiocholanolone 1.4 0.75-1.5 80% Male Estrogens/ Estrogen Ratios Analyte Result 0% 20% 40% 60% 80% 100% %ile Estrone (E1) 3.7 2.4-6.7 43% Male Estradiol (E2) 5.1 0.10-4.4 86% Male Estriol (E3) 3.7 1.7-11 38% Male EQ = E3 / (E1 + E2) 0.42 0.24-2.0 23% Male 2-hydroxyestrone (2OHE1) 2.0 1.0-5.0 39% Male 4-hydroxyestrone (4OHE1) < 0.60 0.10-0.73 < 78% Male 16-hydroxyestrone 7.6 0.65-5.2 94% Male 2-methoxyestrone (2MeOE1) 1.9 0.70-2.3 75% Male 4-methoxyestrone (4MeOE1) < 0.45 0.080-0.51 < 80% Male Sum of Estrogens 24 12-35 62% Male 2MeOE1 / 2OHE1 0.95 0.25-1.1 84% Male Page 3 of 9

Steroid Hormone Pathways Page 4 of 9

WHAT KIND OF HORMONES DOES THIS TEST MEASURE? Steroid hormones are excreted in urine as a mixture of non protein-bound unconjugated hormone plus non protein-bound conjugates. Conjugates are steroids which have had sulphate and/or glucuronide functional groups attached to them to increase water solubility. They may originate in the liver, or may be formed in target tissues such as breast and uterus. Prior to analysis, the urine specimen undergoes a hydrolysis step, to cleave the conjugates. So for example, if the reported value for estrone E1 is 3.7 ug/24 hours that does not mean there were 3.7 micrograms of estrone in the original specimen; we started with more than 3.7 ug of estrone conjugates which after hydrolysis, left us with 3.7 micrograms of estrone. This is an important but subtle distinction. Urinary estrone as measured here, is not directly comparable to serum or salivary estrone. Both those tests measure unconjugated estrone, which may be arising from a different source than conjugates. If the urine level of any particular hormone is markedly low or high, this usually represents markedly decreased or increased production of that hormone; however, if the patient has a congenital inability to form sulphate or glucuronide conjugates, due to a faulty gene SNP for the enzymes in question, or lacks requisite sulphate and/or glucuronide stores to allow conjugation, then hormone levels may be erroneously interpreted as low. This will depend on the relative proportions of non-conjugated and conjugated hormones that are excreted in any given case. Most of the time, steroids must be heavily conjugated to give them enough solubility to allow urinary excretion. 11-beta HYDROXYETIOCHOLANOLONE (11HE) AND 11-KETOETIOCHOLANOLONE (11KE) These two steroids (also known as OHET AND OHAN) can be derived from cortisol as well as from etiocholanolone. If they are considered to be cortisol metabolites, they contribute to the "Catabolic" mass balance;if they are considered to be etiocholanolone metabolites, they contribute to the "Anabolic" mass balance, since etiocholanolone arises from androstenedione. Practice amongst various laboratories is divided in this regard. We have elected to include these steroids in the total for "Anabolic" hormones although they most likely arise from cortisol in the majority of cases. Steroids 2008;73:652-656. 17-Hydroxylase/C17,20-lyase (CYP17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites. Shackleton CH et al. RATIOS WHICH REFLECT 5-alpha/beta REDUCTASE BALANCE We report two ratios which are of the general form: 5-alpha reduced metabolite / 5-beta reduced metabolite. These are: allothf/thf and Androsterone/Etiocholanolone (or A/E), with allothf and THF being the 5-alpha and 5-beta cortisol metabolites, and Androsterone and Etiocholanolone being the 5-alpha and 5-beta Androstenedione metabolites. In our database, for postmenopausal women and for cycling women in the luteal phase, these two ratios correlate fairly well: they both tend to move in the same direction. A ratio near or above unity means that the equilbrium lies toward 5-alpha products. A ratio closer to zero means that 5-beta reduced products are favoured. Since the 5-alpha reduced metabolites are more androgenic, a ratio near or above unity is usually also accompanied by a clinical picture of androgenicity (excess facial hair growth, male pattern scalp hair loss, acne, irritability, oily skin). GENERAL COMMENTS ON THE RATIO: (athf + THF) / THE The ratio (athf + THF) / THE purports to look at the activity of the 11-betahydroxysteroid dehydrogenase (11bHSD) isoenzymes, which interconvert cortisol, cortisone and their metabolites. 11bHSD Type I : cortisone/cortisone metabolites ----> cortisol/cortisol metabolites 11bHSD Type II : cortisol/cortisol metabolites ----> cortisone/cortisone metabolites The ratio mentioned above has received attention recently, as manipulation of the balance of the enzymes in question is being studied as a target to achieve weight loss. The value of the ratio appears to be somewhat method-dependent, but nevertheless, relative changes in the ratio over time in the same patient are still valuable, even if the absolute value of the ratio varies from method to method. A ratio that is getting smaller would indicate a shift toward a greater proportion of cortisone/cortisone metabolites, Page 5 of 9

relative to cortisol/cortisol metabolites. This would be a desirable shift in a patient with clinical signs and symptoms of elevated cortisol. A ratio that is getting larger would indicate a shift toward a greater proportion of cortisol/cortisol metabolites relative to cortisone/cortisone metabolites. This would be desirable in a patient with clinical signs and symptoms of low cortisol. An example of a situation where the ratio should increase would be a patient supplementing with licorice to boost cortisol. HOW CAN A RESULT BE IN THE 100th PERCENTILE? Reference ranges are derived by fitting a statistical model to a relatively small set of results from clinically normal individuals. Depending on the model selected, results which exceed the highest value found in the normal sample may register at the 100th percentile. SELENIUM MORE OR LESS WITHIN MIDDLE TERTILE The 24 hour urinary excretion of selenium was also measured on this specimen, although the report has not yet been modified to display the result graphically. The excretion was 86 ug/24 hours, which is more or less within the middle third of the reference population. Selenium is important to health as it is an important cofactor for conversion of the thyroid hormone T4, to the most active form, T3. Approximately half of ingested selenium appears in urine, so urine reflects selenium intake to a certain extent. For example, selenium measured from a 24 hour urine collection has been shown to reflect dietary intake as well as supplementation. (In the Japanese INTERMAP study, 24 hour urinary excretion of selenium correlated to selenium intake (Yoneyama et al. Eur J Clin Nutr. 2008 Oct;62(10):1187-93.) 24 hour urinary excretion of selenium is also reflective of supplemental selenium (Burk R et al. Cancer Epidemiol Biomarkers Prev 2006;15:804-810). For optimum activity of thyroid hormones in target tissues, it is probably desirable to maintain selenium in the middle tertile, as is the case here. Foods rich in selenium include Brazil nuts (markedly higher than all other foods), all types of fish, liver, oysters, red meat, chicken and pork, as well as garlic and onions. Wheat products and rice also contain some selenium. BMI MAY BE HIGHER THAN OPTIMAL This patients Body Mass Index (BMI) is 28. Patients with a BMI between 25 and 30 are defined as overweight and obesity is defined as BMI greater than 30. Elevated BMI is often associated with elevated estradiol, testosterone and DHEAS, and may also be associated with high urinary cortisol and aldosterone. Metabolic syndrome/insulin resistance are also associated with elevated BMI. Health risks associated with excess weight include increased risk of diabetes, high blood pressure, heart attack, stroke and cancer. Weight reduction and reduced consumption of refined carbohydrates may be worthwhile interventions for some patients with elevated BMI. Note that not all patients with high BMI have metabolic abnormalities. For example, a well-muscled individual may have a BMI in excess of 30, yet be metabolically normal. Note also that not all patients with high androgens have elevated BMI. TETRAHYDRODEOXYCORTISOL (THS) LOW Urinary excretion of THS has been shown to be linked to genetic variation in the CYP11B2 (aldosterone synthase) gene. The clinical significance of this mutation has not been determined. Excretion of the urinary metabolite of deoxycortisol is heritable and influenced by polymorphic variation at the CYP11B2 (aldosterone synthase) locus. Myosi B, Keavney B, Watkins H, et al. Presented at 22nd Joint Meeting of the British Endocrine Societies, Glasgow, UK 24-26 March 2003. Endocrine Abstracts (2003) 5 P223 TOTAL 17-HYDROXYSTEROIDS ELEVATED: WAIST CIRCUMFERENCE The result for total 17-hydroxysteroids is equal to or above the 75th percentile. Collectively, the steroids summed here make up more than half of the metabolites of cortisol; this total therefore gives a good measure of cortisol production and breakdown. Elevated 17-hydroxysteroids may be seen in a wide range of conditions of cortisol overproduction, including Cushing's Disease, adrenal hyperplasias, adrenal adenomas, acute stress, acute illness, depression, hyperthyroidism and obesity. In our database, there is a modest positive correlation between total 17-hydroxysteroids and waist circumference, which in turn is reflective of visceral obesity. Here the waist circumference is 40 inches. In women, a waist Page 6 of 9

circumference around or above 38 inches, or in men 40 inches, may be associated with visceral obesity. Body Mass Index or BMI is 28. BMI equal to or above 27 is also more likely to be associated with increased visceral obesity, but not as strongly as increased waist circumference. Note that visceral obesity is usually associated with Metabolic Syndrome and/or hyperinsulinemia and insulin resistance. This patient should be assessed for the presence of these conditions, and treated accordingly. NO RESULT FOR ALDOSTERONE Aldosterone is not being reported at this time. We apologize for any inconvenience this may cause. The rectangle which would normally correspond to the percentile for aldosterone is positioned at zero for display purposes. The percentile is listed as "<0.001". allo-thb AND/OR allo-thf RANK ABOVE 84th PERCENTILE Both allo-thb and allo-thf are the product of a 5-alpha reductase enzyme. Either or both of the results for these analytes rank above the 84th percentile. These findings point to increased activity of 5-alpha reductase, and this is often accompanied by complaints of excess facial hair growth and/or acne, in some patients, although this is not always the case. PREGNANEDIOL RANKS AT OR ABOVE THE 75%TH PERCENTILE Pregnanediol is elevated. This could be due to supplementation with pregnenolone ( particularly oral pregnenolone) or progesterone. It may also be due to Clomid and/or HCG. Exposure to progesterone may be gained by intercourse with a person who is supplementing with vaginal or labial progesterone. Some older males supplement with progesterone as it is thought to be beneficial for the prostate by some authorities. Excess progesterone can lead to depression and "brain fog". In males, progesterone arises from the adrenal glands. Excessive progesterone may reflect decreased production of downstream metabolites of progesterone. athf / THF ABOVE THE MEDIAN VALUE FOR MALE The meaning of this ratio was explained in the preliminary discussion at the beginning of the report. In our laboratory, for males, the median ratio of allothf (5-alpha reductase cortisol metabolite) to THF (5-beta reductase cortisol metabolite) is 0.47 This patients ratio is 0.59, which is above the median value. This patient might also be expected to have symptoms of excessive conversion ot testosterone to dihydrotestosterone if the 5-alpha reductase system regulating testosterone metabolism is biased in the same way as the cortisol metabolites are biased. An elevation in the ratio athf/thf has been associated with hypothyroidism. (J Endocrinol Invest. 1998 Apr;21(4):219-25. Urinary cortisol metabolites in the assessment of peripheral thyroid hormone action: overt and subclinical hypothyroidism. Vantyghem MC, Ghulam A, Hober C, Schoonberg C, D'Herbomez M, Racadot A, Boersma A, Lefebvre J. (athf + THF/THE) This ratio purports to look at the activity of the 11-betahydroxysteroid dehydrogenase (11bHSD) isoenzymes. 11bHSD Type I favours cortisol over cortisone, whereas 11bHSD Type II favours cortisone over cortisol. Here the ratio is 0.60. In our laboratory, the median ratio, for males is 0.53. In general, a ratio less than 1 may indicate that cortisone/cortisone metabolites are favoured over cortisol/cortisol metabolites. Ratios approaching or greater than 1 may indicate that cortisol metabolites are favoured over cortisone metabolites. This is more likely to occur when there is increased inflammation/visceral fat accumulation. DHEA/S RANKS IN LOWEST TERTILE This result actually reflects the level of DHEAS since most of the DHEA in urine is actually present as the sulphate. The DHEA/S result ranks at or below the 33rd percentile and this patient is not supplementing with DHEA, 7-keto DHEA or pregnenolone. This finding may imply reduced production of DHEA/S from the adrenal glands. Note that the oldest patients in the database from which the reference ranges were derived were 69-70 years of age. DHEA/S tends to decline with age, hence patients older than 70 years may very well have a "low" reading when referenced to this database. Low or low normal levels of DHEA/S in men may be associated with chronic stress, chronic illness (e.g. rheumatoid arthritis) and hypothyroidism are also associated with low DHEA/S. Symptoms associated with low DHEA/S may include decreased libido, feeling "burned out", dry skin, thinning skin, thinning pubic hair and bone loss. Low DHEA/S may be a sign of adrenal fatigue/exhaustion, especially if it is coupled with low cortisol. Older Page 7 of 9

men with low DHEAS may also have low testosterone, although this is not seen in every case. INTERPLAY BETWEEN ANDROGENS AND GLUCOCORTICOIDS Androgens and cortisol balance each other in terms of signaling at nuclear receptors, hence a normal or high normal androgen "input" could be swamped by excessive cortisol "input", leading to symptoms of low androgens in the face of seemingly adequate androgen levels. TOTAL 17-KETOSTEROIDS ABOVE 75th PERCENTILE Total 17-ketosteroids are elevated into the top 25%. This can often be due to supplementation with androgens such as testosterone or androgen precursors such as DHEA or pregnenolone. None of these were listed on the requisition. Elevated Growth Homone such as might be expected in bodybuilders, may also elevate androgens. In males elevated androgens might manifest with symptoms of androgen excess including acne, oily skin, irritability and aggressiveness; however, the actual clinicl picture will also depend on the level of estrogens. High estrogens may oppose the action of androgens at the receptor level in target tissues such as the penis and prostate. RATIO 17HS / 17KS FALLS IN LOWEST TERTILE When the ratio of 17-hyroxysteroids to 17-ketosteroids is low (less than 1 and approaching 0) this indicates disproportionate cortisol output relative to androgen output. In turn, this is a more catabolic state, usually associated with aging, obesity and other low growth hormone states. TOTAL OF ESTROGENS WHEN ONE OR MORE ESTROGENS ARE BELOW DETECTION LIMIT Here, one or more of the estrogen metabolites is below detection limit. The total estrogens or "Sum of Estrogens" reported here only includes the results that are above detection limit. This rarely, if ever, makes a difference to the interpretation of the findings. IMPACT OF HIGH ESTRADIOL The patient is complaining of problems with erections. Although total androgens are elevated, estradiol is also high. Estradiol can interfere with the action of testosterone at the level of the nuclear receptors, blunting the testosterone "signal". Aromatase inhibitors are sometimes useful in this situation, to reduce the level of estradiol formed from aromatization of testosterone. ESTRADIOL ELEVATED Estradiol is at or above the 75th percentile. Since estradiol is the most potent estrogen, in theory a significantly elevated level might pose potential concern about risk of prostate cancer IF the urine conjugated estradiol level reflects the unconjugated estradiol level in prostatic tissue.the patient should be assessed for clinical signs of increased estrogens such as breast enlargement and penis shrinkage. The patient should also be questioned about the possibility of skin-to-skin transfer of estradiol from another individual using topical estrogen replacement, particularly if estrogens are being used in the vagina or on the labia. SIGNIFICANCE OF ESTROGEN QUOTIENT IN MALES In general, the clinical significance of the Estrogen Quotient E3 / (E1 + E2) in males is not known. 4-HYDROXYESTRONE BELOW DETECTION LIMIT 4-hydroxyestrone is a metabolite of estrone that isassociated with increased risk of breast cancer when its level is elevated in women. This metabolite is thought to increase the risk of cancer by covalently bonding with (also referred to as adducting) purine bases on oncogenetic DNA, ultimately pulling them out of the DNA sequence amd causing damage. Very limited, preliminary studies indicate an elevated level of adducts in the urine of men with prostate cancer, but more research is needed. (Prostate. 2006;66:1565-1571. Potential biomarker for early risk assessment of prostate cancer.markushin Y et al.) Here, the 4-hydroxyestrone level is below the detection limit of 0.43 ug/24 hours, which is quite low. In a sample of 100 clinically normal males, approximately 64% of them will have a 4-hydroxyestrone level equal or less than 0.43 ug/24 hours. The marker for this analyte is displayed at zero for display purposes. ELEVATED 16-HYDROXYESTRONE (RANK > 75th PERCENTILE) Formation of 16-hydroxyestrone is catalyzed primarily by the cytochrome enzyme CYP1A2. Inducers of this enzyme include soluble fibre, flaxseed (Nutr Cancer. 2010;62:175-180. Effect of flaxseed consumption on urinary levels of estrogen metabolites in postmenopausal women. Sturgeon SR et al.) and caffeine. Elevated 16-hydroxyestrone has also been noted in SLE and Rheumatoid Arthritis (Cutolo M. J Rheumatology 2004, Editorial: 419-421.) Presumably other high IL-6, high NF-kappaB states might have the same effect, although this has not been well-studied. Page 8 of 9

Very markedly elevated levels of 16-hydroxyestrone (above the 95th percentile) might also be due to testosterone supplementation with excess conversion of testosterone to estrogen metabolites. 4-METHOXYESTRONE BELOW DETECTION LIMIT This metabolite is rarely found in appreciable amounts in women and especially not in men. In a sample of 100 clinically normal men, approximately 80% of them will have a 4-methoxyestrone level below the detection limit of 0.45 ug/24 hours. In other words, a finding of less than detection limit is seen in the majority of cases, for men. The marker for this analyte is positioned at zero for display purposes. WHY ISN'T THE RATIO 2-HYDROXYESTROGENS/16-HYDROXYESTRONE REPORTED HERE? The body of literature on catechol estrogens (measured via ELISA) and the risk of prostate cancer is quite small in comparison to the literature on catechol estrogens and breast cancer risk. The association between breast cancer and the 2-16 ratio is at best weak ((Int J Womens Health 2011;3:37-51. Estrogen metabolite ratio: Is the 2-hydroxyestrone to 16-hydroxyestrone ratio predictive for breast cancer? Obi N et al.) despite a fair number of studies. We feel there is simply not enough data to warrant reporting the 2-16 ratio in males as a measure of risk of prostate cancer, as of April 2012. The original research on the 2-16 ratio was performed with the Estramet ELISA kit which was not able to distinguish between various 2-hydroxylated estrogens. The ratio 2-hydroxyestrone/16-hydroxyestrone measured by mass spectrometric techniques is not the same as the 2-hydroxyestrogen/16-hydroxyestrone ratio measured by ELISA. Any prostate cancer risk threshold arrived at with ELISA cannot be "translated" into a risk threshold arrived at via mass spectrometry. Prospective studies would have to be undertaken, to determine what relationship, if any, exists between prostate cancer and 2- and 16-oxidized estrones measured by GC-MS or LC-MS. For information purposes, a 2009 citation on the Estramet 2-16 ratio and prostate cancer is: J Exp Clin Cancer Res 2009;28:135. Urinary estrogen metabolites and prostate cancer: a case-control study and meta-analysis. Barba M etal. COMMENT ON RATIO 2-METHOXYESTRONE/2-HYDROXYESTRONE Some 2-hydroxyestrone is cleared by conversion to the methoxylated product, 2-methoxyestrone. Examination of the ratio of the methylation product to its parent may be helpful in some cases. Here, the ratio 2-methoxyestrone/2-hydroxyestrone is 0.95. A lower ratio (closer to zero) may indicate a need for support of methylation pathways, since there is relatively less of the methylated product, relative to the precursor. A proportion of these patients may benefit from supplementation with any or all of betaine, methylated tetrahydrofolate, folate, B-12, SAMe, magnesium, B6 and MSM. A ratio close to or above unity indicates a relative excess of the methylated product, relative to the precursor. The ratio is also a function of the activity of the relevant methyltransferase enzyme, COMT. Polymorphisms which result in a loss of COMT function may therefore result in a lower ratio. Supplementation may or may not be helpful in these cases. Catecholamines are also a substrate for COMT. Excessive catecholamines may compete with estrogens for COMT catalytic "space". Measures to normalize catecholamine output may free up COMT to form more methoxylated estrogen metabolites. Note that supplementation with estrogens may increase the demand for methylation cofactors, and in time, deplete them, leading to decreased "yield" of methoxyestrones relative to the parent hydroxyestrones. George Gillson, MD PhD Medical Director Note: The College of Physicians and Surgeons of Alberta considers some laboratory tests to be non-standard, or a form of complementary and alternative medicine.. These interpretation comments have not been evaluated or approved by any regulatory body. Commentary is provided to clinicians for educational purposes and should not be interpreted as diagnostic or treatment recommendations. Page 9 of 9