Do you suspect hormone issues are negatively impacting your patient’s health?

Rocky Mountain Analytical® is now offering Comprehensive Hormone Insights™ (CHI). This urine test provides important information regarding hormone production and metabolism, to provide insights into your patients’ overall hormone balance.

Find out why your colleagues are using CHI

Comprehensive Hormone Insights CHI Provides Insight Into Patient Symptomatology

Comprehensive Hormone Insights provides detailed urine analysis that measures hormones, steroid hormone metabolites and assesses diurnal cortisol levels.

Urine steroid hormone testing can provide important information that may be relevant for many health conditions including:

Women Men • Breast cancer • Metabolic syndrome (MetS) • Endometrial cancer • Benign prostatic hyperplasia (BPH) • Metabolic syndrome (MetS) • Prostate cancer • Polycystic ovary syndrome (PCOS) • Gynecomastia • Endometriosis • Erectile dysfunction (ED) • Osteoporosis/bone loss • Osteoporosis, bone loss • Infertility • Fatigue • Premenstrual syndrome (PMS) • Sleep difficulties • Premenstrual dysphoric disorder (PMDD) • Inflammatory disorders • Fatigue • Sleep difficulties • Inflammatory disorders

Order CHI to receive a comprehensive report to help understand health conditions related to steroid hormone regulation. CHI Provides a Detailed Assessment of Your Patients’ Hormones The following hormones and hormone metabolites are included in CHI Hormone Categories Clinical Utility • Glucose and insulin metabolism Cortisol and cortisone metabolites such as: • Fat deposition and adiposity • 5α-tetrahydrocortisol (5α-THF) • Insight into burnout, chronic illness, acute stress, and • 5β-tetrahydrocortisol (5β-THF) balance between anabolic/catabolic processes • 5β-tetrahydrocortisone (5β-THE) • Insight into thyroid issues

Conjugates such as: • Provide insight into effects of ‘stored’ hormone and liver Cortisol sulfate, cortisol glucuronide, function related to hormone metabolism. glucuronide, sulfate, DHEA sulfate, and DHEA • May provide insight into inflammation glucuronide

Androgens and 17-ketosteroids such as: • Infertility • Testosterone • Polycystic ovary syndrome (PCOS) • (DHT) • Metabolic syndrome (MetS) • Androsterone • Benign prostatic hyperplasia (BPH) • Etiocholanolone • Thyroid issues • (DHEA) Progesterone metabolites: • α-pregnanediol • Mood disorders • β-pregnanediol • Thyroid issues • Allopregnanolone • PCOS • Breast tenderness Precursor steroid: • Uterine fibroids • 17OH-pregnenolone • Menstrual disorders and metabolites such as: • Risk of estrogen-sensitive cancers • Estrone (E1); (E2); (E3) • Prostate cancer risk • (E1S) • Breast enlargement or tenderness • Methylated and hydroxylated • Uterine fibroids • Insomnia Melatonin metabolite: • Premenstrual dysphoric disorder (PMDD) • 6-sulfatoxymelatonin • Breast cancer risk 8-hydroxy-2-deoxyguanosine (8OH2dG) • An important biomarker of oxidative stress

NOTE: This is a summary, rather than a detailed list of analytes reported in CHI.

CHI measures a wide range of metabolites to provide a more complete picture of the hormonal health of your patient. How Does CHI Stack up Against Other Urine Steroid Hormone Tests?

Superior reporting of results • Heat map • Immediate visual identification of which hormone pathways are hot (i.e., overactive) or cold (i.e., underactive) • Allows clinicians to identify hormonal issues at-a-glance

• Custom interpretation of patient results • CHI includes a detailed, patient-specific commentary to guide the interpretation of patient hormone levels and help identify possible causes of symptoms

• Percentiles reported, in addition to individual results • Percentiles show how patients’ results compare to a clinically-well population

• More in-depth analysis of results via ratios

Liquid urine samples • Collection of liquid urine can be done discreetly • Peer-reviewed literature supports liquid urine steroid hormone analysis

4-point pooled urine samples • 4-point samples compared to a 24-hour collection • Cortisol diurnal variation was previously only measurable in saliva and serum. The daytime urine samples show the same pattern as saliva, and also provide information on cortisol metabolism • 4-point samples eliminate the need for a cumbersome 24-hour urine collection • Results from the pooled sample derived from the 4-point samples can be equated to results from a 24-hour collection How to Read a CHI Report

SUMMARY DIALS LEGEND: How to read the graphs Sex Steroid Hormones 50 66 50 66 Middle third of 33 33 84 reference population Hormone Start of 100 100 16 84 16 24 The fi rst page of the report summarizes highest Percentile Precursors Percentile third of Sum of Sum of Estrogens 50 66 reference 0 0 key hormone measures including: population (T, DHT, αβ androstanediol) List of estrogens can be found 33 84 in Estrogens section of report End of 100 androgens, estrogens, progesterone 16 lowest Percentile00 50 66 50 66 third of 33 33 84 metabolites, DHEA & metabolites, reference 0 population Patient’s percentile rank 55 100 2 100 8OH2dG, 6-sulfatoxymelatonin and the compared to reference 16 Percentile 16 Percentile population (see summary) DHEA + Metabolites Sum of Progesterone Metabolites ratio of cortisol to testosterone. 0 (DHEA + A + E) 0 α+β Pregnanediol

CHI Accession: 648479

HORMONE CATEGORY ESTROGENS & METABOLITES Hormone HeatMap Each hormone category has its own HMUS07

Estrogen Quotient (EQ) heatmap and corresponding bar graphs 4-OHE1 E3 EQ = 2-OHE1 E1 + E2 for individual analytes. 2-MeOE1 Methylation Low % High Low % High 2-OHE1 2-MeOE1 Methylation E1 2-OHE1 2-OHE1 4-MeOE1 Low % High Low % High

Methylation HEATMAP DHEAS E1-Sulfate 2-Hydroxylation 4-OHE1 Reactive Intermediate 4-Hydroxylation

3βHSD Aromatase 16-Hydroxylation DHEA Glucuronide DHEA E1 16α-OHE1 DNA Damage Hormone endcap colour immediately cyp19 identifi es which hormone levels are 17βHSD Type 3 17βHSD Type 3 17βHSD Type 1,2 17βHSD Type 1,2 3βHSD Aromatase 16-Hydroxylation Testosterone E2 E3 high, high normal, normal, lowCHI Dials normal cyp19 CHI HeatMap 2-Hydroxylation 4-Hydroxylation and low. Middle third of Not Reported Testosterone Glucuronide 2-OHE2 4-OHE2 reference population Start of Methylation Methylation highest

ESTROGENS

Range RESULTS & RANGES Analyte Result Range Units 0% 16% 33% 66% 84% 100% %ile Applied Estrone (E1) 6.7 5.3 - 15 ng/mL 0.31 31% d21

This section reports the results, ranges Estradiol (E2) 2.4 2.3 - 5.5 ng/mL 0.25 25% d21 and graphs for the percentiles of Estriol (E3) 7.2 6.7 - 39 ng/mL 0.24 24% d21 individual analytes and sums/ratios. E3/(E1 + E2) 0.79 0.65 - 2.7 0.26 26% d21 Range applied is specifi c to patient: 2-Hydroxyestrone (2-OHE1) 6.0 3.0 - 12 ng/mL 0.41 41% d21 male, female and whether luteal, 2-OHE1/E1 0.89 0.38 - 0.99 ng/mL 0.66 66% d21 follicular or post-menopausal. 2-Methoxyestrone (2-MeOE1) 1.5 0.79 - 4.5 ng/mL 0.39 39% d21 2-MeOE1/2-OHE1 0.25 0.21 - 0.55 0.29 29% d21

16a-Hydroxyestrone (16a-OHE1) 2.7 2.3 - 10 ng/mL 0.25 25% d21

2-OHE1/16a-OHE1 2.2 0.43 - 2.6 0.69 69% d21

4-Hydroxyestrone (4-OHE1) 0.98 0.53 - 2.1 ng/mL 0.41 41% d21

4-OHE1/2-OHE1 0.16 0.13 - 0.26 0.40 40% d21

2-Hydroxyestradiol (2-OHE2) 0.94 0.42 - 2.8 ng/mL 0.48 48% d21

2-Methoxyestradiol (2-MeOE2) < 0.30 0.085 - 4.1 ng/mL 0.00

Estrone sulphate (E1-Sulphate or E1S) 0.95 0.45 - 2.0 ng/mL 0.55 55% d21

Sum of Estrogens 28 27 - 87 ng/mL 0.24 24% d21

INTERPRETATION WHEN MULTIPLE ESTROGENS AND ESTROGEN METABOLITES ARE LOW COMMENTARY Many estrogenic hormones and hormone metabolites measured here are low. This pattern may still be quite clinically meaningful; however, prudence should be used when attempting to correlate and compare results via ratios. The clinician Custom interpretation comments has to always bear in mind that the ratio may be less significant when the results making up the numerator and appear after each hormone category. denominator are low compared to ratios constructed from results that are normal or elevated. Unique & Important Metabolites & Ratios

Estrogen Ratios and Metabolites

Measurement, Ratio or Metabolite Clinical Value

Ratio of 4-hydroxyestrone (4OHE1) to Has been proposed as a marker of breast cancer risk.1 2-hydroxyestrone (2OHE1)

Ratio of 2OHE1 to Estrone (E1) Provides insight into 2-hydroxylation: higher levels of 2OHE1 (relative to other hydroxylated estrones) may be associated with decreased cancer risk.2,3,4,5,6

Estrogen Quotient (Estriol (E3)/Estradiol Research suggests that patients with low ratios may be at greater risk for (E2) + E1) estrogen-sensitive cancers.7

Estrone Sulfate (E1S) An important form of stored estrogen that provides insight into the impact of supplemented oral or sublingual estrogens on estrogen stores, and may also inform on sulfatase and sulfotransferase activity.8

Sum of Estrogens Provides a view into overall estrogen production, representing many estrogens and principle metabolites.

Cortisol Metabolism Many of these ratios are unique to CHI, and all contribute to our understanding of the role of cortisol in health.

Measurement, Ratio or Metabolite Clinical Value

Free Cortisol Profile Informs on the cortisol day curve. A positive slope (rising) or a slope near zero (flattened) may be signs of burnout or chronic illness, while a steeply-negative slope (falling) may indicate anxiety or acute stress.

Activity of 11βHSD2 11βHSD2 converts cortisol to inactive cortisone, and when it is inhibited, free cortisol will typically be much greater than free cortisone. 9,10

Ratio of Cortisol Metabolites to Cortisone Provides another view into 11βHSD2 activity. Metabolites (α-THF + β-THF/THE)

Ratio of α-THF to β-THF Provides insight into thyroid function, and decreased adrenocorticotropic hormone (ACTH).11,12,13

Cortisol Conjugation Pattern Provides insight into the proper interpretation of free cortisol results. Ability to conjugate hormones may be affected by liver function, presence of toxins, inflammation14, availability of conjugates, medications15 and genetic polymorphisms.

Morning Cortisol Response The Ratio of Free Cortisol (Morning) to Free Cortisol (Overnight) may be a surrogate for the salivary cortisol awakening response.16

Overnight Cortisol Response Ratio of Free Cortisol (Overnight) to Free Cortisol (Bedtime) provides insight into diurnal rhythm, with low ratios (near 1) found in both Addison’s and Cushing’s patients.17

Ratio of Cortisol to Testosterone Provides insight into the balance of anabolic (repair and growth) and catabolic (wear and tear) hormones. Unique & Important Metabolites & Ratios

Unique or Important Metabolites

Measurement, Ratio or Metabolite Clinical Value

17OH-pregnenolone An intermediate metabolite essential for steroid hormone synthesis. Research shows 17OH-pregnenolone is an important precursor to DHEA and may help identify polycystic ovary syndrome (PCOS).18,19

Allopregnanolone A progesterone metabolite that has been closely linked with premenstrual dysphoric disorder in the luteal phase20, as well as depression in postmenopausal women.21

8-hydroxy-2-deoxyguanosine (8OH2dG ) An important biomarker of oxidative stress, which has been implicated in numerous chronic diseases including: cardiovascular disease, depression, cancers, chronic fatigue and Parkinson’s disease.22,23,24,25,26

6-sulfatoxymelatonin The primary metabolite of melatonin, which has anti-oxidant anti-depressant, anti-inflammatory, pain-modulating, and neuroprotective effects.27,28

Conjugates Provide insight into the effects of supplemented hormones, hormone storage, and liver function.8,11

Unique or Important Ratios

Measurement, Ratio or Metabolite Clinical Value

Ratio of Testosterone to EpiTestosterone Provides insight into whether testosterone is being supplemented in men or (T/Epi-T) women (supplementation increases testosterone, but not epi-testosterone).

Androsterone Excretion Ratio May provide insights into thyroid function and alpha reductase activity.29 (Androsterone/Androsterone + Etiocholanolone)

Methylation Capacity May help inform on catecholamine clearance, neurotransmitter balance and (2-methoxyestrogen (2MeOE1)/2- risk of estrogen-sensitive cancers. hydroxyestrone (2OHE1))

Testing methods and techniques • Liquid chromatography–mass spectrometry (LC-MS/MS) does not use hydrolysis, and measures both conjugated and free forms of the hormones, separately • Gas chromatography–mass spectrometry (GC-MS/MS) numbers represent total hormone. After hydrolysis, all forms of hormone are converted to their unconjugated or ‘free’ form and measured via GC-MS/MS

CHI delivers clinically meaningful insights by reporting ratios, conjugates and sums. CHI Can Benefi t Patients like Nicole

Age: 53 year old female BMI: 25 kg/m2 Current medications: Oral micronized progesterone 200 mg at bedtime Relevant history: Nicole previously had disturbed sleep, which resolved when oral progesterone was prescribed. However, her sleep issues have recurred in the form of night sweats, trouble getting to sleep and frequent waking. Collection Date: 01-Sep-2018 105 - 32 Royal Vista Dr NW Primary complaints: NicoleCollection presented Time(s): to 7:00 her am doctor with complaints of Calgary, AB T3R 0H9 Sample Received: 03-Sep-2018 Website: www.rmalab.com signifi cant sleep disturbance, Reportedmild acne On: 03-Sep-2018and thinning hair. Phone: 403-241-4500 Fax: 403-241-4501 Email: [email protected] Will estrogen therapy help relieve Nicole’s symptoms?

Nicole’s Saliva Hormone Test Results

Hormone Status Result Range Units Range Applied

Estradiol Within range 0.9 0.0 - 4.0 pg/mL Endogenous postmenopausal range Progesterone Within range 110 50 - 150 pg/mL Pg oral 6 to12 hrs (<=200mg) Testosterone Within range 21 15 - 45 pg/mL Endogenous testosterone > 30 yrs DHEAS Within range 4.0 1.0 - 6.5 ng/mL Female DHEAS 51-64 years Cortisol AM Within range 8.0 1.8 - 12 ng/mL First sample:1st hour of waking

Hormone Therapies Last Used

Progesterone oral (200 mg) 9 hrs

Clinical Summary Nicole’s estradiol is at the low end of the normal range, which means she might benefi t from estrogen therapy. Progesterone is within normal range for a post-menopauseal woman taking oral progesterone. Her other hormones are within normal range.

Recommendation: Comprehensive Hormone Insights™ (CHI) CHI measures urine levels of parent hormones and metabolites. Because Nicole is considering supplementing with estrogens, her estrogen metabolism is of particular interest. A CHI test will assess how Nicole metaboilzes estrogens to Co-Signing Physician: Clare Westmacott, MD help Georgemanage Gillson theMD, PhD risks associated with estrogen replacement. CHI also reports Calgary, 6-sulfatoxymelatonin, AB androgens and Medical Director Phone (403) 278-1405 other hormones that may off er insighta Division into ofcauses Lifelabs of sleep disturbances, mild acne and thinning hair. CPSA Accreditation #L0154200 Page 1 of 3

Both saliva and urine can identify low estrogen and diurnal cortisol. However, CHI provides additional insights into estrogen metabolism that could aff ect treatment recommendations. Nicole’s CHI Results

CHI SUMMARY PAGE (HMUS01)

50 66 50 66 VERY LOW Sum VERY HIGH 33 33 84 of Estrogens Sum of 100 100 16 96 16 4 Androgens Percentile Precursors Percentile Sum of Androgens Sum of Estrogens 0 0 (T, DHT,α+β androstanediol) Listed in Interp Guide

50 66 50 66 VERY HIGH Sum VERY HIGH 33 33 84 of Progesterone DHEA & Metabolites 87 100 98 100 Metabolites 16 Percentile 16 Percentile DHEA + Metabolites Sum of Progesterone Metabolites 0 (DHEA + A + E) 0 α+β Pregnanediol OxidativeOxidative Stress Stress

50 5066 66 50 5066 66 ELEVATED ELEVATED 33 33 84 84 33 33 84 84 Cortisol Metabolites 8OH2dG 100100 100100 16 16 8383 16 16 6868 PercentilePercentile PercentilePercentile CortisolCortisol Metabolites Metabolites 8-Hydroxy-2-8-Hydroxy-2- 0 (α+β0 (α+β THF THF + THE) + THE) deoxyguanosinedeoxyguanosine (Overnight)(Overnight)

Clinical Summary Nicole has very high results for Sum of Androgens, DHEA & Metabolites, and Sum of Progesterone Metabolites, consistent with her prolonged use (more than one year) of high dose oral progesterone. Backdoor conversion to and 17-ketosteroid metabolites can occur with excess progesterone, and the elevated levels of these metabolites may be the cause of Nicole’s thinning hair and mild acne. Nicole’s Sum of Estrogens is very low, as is expected based on her age and saliva hormone test results. Her Cortisol Metabolites are high, which might be contributing to sleep issues and her oxidative stress marker 8-OH2dG is elevated, which may contribute to health issues over the longer term. Ascribing clinical signfi cance to estrogen metabolism ratios is ill-advised when estrogen metabolite levels are very low. Possible Clinical Interventions* • Reduce oral micronized progesterone from 200mg to 100mg at bedtime to decrease backdoor conversion of progesterone into testosterone. • Recommend topical estrogens to alleviate night sweats and sleep disturbance. • Undertake measures to reduce oxidative stress such as: antioxidants and hydrogenated water. • Reduce cortisol levels, taking into account results on Cortisol Heat Map (not shown). • It may be worthwhile to order a retest of CHI in 6 to 12 months to determine whether interventions to reduce backdoor conversion and lower cortisol and oxidative stress have been successful. Note, if Nicole uses topical estrogens, the levels of estrogen metabolites may not increase much, if at all. Topical hormones do not form signifi cant metabolites. * Interventions are for discussion purposes - CHI does not provide patient-specifi c treatment recommendations. CHI Can Benefi t Patients like Richard

Age: 64 year old male BMI: 29 kg/m2 Waist 44 inches Current medications: Simvastatin, allopurinol, PPI, SSRI Relevant history: Business executive, stressful career, non-smoker Primary complaints: Richard presented to his doctor with complaints of: loss of interest in usual activities, “grumpiness”, diffi culty losing weight, breast enlargement, constipation, fatigue, weakness, low sex drive, aches and pains.

Is testosterone50 defi66 ciency the cause of Richard’s5 0symptoms?66 33 33 84 100 100 16 2 16 92 Percentile Precursors Percentile Sum of Androgens Sum of Estrogens Richard’s CHI Results0 (T, DHT, α+β a ndrostanediol) 0 Listed in Interp Guide 50 66 50 66 33 33 84 CHI SUMMARY PAGE (HMUS01) 5050 6666 100 1005050 6666 16 9 16 66 3333 Percentile Percentile3333 8484 DHEA + Metabolites Sum of Progesterone Metabolites 100100 100100 HIGH 1616 2 0 (DHEA + A + E) 0 α+β P161reg6 nan92ediol Percentile PrecursorsPrecursors Percentile SumSum ofof An Androgensdrogens SumSum ofof Est Estrogensrogens Sum of Estrogens VERY LOW 0 (T,(T, DHDHT,T, α αβ+β androstanediol)androstanediol) 0 Liste ofd iestrogensn Interp Guid cane be found CortisSumol of Androgens in Estrogens section of report

5050 6666 5050 6666 3333 3333 8484 100100 100100 1616 9 1616 66 Percentile50 66 Percentile VERY LOW 33 DHEA + +8 Me 4Metabolitestabolites SumSum ofof P Progesteronerogesterone M etaMetabolitesbolites 0 (DHEA(DHEA ++ AA + + E E)) Testosterone 0 α+βα+β PPregnanediolregnanediol Cortisol 100 DHEA & Metabolites 16 40 Percentile Cortisol Cortisol Metabolites 0 Testosterone CortisolCortisol (α+β THF + THE) Cortisol/Testosterone provides insight into

Free Cortisol Profile relative catabolic (cortisol) and anabolic (testosterone) states. 50 50 66 HIGH 33 84 50 66 40 Testosterone Cortisol 100 Ratio of Cortisol to 1633 4084 Percentile Testosterone Cortisol 30 100 Cortisol Testosterone 16 40Cortisol Metabolites Percentile0 (α+β THF + THE) CortTestosteroneisol 20 Cortisol Metabolites 0 (α+β THF + THE) Testosterone

10 Cortisol/Testosterone provides insight into Cortisol/Creatinine (ng/mg) Flattened slope relative catabolic (cortisol) and anabolic 0 (testosterone) states. Morning Dinner Bedtime Low AUC A C (Area Under the Curve)

CORTISOL CONJUGATION PATTERN Cortisol (free) Cortisol sulfate Cortisol glucuronide CORTISOLCORTISOL HEAT CONJUGATION MAP (HMUS03) PATTERN Cortisol18.2 - 32.8 (free) % Cortisol20.7 - 34.7sulfate % Cortisol40 - glucuronide53.1 % 18.2 - 32.8 % 20.7 - 34.7 % 40 - 53.1 % Cortisol Conjugation Pattern Reference Population (Male) Reference Population (Male) MARKEDLY ELEVATED LOW Patient Result 16.9% 55.4% 27.7% Patient Result 16.9% 55.4% 27.7%

11βHSD2 Cortisol-Cortisone Metabolite Balance Cortisol Cortisone 11βHSD211βHSD1 Cortisol-Cortisone Metabolite Balance Cortisol Cortisone 11βHSD1

5α-THF 5β-THF 5β-THE 5α-THF 5β-THF 5β-THE Richard’s CHI Results cont.

Activity of 11-β HSD2 CORTISOL HEAT MAP (HMUS03) Activity of 11-β HSD2 This marker informs on the nature of the activity of 11-β HSD2 enzyme. 11-β HSD2 converts Thiscortisol marker to inactive informs cortisone on the nature in kidneys, of the distal activity colon of 11-β and HSD2saliva glands.enzyme. When 11-β thisHSD2 enzyme converts is cortisolmarkedly to inhibited inactive cortisonefree cortisol in kidneys,will be much distal greater colon andthan sal freeiva cortisone. glands. When When this the enzyme enzyme is is markedlyupregulated inhibited free cortisone free cortisol will typicallywill be much be much greater greater than than free cortisone.free cortisol. When the enzyme is upregulated free cortisone will typically be much greater than free cortisol.

Activity of 11-β HSD2 Activity of 11-β HSD2

Inhibited Active Upregulated (MoreInhibited Cortisol) Active (MoreUpregulated Cortisone) (More Cortisol) (More Cortisone)

Alpha Reductase Preference ANDROGEN-KETOSTEROIDS HEAT MAP (HMUS05)

αTHF / βTHF Alpha reductase Androsterone (α) preference may be Androsterone (α) + Etiocholanolone (β) influenced by thyroid 0 Percentile 100% status. 0 100%

Clinical Summary Richard has very low levels of androgens and 17-ketosteroids (DHEA & metabolites) along with signifi cantly elevated estrogens, which suggests aromatase activity is driving the conversion of testosterone to estradiol. Aromatase activity can be driven by cortisol and/or intra-abdominal fat. Indicators of decreased thyroid hormone activity are present including low alpha reductase preference (αTHF/βTHF) and very low Androsterone Excretion Ratio (A/A+E) The high proportion of cortisol sulfate may indicate increased sulfotransferase activity. In this case, a practitioner may speculate that increased sulfotransferase activity for cortisol is also sulfating thyroid hormone, which is known to accelerate thyroid hormone deactivation by deiodinases. Richard has a fl attened curve in his Free Cortisol Profi le, and the area under the curve (amount of free cortisol in profi le) is also low, both of which are consistent with symptoms of fatigue, weakness, and aches and pains. Note that even when Cortisol Metabolites are normal, it is possible to have low free cortisol, and the associated low cortisol symptoms. A high ratio of cortisol to androgens can contribute to loss of muscle mass and aches and pains. Some of Richard’s symptoms may also be related to decreased thyroid hormone activity (e.g. constipation).

Possible Clinical Interventions* • Consider zinc, melatonin, and/or fl avonoid supplementation to decrease aromatase activity and conversion of testosterone to estradiol. • Order confi rmatory thyroid studies (serum thyroid stimulating hormone (TSH) or urine thyroid). • Consider serum binding globulin (SHBG) and homeostasis model assessment of insulin resistance (HOMA-IR) to assess for metabolic syndrome and/or insulin resistance (linked to low androgens, high estradiol in men). • Consider high intensity interval training and/or intermittent fasting to boost testosterone and growth hormone levels, thereby reducing abdominal fat.

The results from CHI provide you with confi dence in treatment decision making.

* Interventions are for discussion purposes - CHI does not provide patient-specifi c treatment recommendations. CHI Can Benefi t Patients like Tamara

Age: 41 year old female BMI: 21.3 kg/m2 Relevant History: Had her fi rst child ten years ago and began serious fi tness training after pregnancy. Non-smoker, healthy diet. Primary complaints: Tamara presented to her doctor with trouble conceiving, insomnia, foggy thinking, anxiety, and irritability.

Is there an underlying hormone issue at the heart of Tamara’s health concerns?

Tamara’s CHI Results CORTISOL CONJUGATION PATTERN Cortisol (free) Cortisol sulfate Cortisol glucuronide CORTISOLCORTISOL HEAT CONJUGATION MAP (HMUS03) PATTERN Cortisol15.6 - 34(free) % Cortisol25.3 - 36.6sulfate % Cortisol36.6 -glucuronide 51.3 % Cortisol Conjugation Pattern 15.6 - 34 % 25.3 - 36.6 % 36.6 - 51.3 %

Reference Population (d21) Reference Population (d21) MARKEDLY ELEVATED LOW LOW Patient Result 53.7% 19.4% 26.9% Patient Result 53.7% 19.4% 26.9%

11βHSD2 Cortisol-Cortisone Metabolite Balance Cortisol Cortisone 11βHSD1 11βHSD2 Cortisol-Cortisone Metabolite Balance Cortisol Cortisone 11βHSD1

5α-THF 5β-THF 5β-THE 5α-THF 5β-THF 5β-THE

Ratio of αTHF + βTHF / βTHE Activity of 11-β HSD2 Ratio of αTHF + βTHF / βTHE Activity of 11-β HSD2 The ratio of aTHF+ βTHF over βTHE provides This marker informs on the nature of the activity of 11-β HSD2 enzyme. 11-β HSD2 converts insight into the relative balance of cortisol cortisol to inactive cortisone in kidneys, distal colon and saliva glands. When this enzyme is Themetabolites ratio of aTHF+over cortisone βTHF over metabolites. βTHE provides Thismarkedly marker inhibited informs free on cortisolthe nature will ofbe the much activity greater of 11-β than HSD2 free cortisone. enzyme. When11-β HSD2 the enzyme converts is insight into the relative balance of cortisol cortisolupregulated to inactive free cortisone cortisone will in typicallykidneys, be distal much colon greater and salthaniva free glands. cortisol. When this enzyme is metabolites over cortisone metabolites. markedly inhibited free cortisol will be much greater than free cortisone. When the enzyme is upregulated free cortisone will typically be much greater than free cortisol. αTHF + βTHF / βTHE Activity of 11-β HSD2 αTHF + βTHF / βTHE Activity of 11-β HSD2 0 Percentile 100% Inhibited Active Upregulated (More Cortisol) (More Cortisone) 0 Percentile 100% Inhibited Active Upregulated (More Cortisol) (More Cortisone)

Cortisol Metabolites Alpha Reductase Preference Cortisol Metabolites Alpha Reductase Preference VERY HIGH 50 66 αTHF / βTHF 33 84 50 66 αTHF / βTHF Cortisol 100 1633 98 84 0 100% Percentile Percentile Metabolites 100 16 Cortisol98 Metabolites 0 Percentile 100% 0Percentile(αTHF + βTHF + βTHE) Cortisol Metabolites 0 (αTHF + βTHF + βTHE)

Free Cortisol Profile Free Cortisol Profile The Free Cortisol Profile provides Free Cortisol Profile 100 Free Cortisol Profile insight into the diurnal rhythm of cortisol. 100 The Free Cortisol Profile provides 80 insight into the diurnal rhythm of cortisol. 6080 Two ratios are calculated: 4060 - Overnight Cortisol Response (D/C) -Two Morning ratios Cortisolare calculated: Response (A/D) - Overnight Cortisol Response (D/C) 2040 Neither ratio is graphed, but both- Morning are commented Cortisol Response on in (A/D) Cortisol/Creatinine (ng/mg) 200 Neitherthe interpretation. ratio is graphed, but both are commented on in Cortisol/Creatinine (ng/mg) Morning0 Dinner Bedtime Overnight the interpretation. MorningA DinnerB BedtimeC OvernightD A B C D CORTISOL CONJUGATION PATTERN Cortisol (free) Cortisol sulfate Cortisol glucuronide 15.6 - 34 % 25.3 - 36.6 % 36.6 - 51.3 %

Reference Population (d21)

Patient Result 53.7% 19.4% 26.9%

11βHSD2 Cortisol-Cortisone Metabolite Balance Cortisol Cortisone 11βHSD1

5α-THF 5β-THF 5β-THE

Ratio of αTHF + βTHF / βTHE Activity of 11-β HSD2

The ratio of aTHF+ βTHF over βTHE provides This marker informs on the nature of the activity of 11-β HSD2 enzyme. 11-β HSD2 converts insight into the relative balance of cortisol cortisol to inactive cortisone in kidneys, distal colon and saliva glands. When this enzyme is metabolites over cortisone metabolites. markedly inhibited free cortisol will be much greater than free cortisone. When the enzyme is upregulated free cortisone will typically be much greater than free cortisol.

αTHF + βTHF / βTHE Activity of 11-β HSD2

0 Percentile 100% Inhibited Active Upregulated (More Cortisol) (More Cortisone)

Cortisol Metabolites Alpha Reductase Preference

50 66 αTHF / βTHF 33 84 100 16 98 0 100% Percentile Percentile Cortisol Metabolites Tamara’s CHI Results0 cont. (αTHF + βTHF + βTHE)

Free Cortisol Profile Free Cortisol Profile 100 The Free Cortisol Profile provides insight into the diurnal rhythm of cortisol. 80

60 Two ratios are calculated: HIGH area under curve 40 - Overnight Cortisol Response (D/C) - Morning Cortisol Response (A/D) STEEP slope 20 Neither ratio is graphed, but both are commented on in Cortisol/Creatinine (ng/mg) 0 the interpretation. Morning Dinner Bedtime Overnight A B C D HIGH Overnight Cortisol Response*(D/C) *Reported in interpretation comments

SUMMARY PAGE (HMUS01)

Oxidative Stress 50 66 50 66 LOW 33 33 84 Sum of Estrogens 100 100 50 16 84 16 24 66 Percentile Precursors Percentile Sum of Androgens Sum of Estrogens 33 84 0 0 (T, DHT, α+β androstanediol) Listed in Interp Guide 100 16 74 HIGH Percentile 8-Hydroxy-2- Sum of 50 66 50 66 0 deoxyguanosine Androgens 33 33 84 (Overnight) 55 100 2 100 16 Percentile 16 Percentile DHEA + Metabolites Sum of Progesterone Metabolites HIGH 8OH2dG 0 (DHEA + A + E) 0 α+β Pregnanediol oxidative stress LOW Progsterone metabolites Clinical Summary Tamara’s BMI is at the low end of normal, and she has a high Sum of Androgens, low Sum of Estrogens, very low Progesterone metabolites and signifi cantly elevated Cortisol Metabolites, all of which may impact her ability to conceive. The steep slope in Tamara’s Free Cortisol Profi le suggests she is “always on”, and her Overnight Cortisol Response suggests the presence of chronic stress, anxiety and/or depression. Tamara’s elevated 8-hydroxy-2-deoxyguanosine (a biomarker of oxidative stress) combined with elevated Cortisol Metabolites, and low proportion of cortisol sulfate suggest the presence of infl ammation. Infl ammation has been shown to induce the sulfatase enzyme,* that removes the sulfate conjugate and creates more free hormone.

*Research has shown that infl ammation induces sulfatase enzymes for DHEA14, and this is also believed to be true for cortisol (physiologically useful to generate free cortisol in the presence of infl ammation).

Possible Clinical Interventions* • Curcumin inhibits 11-beta hydroxysteroid dehydrogenase type 1 (11βHSD1) activity, reduces free cortisol, and also has anti-infl ammatory eff ects. • may help with low estrogen symptoms. • Phosphatidylserine may help reduce cortisol. • Low dose progesterone cream in luteal phase may be benefi cial.

CHI provides you with clinically relevant information about your patient’s condition.

* Interventions are for discussion purposes - CHI does not provide patient-specifi c treatment recommendations. Comparing Hormone Testing Methods

RMA is the only functional medicine laboratory in Canada that offers the top three most common methods of hormone testing: saliva, serum, and urine.

Saliva • Measures levels of hormones that have passed through tissue (i.e. the salivary gland) • Provides insight into levels of hormones that are capable of binding to target-tissue receptors • Is an excellent method for measuring endogenous hormone levels • Measures free hormones and hormones that are loosely bound to albumin • Reflects exposure to supplemented hormones, and may reflect stored hormones • This may be useful for troubleshooting , particularly topical hormones

Serum • Serum tends to underestimate the amount of hormone present when topically applied hormones are used • Most useful for measuring endogenous hormone levels • Serum hormone levels are not reflective of the clinical effects of hormone skin creams • A limited menu of hormones and metabolites are tested in serum

Urine • Provides a view into overall production of each hormone category (estrogens, androgens, progesterone, glucocorticoids) through the measurement of the metabolites • Useful for determining how the body metabolizes supplemented hormones, particularly estrogens, testosterone, and cortisol • Provides a view into hormone conjugation, which may be affected by liver function, availability of conjugates and genetic polymorphisms that impact the ability to conjugate • Like serum testing, urine may under-represent topically applied hormones. • Urine may over-represent orally supplemented hormones as they are rapidly converted to conjugates References

1. Huang, J., et al. “Analysis of multiplex endogenous estrogen metabolites in human urine using ultra-fast liquid chromatography-tandem mass spectrometry: a case study for breast cancer.” Analytica Chimica Acta, 20 January 2012, vol. 711, pp. 60-8, doi: 10.1016/j.aca.2011.10.058. 2. Dallal, C., et al. “Estrogen metabolism and breast cancer risk among postmenopausal women: a case–cohort study within B~FIT” Carcinogenesis, vol. 35 no. 2, February 2014, pp. 346–355, doi: 10.1093/carcin/bgt367. 3. Falk, R., et al. “Relationship of serum estrogens and estrogen metabolites to postmenopausal breast cancer risk: a nested case-control study”, Breast Cancer Research, April 2013, vol. 15, no. 2, R34, pp. 1-14, doi: 10.1186/bcr3416. 4. Fuhrman, B., et al. “Estrogen Metabolism and Risk of Breast Cancer in Postmenopausal Women”, Journal of the National Cancer Institute, 22 February 2012, vol.104, no. 4, pp 326-339, doi: 10.1093/jnci/djr531. 5. Arslan, A., et al, “Circulating estrogen metabolites and risk of breast cancer in postmenopausal women.”, Cancer Epidemiology, Biomarkers & Prevention, July 2014, vol. 23, no. 7, pp. 1290–1297, doi: 10.1158/1055-9965.EPI-14-0009. 6. Ziegler, R., et al. “Epidemiologic Studies of Estrogen Metabolism and Breast Cancer”, , July 2015, vol. 99, Part A, pp 67–75, doi: 10.1016/j. steroids.2015.02.015. 7. Lemon, H.M., “Oestriol and prevention of breast cancer”, Lancet, 10 March1973, vol. 1, no. 7802, pp. 546-7. 8. Rižner, T., “The Important Roles of Steroid Sulfatase and Sulfotransferases in Gynecological Diseases”, Frontiers in Pharmacology, 18 February 2016, vol. 7, no. 30, doi: 10.3389/fphar.2016.00030. 9. Palermo, M., et al. “Urinary free cortisone and the assessment of 11 beta-hydroxysteroid dehydrogenase activity in man”, Clinical Endocrinology(Oxford), 1996, vol. 4, no. 5, pp. 605-611. 10. Yokokawa, A., et al. “The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11beta-hydroxysteroid dehydrogenase 2 activity”, Steroids, 2012, vol. 77, no. 12, pp. 1291-1297, doi: 10.1016/j. steroids.2012.07.008. 11. Vantyghem, M.C,. “Urinary cortisol metabolites in the assessment of peripheral thyroid hormone action: overt and subclinical hypothyroidism.” Journal of Endocrinological Investigation, 1998, vol. 21, no. 4, pp 219-225. 12. Hoshiro, M., et al. “Comprehensive study of urinary cortisol metabolites in hyperthyroid and hypothyroid patients.” Clinical Endocrinology (Oxf) 2006, vol. 64, no. 1, pp 37-45. 13. Gordon, G. and Southren, A. “Thyroid hormone effects on steroid hormone metabolism” Bulletin of New York Academy of Medicine, 1977, vol. 53, no. 3, pp 241-259. 14. Jiang, M., et al. “Inflammatory Regulation of Steroid Sulfatase, A Novel Mechanism to Control Estrogen Homeostasis and Inflammation in Chronic Liver Disease”, Journal of Hepatology, January 2016, vol. 64, no. 1, pp. 44–52, doi: 10.1016/j.jhep.2015.07.022. 15. Ambadapadi, S., et al. “Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1”, Journal of Steroid Biochemistry and Molecular Biology”, August 2015, vol. 52, pp. 101-13, doi:10.1016/j.jsbmb.2015.05.003. 16. Ramachandran, R., et al. “Suboptimal rise in awakening-induced cortisol is an accurate marker of cortisol insufficiency in patients with normal renal function (eGFR >60?mL/min)”, Annals of Clinical Biochemistry, July 2018, vol. 55, no. 4, pp. 496-499. doi:10.1177/0004563217732361. 17. Burch, W., “Using bedtime (PM) and early morning (AM) urine cortisol/creatinine ratios to evaluate pituitary-adrenal function in an office practice”, Endocrine Practice, 2011, vol.17, pp. 591-597, doi: 10.4158/EP10177OR. 18. de Medeiros, S., et al. “Metabolic and endocrine connections of 17-hydroxypregnenolone in polycystic ovary syndrome women”, Endocrine Connections, October 2017, vol. 6, no. 7, pp. 479–488,doi: 10.1530/EC-17-0151. 19. de Medeiros, S., et al. “New insights into steroidogenesis in normo- and hyperandrogenic polycystic ovary syndrome patients.” Arq Bras Endocrinol Metabol. 2013 Aug;57(6):437-44. 20. Bäckstrom, T., et al. “Allopregnanolone and mood disorders”, Progress in Neurobiology, February 2014, vol. 113, pp. 88-94, doi: 10.1016/j.pneurobio.2013.07.005. 21. Bixo, M., et al. “Effects of GABA active steroids in the female brain with a focus on the premenstrual dysphoric disorder”, Journal of Neuroendocrinology, February 2018, vol. 30, no.2, doi: 10.1111/jne.12553. 22. Slopien, R., et al. “Correlation between allopregnanolone levels and depressive symptoms during late menopausal transition and early postmenopause”, Gynecological Endocrinology, February 2018, vol. 34, no. 2, pp. 144-47, doi: 10.1080/09513590.2017. 23. Bonomini, F., “Atherosclerosis and oxidative stress”, Histology and Histopathology, March 2008, vol. 23, no. 3, pp. 381-90, doi: 10.14670/HH-23.381. 24. Sinha, N. and Dabla, P.K. “Oxidative stress and antioxidants in hypertension-a current review”, Current Hypertension Reviews, 2015, vol. 11, no.2, pp. 132-42, DOI : 10.2174/1573402111666150529130922. 25. Tyouz, R.M. and Briones A.M, “Reactive oxygen species and vascular biology: implications in human hypertension”, Hypertension Research, January 2011, vol. 3, no. 1, pp. :5-14. doi: 10.1038/hr.2010.201. 26. Halliwell, B., “Oxidative stress and cancer: have we moved forward?”, The Biochemical Journal, 1 January 2007, vol. 401, no.1, pp.1-11, DOI: 10.1042/BJ20061131. 27. Blesa , J., et al. “Oxidative stress and Parkinson’s disease”, Frontiers in Neuroanatomy, July 2015, vol. 9, no. 91, pp.1-9, doi: 10.3389/fnana.2015.00091. 28. Kennedy, G. et al. “Oxidative stress levels are raised in chronic fatigue syndrome and are associated with clinical symptoms,”Free Radical Biology and Medicine, 1 September 2005, vol. 39, no. 5, pp. 584-9, DOI: 10.1016/j.freeradbiomed.2005.04.020. 29. Eme, M., et al. “A Review of Melatonin, Its Receptors and Drugs”, The Eurasian Journal of Medicine, June 2016, vol. 48, no. 2, pp. 135–141, doi: 10.5152/ eurasianjmed.2015.0267. 30. Rodriguez, C. et al. “Regulation of antioxidant enzymes: a significant role for melatonin”, Journal of Pineal research, January 2004, vol. 36, no. 1, pp. 1-9, doi. org/10.1046/j.1600-079X.2003.00092.x. 31. Skovsted, L. et al. “The androsterone-etiocholanolone excretion ratio in hyper- and hypothyroidism” Acta Medica Scandinavica, September 1966, vol.180, no. 3, pp. 301-6, PMID: 5920076. 32. Xu, G.X., “Curcumin as a potent and selective inhibitor of 11β-hydroxysteroid dehydrogenase 1: improving lipid profiles in high-fat-diet-treated rats.”. 33. Fattah, A. Effect of on Depression and Anxiety in Menopausal Women: A Systematic Review. J Menopausal Med. 2017 Dec; 23(3): 160–165. doi: 10.6118/jmm.2017.23.3.160. Now available through Rocky Mountain Analytical®!

Rocky Mountain Analytical is an accredited Canadian medical laboratory that has provided clear and reliable test reports to preventive and integrative medicine professionals since 2002.

With a commitment to keeping your patients healthy, Rocky Mountain Analytical is proud to offer CHI in our portfolio of laboratory tests—to help you learn what you need to know about the hormonal health of your patients.

Free shipping Tested in Canada RMA reputation • Kits and collected samples • Samples shipped within Canada, • Known for quality hormone testing are shipped anywhere in enable improved turn around times • Dr. Gillson’s extensive knowledge Canada at no charge • All payments are in Canadian of hormones and expert dollars, so the cost does not commentary offers meaningful fluctuate with exchange rates clinical interpretations of results

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