1. Blood Testing for Hormones Ryan Shelton, ND

2. When to Test Always establish a baseline prior to initializing therapy Always establish a baseline prior to initializing therapy Prior to consultation is ideal for patient Prior to consultation is ideal for patient Follow up for safety and maintenance Follow up for safety and maintenance Ensure physiological dosing Ensure physiological dosing Timing varies 4-6 weeks to every 4-6 months; 2-3 months very common Timing varies 4-6 weeks to every 4-6 months; 2-3 months very common Repeat labs when symptoms do not make sense Repeat labs when symptoms do not make sense Phlebotomy timing Phlebotomy timing No sex, exercise, stress prior to morning collection No sex, exercise, stress prior to morning collection Fasting, well hydrated Fasting, well hydrated Change from AM to PM dosing for three days prior to collection for oral or topical Change from AM to PM dosing for three days prior to collection for oral or topical Instruct patient no topical hormones applied to area of venipuncture Instruct patient no topical hormones applied to area of venipuncture

3. Generalities MUST be coupled with clinical picture; treat patient, not labs MUST be coupled with clinical picture; treat patient, not labs Labs may miss ‘metabolic impact’ Labs may miss ‘metabolic impact’ Testing for isolated hormones not typically useful Testing for isolated hormones not typically useful Know if lab reference ranges are adapted for age Know if lab reference ranges are adapted for age Understand testing methodology Understand testing methodology Immunoassay vs. much more sensitive liquid or gas chromatography tandem mass spectrometry Immunoassay vs. much more sensitive liquid or gas chromatography tandem mass spectrometry 20-40% imprecision in immunoassays 20-40% imprecision in immunoassays Use the SAME lab each time Use the SAME lab each time Wide variability in lab reference values (325-350% difference) Wide variability in lab reference values (325-350% difference)

4. Borderline Deficiencies Statistical mean Statistical mean Lower and upper reference values at 2 standard deviations Lower and upper reference values at 2 standard deviations Conventional medicine treats only outside these deviations Conventional medicine treats only outside these deviations Age adjusted Age adjusted Many doctors use reference values for 20-30yo Many doctors use reference values for 20-30yo Optimal value in upper third of reference range Optimal value in upper third of reference range Deficiency vs borderline deficiency Deficiency vs borderline deficiency Pathophysiology Pathophysiology Preliminary Preliminary Decrease in tissues Decrease in tissues Biochemical Biochemical Feedback mechanisms Feedback mechanisms Physiological Physiological Decrease in metabolites and enzyme activity Decrease in metabolites and enzyme activity Clinical Clinical Subclinical Sx Subclinical Sx Anatomical Anatomical Functional disturbances and clinical Sx Functional disturbances and clinical Sx

5. Blood (serum) Advantages Advantages Test directly for hormones, stimulating hormones, releasing hormones Test directly for hormones, stimulating hormones, releasing hormones Test for binding proteins Test for binding proteins Most familiar method for majority of conventional doctors Most familiar method for majority of conventional doctors Other important blood tests can be included Other important blood tests can be included Insurance coverage Insurance coverage Well established normal ranges for frank disease states Well established normal ranges for frank disease states Minimal collection/compliance issues Minimal collection/compliance issues Disadvantages Disadvantages Can vary with circadian/pulsatile/monthly rhythms Can vary with circadian/pulsatile/monthly rhythms Free fractions can vary throughout the day Free fractions can vary throughout the day Large normal reference range, often adapted to age Large normal reference range, often adapted to age May not reflect metabolic impact May not reflect metabolic impact Mostly bound hormones Mostly bound hormones Venous sample Venous sample May be inaccurate with transdermal HRT creams May be inaccurate with transdermal HRT creams Total production missed (only a snapshot) Total production missed (only a snapshot)

6. Blood Tests Overview Standard base Standard base CBC, Chem Panel, Lipids, Insulin, A1c,Ferritin, CRP, Homocysteine (many others) CBC, Chem Panel, Lipids, Insulin, A1c,Ferritin, CRP, Homocysteine (many others) Pituitary Pituitary GH, LH, FSH, ACTH, Prolactin GH, LH, FSH, ACTH, Prolactin Thyroid Thyroid TSH, fT3, fT4, rT3, ATG, ATPO, Thyroglobulin, Calcitonin TSH, fT3, fT4, rT3, ATG, ATPO, Thyroglobulin, Calcitonin Liver Liver IGF-1, IGFBP-3 IGF-1, IGFBP-3 Adrenal Adrenal Cortisol (T), Cortisol (f), DHEA-s, Androstenedione, Pregnenolone-s Cortisol (T), Cortisol (f), DHEA-s, Androstenedione, Pregnenolone-s Female Female Estradiol, Progesterone, Testosterone, SHBG, DHT Estradiol, Progesterone, Testosterone, SHBG, DHT Male Male Testosterone (T), Testosterone (f), SHBG, Androstanediol glucuronide, DHT Testosterone (T), Testosterone (f), SHBG, Androstanediol glucuronide, DHT

7. Basic Panel Female Female CBC CBC Chem Panel Chem Panel Lipid Panel Lipid Panel TSH TSH T3, T4 T3, T4 Test, free & total Test, free & total Estradiol Estradiol Progesterone Progesterone SHBG SHBG IGF-1 IGF-1 Male Male CBC CBC Chem Panel Chem Panel Lipid Panel Lipid Panel TSH TSH T3, T4 T3, T4 Test, free & total Test, free & total Estradiol Estradiol PSA PSA SHBG SHBG IGF-1 IGF-1

8. Comprehensive Panel Female Female CBC, Chem Panel, Insulin CBC, Chem Panel, Insulin Lipid Panel Lipid Panel TSH, T4, T3, rT3 TSH, T4, T3, rT3 Test, free & total Test, free & total Estradiol Estradiol Estrone Estrone Progesterone Progesterone SHBG SHBG DHT DHT IGF-1, IGFBP-3 IGF-1, IGFBP-3 LH LH FSH FSH Ferritin Ferritin DHEA-s DHEA-s CRP, Homocysteine CRP, Homocysteine Vit D Vit D Cortisol Cortisol A1C A1C Prolactin Prolactin Male Male CBC, Chem Panel, Insulin CBC, Chem Panel, Insulin Lipid Panel Lipid Panel TSH, T4, T3, rT3 TSH, T4, T3, rT3 Test, free & total Test, free & total Estradiol Estradiol PSA PSA SHBG SHBG DHT DHT IGF-1, IGFBP-3 IGF-1, IGFBP-3 LH LH FSH FSH Ferritin Ferritin DHEA-s DHEA-s CRP, Homocysteine CRP, Homocysteine Vit D Vit D Cortisol Cortisol A1C A1C Prolactin Prolactin

9. Initial vs Follow-up Testing Initial Initial Schedule labs prior to actual initial visit Schedule labs prior to actual initial visit Establish baseline Establish baseline Information is additional to thorough history and physical exam Information is additional to thorough history and physical exam Follow-up Follow-up Ensure physiologic range Ensure physiologic range Every patient is unique and will require an individualized plan Every patient is unique and will require an individualized plan If initiating a new medication or treatment, may want to evaluate in 6-8 weeks If initiating a new medication or treatment, may want to evaluate in 6-8 weeks If on stable medication protocol, may only need to be tested every 6-12 months If on stable medication protocol, may only need to be tested every 6-12 months Schedule labs prior to actual follow-up visit Schedule labs prior to actual follow-up visit Urine metabolites helpful Urine metabolites helpful

10. Growth Hormone & Secretagogues Blood test should be RIA, not chemoluminescence Blood test should be RIA, not chemoluminescence IGF-1 reflects GH and is stable throughout the day IGF-1 reflects GH and is stable throughout the day GH release is pulsatile, half life only 15-19 min GH release is pulsatile, half life only 15-19 min May take 30-45 days of GH Tx for IGF-1 to increase and stabilize at higher level May take 30-45 days of GH Tx for IGF-1 to increase and stabilize at higher level Stimulated GH (GHRH, insulin, glucagon, arginine, L-Dopa) often required for insurance Stimulated GH (GHRH, insulin, glucagon, arginine, L-Dopa) often required for insurance IGF-1/IGFBP-3 useful for baseline & F/U IGF-1/IGFBP-3 useful for baseline & F/U Reference ranges Reference ranges IGF-1: 115-490ng/ml for 21-30yo IGF-1: 115-490ng/ml for 21-30yo IGFBP-3: 3.4-7.2ng/ml for 21-30yo IGFBP-3: 3.4-7.2ng/ml for 21-30yo IGF-1/IGFBP-3 ratio: 0.07-2.2 IGF-1/IGFBP-3 ratio: 0.07-2.2 IGF-1<84 may be 96% predictive of AGHD IGF-1<84 may be 96% predictive of AGHD Optimal Optimal Male: IGF-1 250-350ng/ml; Stim GH >5ng/ml (>3 if glucagon) Male: IGF-1 250-350ng/ml; Stim GH >5ng/ml (>3 if glucagon) Female: IGF-1 220-300ng/ml; Stim GH >5ng/ml (>3 if glucagon) Female: IGF-1 220-300ng/ml; Stim GH >5ng/ml (>3 if glucagon) IGF-1 166 no AGHD IGF-1 166 no AGHD

11. Thyroid TSH is not sufficient for initial Dx TSH is not sufficient for initial Dx Auto-antibodies important to r/o Hashimoto’s Auto-antibodies important to r/o Hashimoto’s TSH TSH Normal: 0.35-3.5ng/dl Normal: 0.35-3.5ng/dl Optimal: 0.2-2ng/dl Optimal: 0.2-2ng/dl fT4 fT4 Normal: 0.8-1.8ng/dL Normal: 0.8-1.8ng/dL Optimal: 1.3-1.8ng/dL Optimal: 1.3-1.8ng/dL fT3 fT3 Normal: 1.8-3.7ng/dl Normal: 1.8-3.7ng/dl Optimal: 2.5-3.4ng/dl Optimal: 2.5-3.4ng/dl rT3 rT3 Normal: 14.9-26.1ng/dl Normal: 14.9-26.1ng/dl Optimal: 10-16ng/dl Optimal: 10-16ng/dl 25-33% of TSH euthyroid will have elevated rT3 25-33% of TSH euthyroid will have elevated rT3

12. Cortisol Serum(t) Serum(t) Provocative Provocative 500nmol/L or 100% above baseline 500nmol/L or 100% above baseline Morning Morning Ref: 100-250ng/mL; Optimal: 180 Ref: 100-250ng/mL; Optimal: 180 Afternoon Afternoon Ref: 30-100ng/mL; Optimal: 70 Ref: 30-100ng/mL; Optimal: 70

13. Female Hormones Prolactin Prolactin Anti-psychotics, Anti-depressants, Birth Control Pills can cause hyperprolactinemia Anti-psychotics, Anti-depressants, Birth Control Pills can cause hyperprolactinemia Possible issues with utilizing blood tests for F/U if patient is using transdermal cream hormones (vs gels) Possible issues with utilizing blood tests for F/U if patient is using transdermal cream hormones (vs gels) Falsely decreased serum levels may not correlate with endometrial response Falsely decreased serum levels may not correlate with endometrial response DDx DDx LH>FSH c E1>E2 on day 21, Consider PCOS LH>FSH c E1>E2 on day 21, Consider PCOS Elevated FSH may be first indicator of menopause Elevated FSH may be first indicator of menopause SHBG: low may be a sign of E2 def or Test excess or oral estrogen ; high may be sign of E2 def or Test excess SHBG: low may be a sign of E2 def or Test excess or oral estrogen ; high may be sign of E2 def or Test excess Anti-Mullerian Hormone Anti-Mullerian Hormone Best Days Best Days LH (12-14, 21) LH (12-14, 21) E2 & Testosterone (12-14, 21) E2 & Testosterone (12-14, 21) Prog (21) Prog (21)

15. Female Hormones FSH FSH Ref: 30-130mIU/mL; Optimal: 20-50 Ref: 30-130mIU/mL; Optimal: 20-50 E2 E2 Ref: 100-210pg/mL; Optimal: 150 Ref: 100-210pg/mL; Optimal: 150 SHBG SHBG Ref: 41-79pmol/mL; Optimal: 65 Ref: 41-79pmol/mL; Optimal: 65 Prog Prog Ref: 3-27ng/mL; Optimal:15-20 Ref: 3-27ng/mL; Optimal:15-20 Test Test Ref: 100-500pg/mL; Optimal: 350pg/mL Ref: 100-500pg/mL; Optimal: 350pg/mL DHT DHT Ref: 5-30 ng/dL; Optimal 20 ng/dL Ref: 5-30 ng/dL; Optimal 20 ng/dL Androstanediol glucuronide Androstanediol glucuronide Ref: 0.1-6.0ng/mL; Optimal: 3-4 Ref: 0.1-6.0ng/mL; Optimal: 3-4 DHEA DHEA Optimal: 2800ng/mL Optimal: 2800ng/mL

16. Male Hormones No sex, vigorous exercise, emotional stress prior to testing No sex, vigorous exercise, emotional stress prior to testing Bioavailable T=Free T + T loosely bound to albumin Bioavailable T=Free T + T loosely bound to albumin Free T calculator Free T calculator www.issam.ch/freetesto.htm www.issam.ch/freetesto.htm www.issam.ch/freetesto.htm Variables needed: albumin, SHBG, Free Testosterone Variables needed: albumin, SHBG, Free Testosterone Optimal 400-640ng/dl male (250-350pg/ml, <180 considered def); 10-30 ng/dl females (8pg/ml, <5 considered def) Optimal 400-640ng/dl male (250-350pg/ml, <180 considered def); 10-30 ng/dl females (8pg/ml, <5 considered def) Possible issues with utilizing blood tests for F/U if patient is using transdermal hormonal creams Possible issues with utilizing blood tests for F/U if patient is using transdermal hormonal creams Timing Timing Transdermal: 12 hrs p 3 consecutive evening applications Transdermal: 12 hrs p 3 consecutive evening applications Injections: 7 days p an injection in males Injections: 7 days p an injection in males Highest 7am; female peak mid-cycle Highest 7am; female peak mid-cycle

17. Male Hormones Testosterone (total) Testosterone (total) Ref: 3000-10000pg/mL; Optimal: 7000 (700ng/dL) Ref: 3000-10000pg/mL; Optimal: 7000 (700ng/dL) DHT DHT Ref: 25-75 ng/dL; Optimal: 50 Ref: 25-75 ng/dL; Optimal: 50 Androstanediol glucuronide Androstanediol glucuronide 3.4-22ng/mL; Optimal: 15-18 3.4-22ng/mL; Optimal: 15-18 More useful than DHT More useful than DHT Estradiol Estradiol Ref: 10-45pg/mL; Optimal: 25 Ref: 10-45pg/mL; Optimal: 25 SHBG SHBG Ref: 20-55pmol/L; Optimal: 25-30 Ref: 20-55pmol/L; Optimal: 25-30

18. Advanced Testing Nutritional Panel Nutritional Panel Food Allergies Food Allergies Heavy Metals Heavy Metals Cancer Markers Cancer Markers Genomics Genomics Cytokines Cytokines Saliva; Urine Saliva; Urine

19. Case Study 53 yo female, last menses 7 mos ago, hot flashes, insomnia, fatigue, low libido, poor memory, joint pain, sugar cravings, low blood pressure depression, dry eyes, vaginal dryness, droopy breasts, dry hair, seborrheic dermatitis along hairline 53 yo female, last menses 7 mos ago, hot flashes, insomnia, fatigue, low libido, poor memory, joint pain, sugar cravings, low blood pressure depression, dry eyes, vaginal dryness, droopy breasts, dry hair, seborrheic dermatitis along hairline FSH 155mIU/mL; E2 50 pg/mL; Prog 4 ng/mL; Test 100 pg/mL; DHEA 1000ng/mL; Cortisol 30 ng/mL; DHT 15 ng/dL FSH 155mIU/mL; E2 50 pg/mL; Prog 4 ng/mL; Test 100 pg/mL; DHEA 1000ng/mL; Cortisol 30 ng/mL; DHT 15 ng/dL Tx: Bi-Est 1.25 mg/ Test 5 mg gel topical q am; Prog 100 mg cap q hs; E3 0.5 mg vag cream hs; Cortef 5 mg; DHEA 5 mg Tx: Bi-Est 1.25 mg/ Test 5 mg gel topical q am; Prog 100 mg cap q hs; E3 0.5 mg vag cream hs; Cortef 5 mg; DHEA 5 mg

20. Case Study 8 wk F/U 8 wk F/U Hot flashes, insomnia, fatigue, low libido, poor memory, joint pain, sugar cravings, low blood pressure depression, dry eyes, vaginal dryness, droopy breasts, dry hair, seborrheic dermatitis along hairline all greatly improved Hot flashes, insomnia, fatigue, low libido, poor memory, joint pain, sugar cravings, low blood pressure depression, dry eyes, vaginal dryness, droopy breasts, dry hair, seborrheic dermatitis along hairline all greatly improved Pt has noticed oily skin, acne, irritability, hair loss Pt has noticed oily skin, acne, irritability, hair loss FSH 45 mIU/mL; E2 155 pg/mL; Prog 18 ng/mL; Test 345 pg/ml; DHEA 2000 ng/mL; Cortisol 100 ng/mL; DHT 31 ng/dL FSH 45 mIU/mL; E2 155 pg/mL; Prog 18 ng/mL; Test 345 pg/ml; DHEA 2000 ng/mL; Cortisol 100 ng/mL; DHT 31 ng/dL Tx changes Tx changes Bi-Est 1.25 mg gel topical q am; E3 0.5/Test 0.25 mg vag cream hs Bi-Est 1.25 mg gel topical q am; E3 0.5/Test 0.25 mg vag cream hs