Testing Hormones and Neurotransmitters in Different Body Fluids LDN Conference Portland Oregon David T. Zava PhD A Guide to Testing Steroid and Peptide Hormones in Different Body Fluids Hormone therapy is a mainstay in treatment of hormonal imbalances and helps guide and optimize the type, dosage, and delivery of the hormone. Hormone therapy without first testing hormone levels can lead to less than optimal therapy. Hormone testing for steroid hormones is most commonly performed using venipuncture serum or plasma and urine. Other body fluids (saliva and fingertip capillary dried blood spots-DBS) are becoming more commonplace due to convenience of collection and more accurate assessment of bioavailable hormone levels. In this lecture I will discuss the pros and cons of using different body fluids and methods (EIA vs LC-MS/MS) for testing steroid hormones, produced endogenously or taken exogenously by different routes of administration (oral, topical, troche, sc
pellets, etc.). I will explain why most body fluids are appropriate for hormone testing following exogenous hormone supplementation but that serum and urine are less optimal than saliva and capillary blood for detecting hormones following topical hormone delivery. Goals & Objectives 1. Review and discuss pros and cons of using different body fluids for testing steroid hormones 2. Explain why some body fluids are better than others for hormone testing depending on route of hormone administration 3. Show research and clinical studies demonstrating that testing of topically delivered hormones is more accurate using saliva or capillary blood (dried blood spot) and NOT serum or urine Different types of body fluids commonly used for testing hormones Serum and Plasma (Venipuncture)
Urine (Liquid and Dry) Saliva Capillary Blood (Finger Stick Dried Blood Spots) A Guide to Steroid Hormone Testing in Different Body Fluids with Different Routes of Hormone Administration Type of Body Fluid None Endogeno us Steroids
Oral Steroids Topical Gels/ Creams Steroids Vaginal Steroids Troche/ Sublingua l Steroids Transderm al Patch Occluded Pellet/IM
Steroids Seru m Yes Yes (1) No (2) Yes Yes Yes Yes Saliva Yes
Yes Yes (3) Yes No (4) Yes Yes Urine Yes Yes (1) No (2) No (4)
Yes Yes Yes (1) 1) Overestimation: Metabolites likely to interfere with Yes Yes Yes (5) Yes Yes Yes Yes DBS immunoassays 2) Underestimation: Hormone levels not reflective of tissue uptake 3) Caution: Requires range adjustment 4) Overestimation: Direct contamination of body fluid (saliva/ urine) 5) Caution: Direct contamination of capillary blood can occur
if ungloved hands used to apply topical hormones. Apply Saliva Testing How do steroid hormones enter saliva? Steroid hormones (about 2% of total) are released from binding proteins in bloodstream and passively diffuse into tissues (salivary gland) and accumulate in saliva S steroid hormone carrier protein (SHBG, CBG, albumin) red blood cell S S S S SALIVA
S S S S SS S S S Salivary Gland S capillary Clinical Relevance Do the hormone results make sense from a clinical perspective?
Salivary Estradiol: The Menopausal Transition Premenopaus al Estradiol Median vs. Age n = 49,769 Estradiol Median (pg/ml) 2.0 1.5 Postmenopau sal 1.0 0.5 25 30
35 40 45 50 Age 55 60 65 70 75 Salivary Estradiol & Hot Flashes
Estradiol vs. Hot Flash Severity n = 39,570 1.6 Average Hot Flash Severity (0-3) 1.5 1.4 1.3 1.2 1.1 1 OPTIMAL REFERENCE RANGE 0.9 0.8 0.7 0.6 0
1 2 3 4 5 Salivary Estradiol (pg/ml) 6 7 8 9 Bottom Line: Saliva An excellent test to measure bioavailable levels of steroid
hormones, and is more reflective of tissue uptake (in salivary gland). Disadvantage is its limited to low molecular weight nonpolar molecules like steroid hormones. Cannot test high molecular weight peptide hormones. Blood Testing Serum/Plasma vs Dried Blood Spot (DBS) Dried Capillary Blood Dried Blood Spot (DBS) Minimally invasive testing Collection timing convenient Convenient at home collection Quick and easy Less infectious pathogens with drying
Dried sample stable for weeks at RT No refrigeration required for shipment Fewer shipment restrictions Labeling & Storage convenient Venipuncture Serum Invasive testing Inconvenient collection timing Requires driving to clinic Time consuming Wet sample contains infectious pathogens Sample unstable-overnight shipment Shipment requires refrigeration Restrictions for wet blood shipments Labeling and Storage difficult Correlation of Finger-Stick Capillary Dried Blood (DBS) with
Serum Are DBS results quantitatively equivalent to venipuncture serum results? Menstrual Cycle Premenopausal vs Postmenopausal Symptoms of Estrogen Imbalance-Deficiency and Excess Estrogen Supplementation Progesterone Blood Spot/Serum Correlation 18 16 Serum Progesterone (ng/ml)
14 12 10 8 6 y = 1.05x - 0.6 R2 = 0.99 4 2 0 0 2 4 6 8 10
12 Blood Spot Progesterone (ng/ml) 14 16 18 Menstrual Cycle Mapping Blood Spot and Plasma Levels of Estradiol, Progesterone, LH, and FSH Over a Normal Menstrual Cycle Estradio l Validation of Blood Spot Sampling for Gonadotropins and Ovarian Hormone Levels in Reproductive Age Women. Fertility and Sterility, November 2007 A. Edelman, R. Stouffer, D. Zava, J. Jensen Menstrual Cycle Mapping
Blood Spot and Plasma Levels of Estradiol, Progesterone, LH, and FSH Over a Normal Menstrual Cycle Progesteron e Validation of Blood Spot Sampling for Gonadotropins and Ovarian Hormone Levels in Reproductive Age Women. Fertility and Sterility, November 2007 A. Edelman, R. Stouffer, D. Zava, J. Jensen CONCLUSION: WITH ENDOGENOUSLY PRODUCED HORMONES VENIPUNCTURE SERUM = CAPILLARY BLOOD (DBS) Bottom Line: Serum Serum steroids-good test to measure total levels of steroid hormones in the bloodstream, but is less reflective of tissue uptake, especially if serum binding proteins (e.g. SHBG,
CBG) are elevated Bottom Line: Capillary Whole Blood (DBS) Capillary Blood-good test to measure tissue levels of steroid hormones. Advantage: its a good body fluid to also measure other peptide hormones and other analytes-near With Some Exogenously Supplemented Hormones, Especially Topical Hormones VENIPUNCTURE SERUM = CAPILLARY BLOOD (DBS) Topical Hormone Therapy and Testing in Different Body Fluids A Guide to Steroid Hormone Testing in Different Body
Fluids with Different Routes of Hormone Administration Type of Body Fluid None Endogeno us Steroids Oral Steroids Topical Gels/ Creams Steroids Vaginal Steroids
Troche/ Sublingua l Steroids Transderm al Patch Occluded Pellet/IM Steroids Seru m Yes Yes (1) No (2) Yes
Yes Yes Yes Saliva Yes Yes Yes (3) Yes No (4) Yes Yes
Urine Yes Yes (1) No (2) No (4) Yes Yes Yes (1) 1) Overestimation: Metabolites likely to interfere with Yes Yes Yes (5) Yes
Yes Yes Yes DBS immunoassays 2) Underestimation: Hormone levels not reflective of tissue uptake 3) Caution: Requires range adjustment 4) Overestimation: Direct contamination of body fluid (saliva/ urine) 5) Caution: Direct contamination of capillary blood can occur if ungloved hands used to apply topical hormones. Apply Important take home Topical delivery of ALL steroid hormones is not monitored accurately with serum or urine Topical Naltrexone? Is it more effective than serum or urine tests for naltrexone would make you believe?
Urine Testing: Steroid Metabolites Neurotransmitters Elements Dried vs Liquid Urine Collection Simple and convenient at home collection Multiple collections throughout day allows monitoring of circadian rhythms Flexibility in testing, storage, and shipment to laboratory Inconvenient collection & shipment 24 hr collection good for average hormone metabolite production but not possible to evaluate circadian rhythms GC/MS/MS Testing of Estrogen Metabolites in Dried
Urine ESTROGEN METABOLISM AND BREAST CANCER RISK Sulfotransferase E1-S04 E1 Sulfatase INACTIVE ESTROGEN METABOLITES 2-MeOE1(E2) 4MeO-E1(E2) 2-GS-E1(E2) 4-GSE1(E2) CANCER CYP 1B1
4-OH-E1(E2) 17 -HSD-Type II E2 17-HSD-Type I Cyp-1A1 2-OH-E1(E2) 16-OH-E1 E3 COMT PROTEIN 4-QUINONE-E1(E2) 2-QUINONE-E1(E2) BINDING GSH Transferase DNA ADDUCT FORMATION
DNA DAMAGE MUTATION DNA ADDUCT FORMATION DNA REPAIR NO FURTHER EFFECT E1 = ESTRONE E2 =ESTRADIOL E3 = ESTRIOL Stress-HPA Axis-Cortisol-Inflammation and Breast Cancer Risk OVARIAN DYSFUNCTION XENOBIOTICS (eg, PCBs) POLLUTANTS
ESTROGEN S (eg, DDT) PROGESTERONE E ER SHBG ADRENAL GLAND Iodine (I2) INSULIN POOR DIET THYROID (refined
carbohydrates) MELATONIN BREAST STROMA CADMIUM DHEA INSULIN RESISTANCE OBESITY 17B-HSD TYPE II (E1E2) E1-SULFATASE (E1-S04 E1) AROMATASE (A E1) ZINC CORTISOL LH/FSH
Vitamin D3 PROLACTIN Physical Surgical ERT HRT TESTOSTERONE XENOESTROGENS (AROMATASE) (E1, E2) STRES S Emotional POLYCYSTIC OVARIES IMMUNE CYTOKINES
SYSTEM TH1 TH2 SMOKING Dried Urine Ideal for Tracking Circadian Patterns of Free Cortisol, Free Cortisone, and Melatonin Healthy Adult: Normal Cortisol and Melatonin Circadian Rhythm Healthy Adult: Normal Cortisol and Melatonin Circadian Rhythm Typical Cancer Patient: Flattened Cortisol and Melatonin Circadian Rhythm Urinary Diurnal Patterns of Stress Hormones and Melatonin
Bottom Line: Urine Urinary Metabolites-good test to evaluate excreted steroid metabolites which partially reflect total production of steroid hormones, but is NOT reflective of circulating levels of, or tissue uptake, of bioactive steroid Bottom Line: Urine Urine steroid metabolitesminimal increase in urine steroid metabolites with topical delivery, even with pharmacological hormone delivery to the skin: 10-20x higher relative increase in levels seen in DBS and saliva with same dosing!! Bottom Line: Urine = Serum Not Reflective of Tissue Levels Capillary Blood = Saliva
Reflective of Tissue levels Conclusion: All topical steroids result in a marked increase in salivary and DBS levels of hormone, but little increase in serum or urine hormone Tissue uptake of steroid indicate salivary and DBS hormone levels more accurately reflect tissue Conclusions Know what body fluid is best for detecting steroids and monitoring hormone replacement therapies Otherwise you may not optimize hormone replacement therapies and patient outcome A Guide to Steroid Hormone Testing in Different Body Fluids with Different Routes of Hormone Administration
Type of Body Fluid None Endogeno us Steroids Oral Steroids Topical Gels/ Creams Steroids Vaginal Steroids Troche/ Sublingua
l Steroids Transderm al Patch Occluded Pellet/IM Steroids Seru m Yes Yes (1) No (2) Yes Yes
Yes Yes Saliva Yes Yes Yes (3) Yes No (4) Yes Yes Urine
Yes Yes (1) No (2) No (4) Yes Yes Yes (1) Yes Yes Metabolites Yes (5) likely Yes to interfere Yes Yes Yes
DBS 1) Overestimation: with immunoassays 2) Underestimation: Hormone levels not reflective of tissue uptake 3) Overestimation: Requires range adjustment 4) Overestimation: Direct contamination of body fluid (saliva/ urine) 5) Overestimation: Direct contamination of capillary blood if QUESTIONS ? [email protected] m
