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Testing Hormones and Neurotransmitters in Different Body Fluids

This lecture discusses the pros and cons of using different body fluids for testing steroid hormones. It explains why some fluids are better than others depending on the route of hormone administration. Research and clinical studies demonstrating the accuracy of testing topically delivered hormones using saliva or capillary blood are also presented.

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Testing Hormones and Neurotransmitters in Different Body Fluids

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  1. Testing Hormones and Neurotransmitters in Different Body Fluids LDN Conference Portland Oregon David T. Zava PhD

  2. 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.

  3. Goals & Objectives • Review and discuss pros and cons of using different body fluids for testing steroid hormones • Explain why some body fluids are better than others for hormone testing depending on route of hormone administration • 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

  4. 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)

  5. A Guide to Steroid Hormone Testing in Different Body Fluids with Different Routes of Hormone Administration 1) Overestimation: Metabolites likely to interfere with immunoassays Underestimation: Hormone levels not reflective of tissue uptake Caution: Requires range adjustment Overestimation: Direct contamination of body fluid (saliva/urine) Caution: Direct contamination of capillary blood can occur if ungloved hands used to apply topical hormones. Apply hormones as usual but avoid hand contact for at least 2 days prior to collection

  6. Saliva Testing

  7. 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

  8. Clinical Relevance • Do the hormone results make sense from a clinical perspective?

  9. Salivary Estradiol: The Menopausal Transition Premenopausal Postmenopausal

  10. Salivary Estradiol & Hot Flashes OPTIMAL REFERENCE RANGE

  11. Bottom Line: SalivaAn excellent test to measure bioavailable levels of steroid hormones, and is more reflective of tissue uptake (in salivary gland). Disadvantage is it’s limited to low molecular weight nonpolar molecules like steroid hormones. Cannot test high molecular weight peptide hormones.

  12. Blood TestingSerum/Plasma vs Dried Blood Spot (DBS)

  13. Dried Capillary Blood Dried Blood Spot (DBS) 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 • 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

  14. 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

  15. Progesterone Blood Spot/Serum Correlation

  16. Menstrual Cycle Mapping Blood Spot and Plasma Levels of Estradiol, Progesterone, LH, and FSH Over a Normal Menstrual Cycle Estradiol 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

  17. Menstrual Cycle Mapping Blood Spot and Plasma Levels of Estradiol, Progesterone, LH, and FSH Over a Normal Menstrual Cycle Progesterone 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

  18. CONCLUSION: WITH ENDOGENOUSLY PRODUCED HORMONESVENIPUNCTURE SERUM=CAPILLARY BLOOD (DBS)

  19. Bottom Line: SerumSerum 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

  20. Bottom Line: Capillary Whole Blood (DBS)Capillary Blood-good test to measure tissue levels of steroid hormones. Advantage: it’s a good body fluid to also measure other peptide hormones and other analytes-near equivalence to serum.

  21. With Some Exogenously Supplemented Hormones, Especially Topical HormonesVENIPUNCTURE SERUM=CAPILLARY BLOOD (DBS)

  22. Topical Hormone Therapy and Testing in Different Body Fluids

  23. A Guide to Steroid Hormone Testing in Different Body Fluids with Different Routes of Hormone Administration 1) Overestimation: Metabolites likely to interfere with immunoassays Underestimation: Hormone levels not reflective of tissue uptake Caution: Requires range adjustment Overestimation: Direct contamination of body fluid (saliva/urine) Caution: Direct contamination of capillary blood can occur if ungloved hands used to apply topical hormones. Apply hormones as usual but avoid hand contact for at least 2 days prior to collection

  24. Important take home Topical delivery of ALL steroid hormones is not monitored accurately with serum or urine

  25. Topical Naltrexone?Is it more effective than serum or urine tests for naltrexone would make you believe?

  26. Urine Testing: Steroid MetabolitesNeurotransmittersElements

  27. Dried vsLiquid 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

  28. GC/MS/MS Testing of Estrogen Metabolites in Dried Urine

  29. ESTROGEN METABOLISM AND BREAST CANCER RISK Sulfotransferase 17 ß-HSD-Type II E1-S04 E1 E2 Sulfatase 17ß-HSD-Type I Cyp-1A1 CYP-1B1 INACTIVE ESTROGEN METABOLITES 16-OH-E1 4-OH-E1(E2) E3 2-OH-E1(E2) 2-MeO-E1(E2) 4-MeO-E1(E2) COMT PROTEIN BINDING 2-GS-E1(E2) 4-GS-E1(E2) 2-QUINONE-E1(E2) 4-QUINONE-E1(E2) GSH Transferase DNA ADDUCT FORMATION DNA ADDUCT FORMATION NO FURTHER EFFECT E1 = ESTRONE E2 =ESTRADIOL E3 = ESTRIOL DNA DAMAGE MUTATION DNA REPAIR CANCER

  30. Stress-HPA Axis-Cortisol-Inflammation and Breast Cancer Risk POLYCYSTIC OVARIES OVARIANDYSFUNCTION XENOBIOTICS (eg, PCBs) ERT HRT POLLUTANTS ESTROGENS (E1, E2) TESTOSTERONE XENOESTROGENS (eg, DDT) (AROMATASE) LH/FSH PROGESTERONE Vitamin D3 Iodine (I2) E ER PROLACTIN INSULIN RESISTANCE INSULIN SHBG OBESITY POOR DIET (refined carbohydrates) THYROID STRESS 17B-HSD TYPE II (E1→E2) E1-SULFATASE (E1-S04 →E1) AROMATASE (A→E1) Emotional Physical Surgical MELATONIN ZINC SMOKING CORTISOL BREAST STROMA ADRENAL GLAND CADMIUM DHEA IMMUNE CYTOKINES SYSTEM TH1 ↓ TH2 ↑

  31. Dried Urine Ideal for Tracking Circadian Patterns of Free Cortisol, Free Cortisone, and Melatonin Healthy Adult: Normal Cortisol and Melatonin Circadian Rhythm

  32. Healthy Adult: Normal Cortisol and Melatonin Circadian Rhythm Typical Cancer Patient: Flattened Cortisol and Melatonin Circadian Rhythm

  33. Urinary Diurnal Patterns of Stress Hormones and Melatonin

  34. Bottom Line: UrineUrinary 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

  35. Bottom Line: UrineUrine steroid metabolites- minimal 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!!

  36. Bottom Line: Urine = SerumNot Reflective of Tissue LevelsCapillary Blood = Saliva Reflective of Tissue levels

  37. Conclusion:All topical steroids result in a marked increase in salivary and DBS levels of hormone, but little increase in serum or urine hormoneTissue uptake of steroid indicate salivary and DBS hormone levels more accurately reflect tissue response

  38. 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

  39. A Guide to Steroid Hormone Testing in Different Body Fluids with Different Routes of Hormone Administration 1) Overestimation: Metabolites likely to interfere with immunoassays Underestimation: Hormone levels not reflective of tissue uptake Overestimation: Requires range adjustment Overestimation: Direct contamination of body fluid (saliva/urine) Overestimation: Direct contamination of capillary blood if ungloved hands used to apply topical hormones < 2 days prior to collection

  40. QUESTIONS ? dzava@ZRTLAB.com

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