Hormone Testing Jim Paoletti, B.S. Pharmacy, FAARFM

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1 Hormone Testing Jim Paoletti, B.S. Pharmacy, FAARFM

2 Establish the Need Lab tests Correlate patient assessment with testing results Lab tests alone do not always tell the whole story Lab values are guides to which direction therapy should be considered Lab values should be used to confirm the diagnosis

3 Testing Considerations Limitations Timing of cycle No individual baseline in many cases Lack of correlation of symptoms to levels No consideration of influences on free hormone levels (such as SHBG) Dosage form differences Don t rely on lab tests alone Treat the patient, not the labs

4 Testing Considerations Are you comparing to the range of a person of the reported life stage or the range of the age you are attempting to replicate? We don t want menopausal levels in most cases

5 Testing Considerations When was the test done compared to the timing of the last dose? Make sure patient is at steady state if possible 8-24 hrs post topical application 4-8 hrs post oral dosing (SR capsules) Be consistent with subsequent testing Pay attention to changes in: Site of application Base Volume applied

6 Body Fluids Commonly Used for Testing Steroid Hormones Serum/plasma 24-hour urine Saliva Capillary blood (dried blood spot)

7 Serum Testing Advantages Wide range of hormones available Familiar reference ranges Many laboratories to choose from Standard automated methods with appropriate proficiency testing Insurance coverage

8 Serum Testing Considerations Established gold standard based on evidence for endogenous values, not exogenous administration Invasive to the patient - phlebotomist required Difficult to measure multiple times during day or month Unable to distinguish between bound and unbound hormones Progesterone assays do not distinguish between progesterone and its metabolites Serum E2 less reflective of loose bound hormone in women

9 Serum Testing Considerations Large normal ranges Ranges for free and bio-available testosterone established for men, not sensitive enough for women Normal testosterone range is often the same for all adult women, no matter of age Free Androgen Index (Total T SHBG) often inaccurate Hidden costs in patient obtaining sample Adequate for endogenous hormone measurement Overestimation for oral supplementation Underestimation for topical supplementation

10 Serum Testing Considerations Cortisol in serum Elevations due to fear of being stuck Timing of test vs. normal range timing Inconvenient to do multiple tests Unrepresentative of normal situation Did anyone pay any attention to the time of her cycle?!! Follicular or Luteal phase Current hormone therapy Not correlated in literature

11 24-Hour Urine Testing Advantages Non-invasive Wide range of hormones available Good estimation of total daily production Not as subject to the daily ebb and flow of steroid production as serum/saliva A good reflection of endogenous steroid production if no supplementation given

12 24-Hour Urine Testing Considerations Urine Estradiol is not the same as serum estradiol Serum measures the unconjugated hormone bound to protein Urine measures conjugated estradiol Most of conjugates in urine are glucoronides; the kidney excretes estrogen sulphates

13 24-Hour Urine Testing Considerations Expensive Inconvenient-collection over 24 hrs Measurement of metabolites Assay may not differentiate between a hormone a it s metabolites Measurement of what is being thrown away and not what is bioavailable or being utilized by tissues Not getting representation of the active hormone delivered to the tissue with exogenous administration

14 Urine Testing Not Representative of Delivery to Tissue Progesterone 100mg p.o % metabolized on first pass effect 5-10 mg delivered to tissues (bioavailable: 5-10 mg) Metabolites measured reflect 100mg progesterone Progesterone 10 mg topical 90% delivered to tissues 5-10 mg delivered to tissues (bioavailable: 5-10 mg) Metabolites measured reflect 10 mg progesterone

15 Saliva Testing First citations in 1960 s Commercially available in 1990 s Steadily increasing number of citations: cortisol, estradiol, progesterone, testosterone, DHEA

16 Saliva Testing Considerations Have to use extracted testing for estradiol Direct measurement of E2 is inaccurate and imprecise, and provides no useful clinical results Testing procedures must be sensitive enough to differentiate different hormones To be clinically useful, need to have established valid reference ranges that consider route, timing of dose, and symptom relieve relative to the level.

17 Saliva Testing Considerations Poor Technique in sample collection or handling can compromise results Blood contamination Spurious results with periodontal disease (more problem with chewing gum) Contamination from supplementation products Saliva easily contaminated with topical hormones on lips or hands Local pooling effect with sublingual administration Smaller volumes of saliva will be more variable Saliva samples should not be pooled for analysis

18 Saliva Testing Considerations Sublingual/Buccal use of hormones leads to spurious high test results Direct contamination of the oral mucosa Pooling of hormones in oral cavity Blood Spot Testing is an excellent alternative for sublingual supplementation Makes no sense to stop hormone for a certain period of time and then test (my opinion) Makes no sense to give an exaggerated normal range and try to correlate to tissue levels (my opinion)

19 SALIVARY HORMONE LEVEL (pg/ml) Dose-Dependent Increase in Salivary Progesterone and Testosterone Following Topical Delivery (Mean ± 2 SD) 7000 ( ) (n = 238) ( ) (n = 319) PROGESTERONE (female) ( ) (n = 122) ( ) (n = 267) TESTOSTERONE (male) ( ) (n = 39) ( ) (n = 31) ( ) (n = 90) ( ) (n = 17) TOPICAL DOSE (mg)

20 Measurement of Steroid vs. Background

21 Self-Reported Hot Flash Severity Salivary Estradiol & Hot Flashes in 39,000 women ESTROGEN DEFICIENT ESTROGEN EXCESS Salivary Estradiol Concentration as determined by extracted EIA

22 Topical Hormone Delivery - The Controversy Venipuncture blood (serum or whole blood) suggests poor topical absorption of hormones Saliva suggests topical hormones rapidly and efficiently absorbed Capillary blood suggests topical hormones rapidly and efficiency absorbed

23 Saliva Testing and Topical Administration of Hormones Demonstrates increase in tissue levels after topical application of hormones Linear correlation seen with increasing doses Illustrates cumulative effect of topically applied supra-physiological doses

24 ZRT Laboratory.LLC January 20,

25 Chang KJ Influences of Percutaneous Administration of E2 & Progesterone on Human Breast Epithelial Cell Cycle in vivo. Application of E2 or Progesterone (20 mg) directly to breasts Intraglandular Steroid Concentration Placebo Pg (n=8) (n=7) Intraglandular Pg (ng/g) 0.6 +/ /-120 Progesterone levels increase 100X at the tissue level

26 Topical Progesterone applied to the breast resulted in significant increase in breast tissue, breast cyst fluid and saliva. Serum and urine levels remained relatively unchanged

27 HOURS POST A PPLICA TION 20 SALIVA AND SERUM PROGESTERONE FOLLOWING PERCUTANEOUS APPLICATION OF 20 MG PROGESTERONE PROGESTERONE (ng/ml) SALIVA SERUM WEEK :00 8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 6: TIME OF DAY ZRT Laboratory.LLC January 20,

28 Concentration (ng/dl) Testosterone Topical Supplementation: Serum Underestimation & Potential Overdosing Testosterone Measurements Post-Supplementation 5mg Topical Application Venous Serum Venous Blood Spot Capillary Serum Capillary Blood Spot Baseline 6 Hours Post Supplementation

29 Review of Articles Articles examined whether topical progesterone (20-30mg) protected the estrogen-stimulated endometrial lining All the articles which claimed that topical progesterone failed to provide protection looked at the serum level and assumed it was too low for the progesterone to provide protection The two articles that demonstrated protection biopsied the endometrial tissue and looked at histological changes Stanczyk, FZ et al. Percutaneous administration of progesterone: blood levels and endometrial protection. Menopause 12(2): , 2005

30 Continued Studies investigating the effect of topical cream on the endometrium should not be based on serum progesterone levels but on histologic examination of the endometrium. Stanczyk, FZ et al. Percutaneous administration of progesterone: blood levels and endometrial protection. Menopause 12(2): , 2005

31 Conclusion Conclusion from Stanczyk article: You cannot use serum value to judge the tissue effect on the endometrium for topically applied progesterone Since topical progesterone concentrates more in the uterus than almost all other tissue, one would have to assume the same conclusion on serum levels would apply to all tissues unless and until proven otherwise

32 Serum progesterone levels may not reflect progesterone levels in a particular tissue.. It is now recognized that salivary progesterone levels can increase from baseline levels by at least two orders of magnitude after topical cream application, depending on dose and time of saliva sampling. These findings are consistent with rapid uptake of progesterone by salivary glands. Presumably there is also rapid uptake of progesterone by other tissues, eg, the endometrium.. Stanczyk, FZ et al. Percutaneous administration of progesterone: blood levels and endometrial protection. Menopause 12(2): , 2005

33 Average Salivary Progesterone (ng/ml) Salivary Progesterone with Topical Supplementation (n=1,863) Hours Post Supplementation 12-Hours Post Supplementation Physiological Luteal Levels of Progesterone (75-270) Daily Progesterone Dosage (mg)

34

35 Conclusions When steroid hormones are delivered through the skin, saliva and blood spot hormone levels are more reflective of tissue uptake of steroids than serum hormone levels, but supplementation reference ranges are needed.

36 Epidermis Dermis RBC Binding Protein Hormone

37 Tissue Cells Interstitial Fluid Capillary Bed RBC Binding Protein Hormone

38 Tissue Cells Interstitial Fluid Blood Spot Serum RBC Binding Protein Hormone

39 HORMONE TESTING IN CAPILLARY BLOOD SPOTS

40 Dried Blood Spot Convenient-Simple at home collection procedure Wide range of analytes can be tested-similar to serum/plasma More latitude in collection timing Results equivalent to serum/plasma (except when supplementing topically) Dried serum analytes very stable for weeks at ambient temperature-shipping simplified

41 Blood Spot Testing: Not a New Concept

42 Blood Spot Testing: Not a New Concept

43 Blood Spot How to?

44

45 Advantages (blood spot vs serum) No special processing prior to shipment Convenient shipment-no biohazard precautions required as per CDC International Shipments Correlates well with serum endogenous levels

46 Disadvantages Vs. serum: limited testing available in blood spot compared to serum/plasma Vs. Saliva: not as much data at this point

47 Why Blood Spot and Not Saliva? Allows for testing of larger protein (peptide) molecules Thyroid, Vitamin D, FSH, LSH, PSA, Cardiometabolic risk factors Reasonable alternative for testing steroid hormones in individuals supplementing as a sublingual or troche When steroid hormone test results are combined with hormone binding proteins (eg. SHBG) the ratio provides information on the bioavailable fraction

48 Hormones Tested By Blood Spot Free T3 Free T4 TSH TPO Estradiol Progesterone Testosterone SHBG PSA Vitamin D

49 Testosterone Correlation Blood Spot vs. Serum

50 Estradiol Correlation Blood Spot vs. Serum

51 Progesterone Correlation - Blood Spot vs. Serum

52 80 Blood Spot Progesterone with Topical Supplementation After 8-12 Hours (n=99) Median Blood Spot Progesterone (ng/ml) Daily Progesterone Dosage (mg)

53 Thanks for Listening! Jim Paoletti

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