Hormone Testing Jim Paoletti, B.S. Pharmacy, FAARFM 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” 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 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 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 Body Fluids Commonly Used for Testing Steroid Hormones

• Serum/plasma • 24-hour urine • • Capillary (dried blood spot) 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 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 • assays do not distinguish between progesterone and its metabolites • Serum E2 less reflective of loose bound hormone in women Serum Testing

Considerations • Large normal ranges • Ranges for free and bio-available established for men, not sensitive enough for women • Normal testosterone range is often the same for all adult women, no matter of age Free 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 Serum Testing

Considerations • 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 • Current hormone therapy – Not correlated in literature 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 24-Hour Urine Testing

Considerations • Urine 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 sulphates 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 Urine Testing

• Not Representative of Delivery to Tissue • Progesterone 100mg p.o. – 90-95% 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 Saliva Testing

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

Considerations • Poor Technique in sample collection or handling can compromise results – Blood contamination • Spurious results with (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 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) Dose-Dependent Increase in Salivary Progesterone and Testosterone Following Topical Delivery (Mean ± 2 SD)

(4040(4040--6602)

7000 (n = 238)

/ml) pg 6000 PROGESTERONE

(2757(2757--4581) (female) 5000 (n = 319)

4000

(1054-2580) 3000 (n = 267) TESTOSTERONE (680-2152) (n = 122) (male) 2000

(1654-2698) (n(n == 90)90) SALIVARY ( LEVEL HORMONE SALIVARY 1000 (1218-3254) (n = 17) (571-1747) (715-2057) (n = 31) 0 (n = 39) 5 10 25 50 100 TOPICAL DOSE (mg) Measurement of Steroid vs. Background Salivary Estradiol & Hot Flashes

in 39,000 women

Hot Flash Severity Hot

Reported

- Self

ESTROGEN DEFICIENT ESTROGEN EXCESS

Salivary Estradiol Concentration as determined by extracted EIA Topical Hormone Delivery - The Controversy

• 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 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 © ZRT Laboratory.LLC January 20, 2016 23 Chang KJ. 1995. 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 +/-0.3 66 +/-120

• Progesterone levels increase 100X at the tissue level

2002 04 16 058 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 PPLICATION A HOURS POST HOURS PROGESTERONE (ng/ml) 10 12 14 16 18 20 0 2 4 6 8

600

630 PROGESTERONE MG 20 OF APPLICATION PERCUTANEOUS 7:00 700 -1 SALIVA AND SERUM PROGESTERONE FOLLOWING FOLLOWING PROGESTERONE SERUM AND SALIVA 730 8:00

800 0 830 9:00 SALIVA 900 1 930 10:00 © ZRT Laboratory.LLC © ZRT 1000 2 1030 11:00

1100 3 TIME OF OF DAY TIME 1130 SERUM 12:00

1200 4

123013:00 January 20, 2016 20, January 1300 5 1330 14:00

1400 6 1430 15:00

1500 7 26 1530 WEEK 1 WEEK 16:00 1600 8 1630 17:00 1700 9 1730 18:00 1800 10 1830 6:30 630 22 Testosterone Topical Supplementation: Serum Underestimation & Potential Overdosing

Testosterone Measurements Post-Supplementation 5mg Topical Application 700

600 Venous Serum Venous Blood Spot 500 Capillary Serum

400 Capillary Blood Spot

300

200 Concentration (ng/dl) 100

0 Baseline 6 Hours Post Supplementation 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. 12(2): 232- 237, 2005 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): 232- 237, 2005 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 “ 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): 232- 237, 2005 Salivary Progesterone with Topical Supplementation (n=1,863)

3000

2500 24-Hours Post Supplementation

2000 12-Hours Post Supplementation

1500

1000

Average Salivary Progesterone(ng/ml)Average 500

Physiological Luteal Levels of Progesterone (75-270) 0 0 25 50 75 100 Daily Progesterone Dosage (mg)

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. Epidermis

Dermis

RBC

Binding Protein

Hormone Tissue Cells

Interstitial Fluid

Capillary Bed

RBC

Binding Protein

Hormone Tissue Cells

Interstitial Fluid

Blood Serum Spot

RBC

Binding Protein

Hormone HORMONE TESTING IN CAPILLARY BLOOD SPOTS 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 Blood Spot Testing: Not a New Concept Blood Spot Testing: Not a New Concept Blood Spot How to?

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 Disadvantages

• Vs. serum: limited testing available in blood spot compared to serum/plasma

• Vs. Saliva: not as much data at this point 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 test results are combined with hormone binding proteins (eg. SHBG) the ratio provides information on the bioavailable fraction Hormones Tested By Blood Spot

• Free T3 • Progesterone • Free T4 • Testosterone • TSH • SHBG • TPO • PSA • Estradiol • Vitamin D Testosterone Correlation Blood Spot vs. Serum Estradiol Correlation Blood Spot vs. Serum Progesterone Correlation - Blood Spot vs. Serum Blood Spot Progesterone with Topical Supplementation After 8-12 Hours (n=99) 80

70

60

50

40

30

20

10

Median Blood Spot Progesterone (ng/ml) Progesterone Spot Blood Median

0 0 10 20 30 40 50 60 70 80 Daily Progesterone Dosage (mg) Thanks for Listening! Jim Paoletti [email protected]