Why do we test hormones? Rocky Mountain Analytical 4/21/2011. Copyright Saliva HormoneTesting. Endogenous hormones

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1 Saliva HormoneTesting George Gillson MD PhD April 26, 2011 Rocky Mountain Analytical Calgary, Alberta O Sullivan s Rule If you don t know what s wrong with your patient before you send them to the specialist, you won t be any farther ahead after they come back John O Sullivan MD If you don t understand the lab tests you are using, you won t understand the patient any better after their test results come back. Why do we test hormones? Endogenous hormones Identify overt deficiency or excess Understand situations where there are symptoms of hormone deficiency or excess, but one or more hormone levels are normal Copyright

term complications such as breast or endometrial cancer Avoid side effects All these different forms are not the same!

2 Why do we test hormone levels? Supplemented hormones Troubleshooting: therapy isn t working Make sure the patient is not over- or under-dosed Avoid long term complications such as breast or endometrial cancer Avoid side effects All these different forms are not the same! Capillary Only the free steroid is supposed to be able to get out of blood into tissue, as it is the only form small enough to fit through the cracks between the cells which line the capillary walls Copyright

Junctions between cells lining the capillary are wider

salivary ducts Saliva duct endplate So how do steroid

Hormones are thought to get into saliva by partitioning

endplate In order to see high levels of hormone in saliva,

3 Junctions between cells lining the capillary are wider than the junctions of the cells making up the endplate of salivary ducts Saliva duct endplate So how do steroid hormones get into saliva? Hormones are thought to get into saliva by partitioning from the cell membranes of the cells that line the duct endplate In order to see high levels of hormone in saliva, you have to establish high levels of hormone in the tissue that makes up the duct endplate Copyright

4 [Hormone] in saliva is approximately the same as [Hormone] in the capillary But the two measurements are not identical Saliva reflects unconjugated free hormone in blood, but is not identical to it Sublingual Delivery Sublingual hormone preparations elevate serum levels quite well Don t even think about doing saliva tests after sublingual hormone use! Levels of supplemented hormone(s) will be high and meaningless due to diffusion of hormone locally Will also see high levels of metabolites formed in the saliva glands Sublingual Hormone Administration 49 y.o female on sublingual estrogens and progesterone (Phyto B) No hormone excess symptoms Estradiol 850 pg/ml Progesterone pg/ml Testosterone 2150 pg/ml (Metabolite) Cortisol 26 ng/ml (Metabolite) Saturation of salivary apparatus due to local application of hormones Local conversion: progesterone cortisol progesterone testosterone See this due to high local levels of progesterone Copyright

5 Don t do saliva testing after sublingual hormone use!! 2/19/2011 Copyright Rocky Mountain Analytical 13 Reference Ranges There is general agreement that saliva works as well as any other sample type for steroid hormone testing when people are making their own hormones (Endogenous hormones) There is no issue about the Endogenous Ranges (except that they vary from lab to lab, depending on methodology, and that is also true for many other types of tests) The Endogenous Ranges are clinically valid; they correlate to the clinical picture: Low E2 in saliva symptoms of low E2 High E2 in saliva breast tenderness, migraines Low noon, supper and bedtime cortisols symptoms of low cortisol High T and DHEAS symptoms of high androgens Month-Long Profiles E2 and Pg every 3 rd day (11 specimens) Testosterone on a specimen pooled from all 11 tubes $250 vs $176 (Bronze level provider) Useful for getting to the bottom of erratic bleeding Useful for women trying to conceive Don t use in postmenopause if not bleeding Don t use if supplementing E2 and Pg and T Please do all 11 tubes!! Copyright

6 Copyright

7 Mood swings Paranoid Impatient Intolerant Heavy mucus discharge First day of bleeding Cessation of menses c/o hot flashes during this time -Sky high progesterone also measured two years prior at another lab -General complaints of fatigue and foggy thinking Breast tenderness Water retention No bleeding Copyright

8 Estrogen dominant E2 fits with her BMI Anovulatory 49 year old with regular menses! Cycle length: days Month-Long Profiles Can yield insight into confusing presentations Progesterone isn t the solution to everything Progesterone isn t the solution to everything Average testosterone across the month enhances the utility of this profile Copyright

MetS and Saliva Hormone Testing Overweight female with irregular menses: Estradiol 15 pg/ml (1-9 pg/ml) : Hi Progesterone 22 pg/ml (50-250 pg/ml) : Low Testosterone 63 pg/ml (15-45 pg/ml) : Hi DHEAS

high insulin, pregnenolone is diverted to DHEA and progesterone is diverted to androstenedione.

9 MetS and Saliva Hormone Testing Overweight female with irregular menses: Estradiol 15 pg/ml (1-9 pg/ml) : Hi Progesterone 22 pg/ml ( pg/ml) : Low Testosterone 63 pg/ml (15-45 pg/ml) : Hi DHEAS 18 ng/ml (2-11 ng/ml) : Hi Saliva Hormone Testing and MetS High insulin upregulates several key enzymes in the steroidogenic pathways in the ovaries and adrenals Abnormal fatty acid metabolites -With high insulin, pregnenolone is diverted to DHEA and progesterone is diverted to androstenedione. -The net result is increased DHEA and androstenedione (leading to higher testosterone and estradiol) Tracking Metabolites: Pregnenolone 56 y.o. female on 150 mg/day pregnenolone Testosterone 204 pg/ml (15-45 pg/ml) Estradiol 13 pg/ml (1 9 pg/ml) Estradiol 13 pg/ml (1-9 pg/ml) DHEAS 15 ng/ml (2-11 ng/ml) Progesterone 48 pg/ml (20-50 pg/ml) Cortisol 8.2 ng/ml (3-10 ng/ml) Copyright

10 Tracking Metabolites: DHEA 56 y.o. female on 25 mg/day DHEA Testosterone 75 pg/ml (15-45 pg/ml) Estradiol 8 pg/ml (< 4 pg/ml) DHEAS 6 ng/ml (2-11 ng/ml) DHEAS level isn t high, but downstream metabolites of DHEA are increased Well documented in literature for serum: serum DHEAS does not always reflect supplementation with DHEA Dihydrotestosterone Dihydrotestosterone (DHT) is a direct metabolite of testosterone Active in many tissues but especially in skin, hair follicles Salivary DHT correlates to increased severity of complaints of facial hair growth and loss of scalp hair Sometimes helpful when testosterone is normal, yet patient c/o high androgen symptoms (DHT high in those cases) Salivary Estrogens and Breast Cancer Based on work by Rebecca Glaser Did saliva hormone tests on women newly diagnosed with breast cancer but not treated Women who have just been diagnosed with breast cancer have more estrogen in their saliva than disease-free women Dr. Glaser s published analysis indicates higher group mean E1 and E2 in cases compared to controls Glaser says premenopausal women with a new diagnosis of breast cancer have lower E3 compared to controls Copyright

11 The data are noisy! Group means for E3 are lower for premenopausal cases BMC Cancer 2010;10:547. Low salivary testosterone levels in patients with breast cancer. Dimitrakakis C, Zava D, Marinopoulos S et al. Salivary Estrogens and Breast Cancer Glaser says premenopausal women with a new diagnosis of breast cancer have lower E3 compared to controls We don t know if these women had elevated estrogens years prior to diagnosis Once the tumour is established, it may generate excess estrogens, as opposed to excess estrogens laying the foundation for a tumour Salivary Estrogens and Breast Cancer Bottom line: There are enough other lines of evidence building the case that excess antecedent estrogens (E1 and E2) probably are a risk factor for breast cancer Measurement of E1, E2 and E3 in women making their own hormones is likely worthwhile, in women at increased risk of breast cancer Copyright

12 What does a 24 hour urine hormone level measure? Urine mostly contains conjugated steroids Glucuronides, sulphates, mixed conjugates The conjugates are hydrolyzed before analysis The results are reported as estradiol, testosterone, etc, but we are really measuring conjugates e.g. estrone sulphate Urinary Estrogens % of Total Estradiol 5.3 Estrone 15.0 Estriol OH estrone 7.3 4OH estradiol 1.2 4OH estrone 3.1 2OH estradiol 3.6 2OH estrone methoxyestradiol 0.2 4methoxyestrone 0.5 2methoxyestradiol 0.7 2methoxyestrone 8.0 Xu X et al. J Clin Endocrinol Metab 1999;84: EQ: Estrogen Quotient EQ = E3/(E1 + E2) EQ = 11.1/( ) = 0.55 (Caucasian population) EQ doesn t look at the major form of estrogen in the urine Estrogen Quotient in Urine Old studies by Henry Lemon (1970 s) indicated that populations with a low incidence of breast cancer have a urinary EQ > 1 (lots of conjugated estriol in urine) Caucasians normally have a urinary EQ <1 and are at increased risk for breast cancer compared to other races The thinking has been that if the urinary excretion of estriol (E3) can be increased relative to E1 and E2 (make EQ >1), this may decrease the risk of breast cancer Can we port this logic over to salivary estrogens? Copyright

13 Saliva Urine E1 = 1.0 pg/ml E1 = 1770 pg/ml E2 = 1.0 pg/ml E2 = 1600 pg/ml E3 = 2.5 pg/ml E3 = 2000 pg/ml EQ = 1.25 EQ = 0.6 Normal, healthy Increased risk of breast postmenopausal cancer when EQ <1 women have an EQ >1 Normal risk of breast cancer You can t blindly substitute estrogen numbers from saliva into a formula which was developed for urine estrogens Serum Cortisol Day Curve X X X X Older Younger Salivary Cortisol Salivary cortisol is not perturbed by anticipatory stress associated with venipuncture Saliva samples can be given at home, and mailed directly to the laboratory. It is much easier to measure a day curve in saliva than in serum It is much easier to study cortisol in children/adolescents via saliva Copyright

Salivary Cortisol Day Curve X X X Psychosom Med 2003;65:836-841. Peeters F, Nicholson NA, Berkhof J.

The morning point is typically collected across the first hour after waking (before eating, toothbrushing, exercise) It takes many people that long to

14 Salivary Cortisol Day Curve X X X Psychosom Med 2003;65: Peeters F, Nicholson NA, Berkhof J. X End-stage adrenal fatigue Rhetorical question #1: Is there anything special about the morning cortisol point? The morning point is typically collected across the first hour after waking (before eating, toothbrushing, exercise) It takes many people that long to get a decent sample If you look closer cortisol is not static in that first hour after you open your eyes What happens if you take a sample as soon as your eyes pop open and every 15 minutes after that for the first hour? Copyright

Cortisol Awakening Response (CAR) 30 min after waking, we regain the same global cerebral blood flow we had the night before -prefrontal cortex up

the rest of the day Neurosci Biobehav Rev 2010;35:97-103. The cortisol awakening response: more than a measure of HPA axis function.

Non HC brain damage Normal controls HC damage Caregivers The CAR is regulated by the hippocampus Abolishment of the hippocampus (e.g. due to stroke) flattens the CAR -Buchanan TW et al.

15 Cortisol Awakening Response (CAR) 30 min after waking, we regain the same global cerebral blood flow we had the night before -prefrontal cortex up and running -full alertness -reduction in fatigue Cortisol Awakening Response (CAR) The cortisol awakening response is what the name implies: a physiological response to awakening. It is a discreet and distinct component of the cortisol circadian cycle, with characteristics unrelated to those of cortisol secretion throughout h t the rest of the day Neurosci Biobehav Rev 2010;35: The cortisol awakening response: more than a measure of HPA axis function. Clow A, Hucklebridge F, Stalder T, Evans P, Thorn L. Non HC brain damage Normal controls HC damage Caregivers The CAR is regulated by the hippocampus Abolishment of the hippocampus (e.g. due to stroke) flattens the CAR -Buchanan TW et al. Circadian regulation of cortisol after hippocampal damage in humans. Biol Psychiatry 2004;56: Hippocampal volume on MRI is associated with the magnitude and shape of the CAR: smaller HC volume = smaller and flatter CAR HC damage had no effect on the rest of the day curve The caregivers are as bad off as the patients! Copyright

16 Stuff you need to know about the Hippocampus Consolidation of information from short-term memory of events to long-term memory Attaches emotion to memories, attaches location to memories Stores spatial information: navigation, mental maps (London taxi drivers have bigger hippocampi) Involved with imagination Exclusive location of mineralocorticoid receptors (MR) MR is 6-10x more sensitive to cortisol than GR The role of the awakening surge may be to prime the hippocampus Salivary Cortisol Testing The first morning point is different! It is regulated differently than the rest of the cortisol profile It is much more sensitive to stress and the overall emotional landscape There is a sizeable mound of literature on the CAR ; we will likely be offering a panel looking at the CAR, depending on interest Accurate Cortisol Sampling If you are doing serial 4 point cortisol tests, take care to ensure that the morning point is collected in the same fashion each time! The patient must be sampled at the same time relative to waking, not at a set time, e.g. 8 AM Have the patient give the first sample across the first hour after waking If you don t take the variation within the first hour into account, you could see a large day-to-day variation in the first morning cortisol level. Copyright

17 Monitoring of Oral Cortisol Therapy via Salivary Cortisol Main issue is spurious results from direct contamination of the oral cavity from HC tablets This is less likely if use compounded HC capsules as opposed to manufactured HCA tablets Physiologic levels measured 60 mg pregnenolone 30 mg oral HCA 10 mg of oral HCA 3.5 mg of oral HCA at 8:00 PM 43.5 mg HCA Supraphysiologic level at 10:00 may be due to conversion of pregnenolone to cortisol Salivary Cortisol (ng/ml) Time (Hours) 04-Apr Feb mg pregnenolone 30 mg oral HCA 100 mg 7-keto DHEA 15 mg of oral HCA 5 mg of oral HCA 10 mg of oral SR HCA 50 mg IR HCA + 10 mg SR HCA Copyright

Hydrocortisone Skin Cream Hydrocortisone = Cortisol Hydrocortisone is in lots of topical products, and is available OTC as hydrocortisone acetate Use of hydrocortisone

A Typical Question The sex hormones peak around the age of 25. Is there a benefit in keeping the hormones at that levelthroughout someone s s life?

18 Hydrocortisone Skin Cream Hydrocortisone = Cortisol Hydrocortisone is in lots of topical products, and is available OTC as hydrocortisone acetate Use of hydrocortisone skin cream can easily send salivary cortisol levels through the roof If the patient has markedly high levels, but is not anxious, think HC skin cream! A Typical Question The sex hormones peak around the age of 25. Is there a benefit in keeping the hormones at that levelthroughout someone s s life? Any side effects if the hormones are balanced perfectly at age 25 levels? We would like hormone replacement to go like this: test, supplement, retest, adjust sliders i.e. dose based on test results Copyright

19 Salivary Hormone Levels: Two Cases 37 yr old female with regular menses: E2 = 5 pg/ml, Pg = 168 pg/ml, Pg/E2 = 34 (range midluteal) Salivary numbers would correlate to serum numbers in this case 55 yr old female on BiEst and Pg creams: E2 = 27 pg/ml and Pg = 3300 pg/ml, Pg/E2 = 122 What do these numbers mean? Do they mean that the pt is overdosed by a factor of 5 for E2 and by a factor of 20 or more for Pg? Patient feels fine!! Why?? Version I A portion of the hormone measured in saliva after topical hormone application may be getting g to the salivary glands by travelling over or across the body surface (via shallow subdermal lymphatics or through subcutaneous fat) This portion never sees the systemic circulation and doesn t reflect the clinical picture Why?? Version II Supposition is that after hormone skin cream use, hormones ride on the red blood cells, not in the serum Tissues have a unique propensity to extract hormone carried on red blood cells (after topical hormone use) Saliva is therefore a surrogate marker for deposition of this supplemented hormone in other tissues Copyright

20 Why the discrepancy between endogenous and on-cream levels of hormones in saliva? Bottom line: We don t really know what is going on from a theoretical standpoint We have to look at the available data to decide how to use these on-cream numbers How closely do On-Cream salivary hormone levels track symptoms? On cream levels track applied dose, but RMA database analysis indicates that they do not track the clinical picture very well On cream Salivary Hormone Levels after Cessation of Topical Therapy Loss of clinical effect e Level Salivary Hormone Return to baseline could take months The rate of fall-off is a function of the dose, the hormone affinity for fatty tissue, and the former site(s) of application Physiologic range Stop cream Time Days to months Copyright

21 Severity of Breast Tenderness vs On-cream Salivary Estradiol BreastTenderness Sx Score Avg Endogenous Range Salivary E2 after cream Salivary supplementation E2 (pg/ml) Symptom severity is not proportional to On-cream salivary hormone level ( a score of 3 indicates severe breast tenderness) Severity of Night Sweats vs On-cream Salivary Estradiol NightSweats x Score Avg Sx Endogenous Range Salivary E2 after cream Salivary supplementation E2 (pg/ml) Symptom severity is not proportional to On-cream salivary hormone level ( a score of 3 indicates severe night sweats) Severity of Excess Facial Hair vs On-cream Salivary Testosterone ExcessFacialBodyHair Sx Score Avg S 2Endogenous Range Salivary T after Salivary cream Testosterone supplementation (pg/ml) (pg/ml) Symptom severity is not proportional to On-cream salivary hormone level ( a score of 3 indicates excessive facial hair) Copyright

22 Severity of Oily Skin vs On-cream Salivary Testosterone OilySkin Sx Score Avg S 2 Endogenous 1.5 Range Salivary T after Salivary cream Testosterone supplementation (pg/ml) (pg/ml) Symptom severity is not proportional to On-cream salivary hormone level ( a score of 3 indicates excessively oily skin) Observed Range vs Normal Range This breakdown of clinical correlation leads us to call our On-cream ranges Observed Ranges instead of Normal Ranges All saliva testing labs do this. An Observed Range is what is commonly OBSERVED Observed ranges are not clinically validated targets Observed Range What we observe is a function of many different variables: Hormone in question Base used (e.g. Gel vs Lipoderm vs VanPen vs Cosmetic Base vs Petroleum Jelly vs Emu Oil) Site of application Sampling interval Duration of use Schedule i.e qd, bid Copyright

The measured level drops quite steeply between 8 and 12 hours after application of

com/download-document/42-observed-salivary-supplementation-ranges.

application tracks applied dose. http://www.zrtlab.

an Observed Range, it doesn t necessarily mean that is the right dose for the patient

23 The measured level drops quite steeply between 8 and 12 hours after application of cream Reproduced with permission Clearly, the salivary level after topical hormone application tracks applied dose. Reproduced with permission Observed Range vs Normal Range If a result is within an Observed Range, it doesn t necessarily mean that is the right dose for the patient (if supplementing with hormone cream/gel) The appropriateness of the dose has to be established primarily on clinical grounds Copyright

24 0 Rocky Mountain Analytical 4/21/2011 Effect of Application Site on Appearance of Hormone in Saliva GG applied 25 mg testosterone (Androgel) via spatula One application to a different body location every 2 weeks or so (salivary T washed out to baseline between experiments) Profiled testosterone in saliva for the next 24 hours or so after each application Application sites: dorsal foot, buttock, abdomen, medial forearm, supraclavicular area SALIVA Supraclavicular Huge elevation over baseline e (pg/ml) Tesosterone hours Same oscillations as those published by Lewis (saliva) and Mazer (serum) Level eventually stabilizes Time after application (minutes) [Pg] in saliva as a function of time since application of Pg cream SALIVA Lewis J, McGill H, Patton V, Elder P. Maturitas 2002;41:1-6. Copyright

25 0 0 Rocky Mountain Analytical 4/21/2011 SERUM Androderm patch Androgel Day 7 Day 7 Day 14 Day 14 Mazer N et al. J Sex Med 2005;2: SALIVA Supraclavicular Tesosterone e (pg/ml) Is this getting to saliva via blood or some other route? Time after application (minutes) Abdomen Tesostero one (pg/ml) Where is this coming from? hours after application Time after application (minutes) Copyright

26 0 Rocky Mountain Analytical 4/21/2011 Salivary T after application of Androgel to various body parts Tesosterone (pg/ml) The closer the application site is to the head, the higher the wave Time after application (minutes) mg Androgel to L foot 25 mg Androgel to supraclavicular area 25 mg Androgel to L forearm 25 mg Androgel to R Buttock 25 mg Androgel to Abdomen Peak Salivary T vs Distance from Application Site Supraclavicular Peak Salivary Testosterone (pg/ml) Forearm Abdomen and Buttock Foot Approximate distance from application site to saliva gland (inches) This implies that some component of what is seen in saliva gets there literally by travelling over the body surface Other RMA Experiments Made up 1 mg/ml Pg topicals in a variety of different bases Did same for 1 mg/ml Testosterone Same female applied one dose of T in selected base to skin Followed salivary T for 24 hours afterward Waited 2-3 weeks Repeated experiment with same person and same application site, but different base Repeated until all bases tested Did same series of experiments with a male, using 1 mg/ml Pg in same suite of bases Copyright

27 Copyright

28 So what does all this mean? We are still working on understanding salivary hormone levels after topical hormone application Don t get hung up on On-Cream reference ranges There are too many uncontrolled variables DO NOT OVERINTERPRET ON CREAM SALIVA HORMONE LEVELS!!! What does Over-Interpret Mean? Don t tinker with hormone doses to get the salivary hormone levels after supplementation into the ranges we provide you with They are not clinically validated ranges! We don t know the level of salivary Pg at which you can say OK, the endometrium is protected It will be different for each woman, each base, each site of application, each sampling interval, and each type of estrogen therapy What does Over-Interpret Mean? Don t tinker with dosing, trying to achieve a particular ratio: e.g. Pg/E2 This approach has never been scientifically validated Salivary progesterone measurements confirm transdermal absorption, but to our knowledge, there is no empirical association between progesterone dosage and salivary concentrations that can be used to treat menopausal symptoms. O Leary P et al. Clin Endocrinol 2000;53: Copyright

29 So am I saying we shouldn t measure hormone levels in saliva after we use topical hormones? No. Do it!!!! Understand the limitations and strengths of your tests Use each tool in your toolbox in appropriate fashion Salivary hormone levels after topical hormone use can be very helpful for troubleshooting Avg Pg Result vs Evening Pg Cream Dose bid dosing only y = x R 2 = Avg Pg (pg/ml) Evening Pg Dose (mg) The finding of a low/high level of a hormone despite an average dose of hormone might point to: Poor absorption/excessive absorption Non-compliance/over-compliance Problem with composition of topical (actual dose lower/higher than labelled) So am I saying we shouldn t measure hormone levels in saliva after we use topical hormones? No. Do it!!!! Some practitioners advocate doing a saliva Some practitioners advocate doing a saliva test in the off period, several days or a week after discontinuation of the topical hormone, to gauge accumulation of hormone in the body I think this has merit; the saliva level does track body burden of hormone Copyright

30 Do and Don ts If you are doing serial testing of a patient, PLEASE use the same sampling interval each time!!! Don t test a level until the person has been using the topical for at least 2 weeks (the levels increase with repeated usage) If you change dose, wait one or two cycles before retesting, to let the new dose equilibrate with tissues Take all the precautions to avoid contaminating the specimen How to avoid contaminating the saliva specimen If you wash your face and dry it in the morning, before giving the specimen Be very aware of the history of the towel used to dry your face!! Use a clean, unused towel Be aware that if you are putting hormone cream on your face and neck, you increase direct deposition of hormone into the saliva glands How to avoid contaminating the saliva specimen Be aware of the possibility of skin to skin transfer! Women getting testosterone from a person who is using testosterone cream Men getting estrogens from a person who is using estrogen cream (especially vaginally!) This could possibly be used in divorce proceedings! Copyright

31 Closing Thoughts Saliva is a great way to test hormones! More and more papers are being produced every year Understand the limitations of the tests and they will serve you well If in doubt, call us!! Copyright

Month-Long Hormone Assessment: What goes up must come down. Disclaimer. Aims and Objectives. George Gillson MD PhD

Month-Long Hormone Assessment: What goes up must come down George Gillson MD PhD 1 Disclaimer The information presented in this Webinar is the opinion of Dr. Gillson based on his research and experience.