The ZRT Laboratory Blog

Convenience of Collecting Dried Blood Spots at Home for Hormone Testing in the Laboratory

Fri, 03 Apr 2020 14:46:18 -0700

COVID-19 is Making Blood Draw Clinics a Scary Place to Be

In today’s scary world of rapidly spreading COVID-19 infections, most individuals are hesitant to have their health care provider, or worse, a stranger at a blood draw clinic, violate their mandated social space of 6 feet to allow them to stick a needle in their vein and draw blood for testing hormones. It is understandable why during the SARS-CoV-2 pandemic both the physician and staff would rather limit physical contact as much as possible with a potentially infectious COVID-19 patient, and vice versa. Some patients suffering from severe hormonal imbalances that affect their quality of life and ability to cope through these difficult times may just choose to suffer through it needlessly, because they consider the risk of social contact to have their blood drawn too great.

Solving the Problem of Blood Draws for Hormone Testing

There is a solution to this problem for both the health care provider and patient, and it’s something ZRT has been doing for the past 15 years. It’s collection of blood by the patient at home from a simple finger-prick. Kits containing a lancet and filter card along with directions are given to the patient at the doctor’s office or drop-shipped by order of the physician to the patient. At the patient’s convenience the finger is pricked with a small lancet, and blood drops are collected on a filter card and allowed to dry for at least 4 hours. Once dried, the filter card is then safely packaged at the patient’s home and from there sent by mail directly to ZRT Laboratory for testing. Special preservation on ice, as required for liquid blood serum or plasma, is not necessary for shipping since the hormones and other analytes in dried blood are stable for weeks at room temperature. Better yet, the drying of blood kills infectious pathogens like viruses and bacteria. That’s good news for everyone – the physician who doesn’t have to draw blood, the patient who doesn’t have to leave their home to have blood drawn, and the laboratory staff who process the dried blood spots.

Getting the Hormone Test Results to You and Your Doctor without Leaving Home

Once the requested tests are performed by ZRT Laboratory, the results are then provided to the practitioner and patient electronically. On average turnaround time once the sample reaches the laboratory takes about 2-4 days depending on the tests requested and day of arrival. Physician consultations regarding test results and recommendations for therapy can then be done over the phone. The patient never has to leave home through the entire process and physician-patient contact is limited, at least while we’re all hunkered down at home during the COVID-19 pandemic.

Finger-stick Dried Blood Spot Test Results Equivalent to Venipuncture Serum or Plasma

Many research studies have shown the advantages of dried blood over liquid blood for testing hormones (see references). Over the past 15 years ZRT has developed, validated, and put to the test through publications in the scientific literature and presentations at scientific conferences a broad array of tests commercialized for hormones (estrogens, progesterone, testosterone, cortisol, thyroid hormones, etc.) as well as essential elements and toxic heavy metals using the dried blood method. Hormones in capillary blood drawn from the finger and dried as blood spots have been shown to be quantitatively equivalent to levels seen in serum or plasma derived by venipuncture and the units of measurement and ranges are also near equivalent. This makes the transition to dried blood spot testing from serum/plasma testing simple as well as convenient for these difficult times.

To find out more about ZRT’s innovative, simple, and convenient DBS testing please visit https://www.zrtlab.com/sample-types/blood-spot/ or contact ZRT at 866.600.1636.

References

A comparison of blood spot vs. plasma analysis of gonadotropin and ovarian steroid hormone levels in reproductive-age women. Edelman A, Stouffer R, Zava DT, Jensen JT. Fertil Steril 2007; 88:1404-7.
Percutaneous progesterone delivery via cream or gel application in postmenopausal women: a randomized cross-over study of progesterone levels in serum, whole blood, saliva, and capillary blood." Du JY, Sanchez P, Kim L, Azen CG, Zava DT, Stanczyk FZ. Menopause 2013; 20:1169-1175.
Analysis of the Use of Dried Blood Spot Measurements in Disease Screening. Lakshmy R. J Diabetes Sci Technol 2008; 2: 242-243.
Cardiometabolic risk factors assessed by a finger stick dried blood spot method." Kapur S, Kapur S, Zava D. J Diabetes Sci Technol 2008; 2: 236-241.
A liquid chromatography/tandem mass spectrometry method for determination of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in dried blood spots: a potential adjunct to diabetes and cardiometabolic risk screening. Newman MS, Brandon TR, Groves MN, Gregory WL, Kapur S, Zava DT. J Diabetes Sci Technol 2009; 3:156-62.
Challenges of Blood Spot Heavy Metal Testing – Using Blood Spot Area to Correct for Abnormal Sample Spread when Sample Matrix is Unknown. Zava TT. 71st American Association for Clinical Chemistry Annual Scientific Meeting, Anaheim, CA, August 4-8 2019. See the poster presented.
Dried Urine and Blood Spot Analysis of Essential and Toxic Elements by ICP-DRC-MS with an Emphasis on Inter-Assay Stability of Samples Kept at Room Temperature. Zava TT. 69^thAmerican Association for Clinical Chemistry Annual Scientific Meeting & Clinical Lab Expo, San Diego, CA, July 30-August 3 2017. Abstract A402. See the poster presented.
Dried Urine and Blood Spot Analysis of Essential and Toxic Elements by ICP-DRC-MS with an Emphasis on Inter-Assay Stability of Samples Kept at Room Temperature. Zava TT. 69^thAmerican Association for Clinical Chemistry Annual Scientific Meeting & Clinical Lab Expo, San Diego, CA, July 30-August 3 2017. Abstract A402. See the poster presented.
Gonadotropin and steroid hormone testing by conventional serum venipuncture and finger-stick dried blood spot. Zava D, Kapur S, Kashi ZM. Endocrine Society Annual Meeting, Boston, MA, June 24-26 2006. See the poster presented.
Filter paper dried blood spot assay of insulin measurement. Zava D, Kapur S, Kashi ZM. American Diabetes Association 66th Scientific Sessions, Washington DC, June 9-13 2006; Poster 600-P. See the poster presented.
Gender differences in high-sensitivity C-reactive protein as determined by dried blood spot assay. Kapur S, Kapur S, Zava D. The Endocrine Society’s 89th Annual Meeting, Toronto, June 2-5, 2007. See the poster presented.
Metabolic syndrome wellness screening program using dried blood spot. Kapur S, Meyer W, Kapur S, Zava D. American Diabetes Association 67th Scientific Sessions, Chicago, IL, June 22-26, 2007; Abstract 2289-PO.
Filter paper-dried blood spot assay of hemoglobin A1c. Kapur S, Kapur S, Zava D. American Diabetes Association 67th Scientific Sessions, Chicago, IL, June 22-26, 2007; Abstract 2287-PO.
Dried blood spot screening of cardiometabolic risks. Kapur S, Kapur S, Zava D. American Academy of Clinical Cardiology Annual Meeting, San Diego, July 15-19, 2007.
Cardiometabolic health monitoring using dried blood spot. Kapur S, Kapur S, Zava D. J Diabetes Sci Technoland 7th Annual Diabetes Technology Meeting, San Francisco, October 25-26, 2007.
Quantitative analysis of 25-hydroxyvitamins D2 and D3 in dried bloodspot by liquid chromatography/tandem mass spectrometry. Newman MS, Zava DT, Brandon TR, Groves MN. Pediatric Academic Societies and Asian Society for Pediatric Research Joint Meeting, Honolulu, May 2-6, 2008. See the poster presented.
Dried blood spot screening for cardiometabolic risk markers shows benefit of exercise. Kapur S, Kapur S, Groves M, Zava D. American Diabetes Association 68th Scientific Sessions, San Francisco, June 6-10, 2008; Abstract 1046-P. See the poster presented.
Dried blood spot study shows improvement of cardiometabolic risk with higher testosterone levels in men. Kapur S, Kapur S, Norris W, Groves M, Zava D. Diabetes Science and Technology Meeting, Bethesda, Maryland, Nov. 13-15, 2008.
Cardiovascular risk screening using simple and convenient dried blood spot technology. Kapur S, Kapur S, Groves M. Preventive Cardiovascular Nurses Association 16th Annual Scientific Meeting,Chicago, IL, April 15-17, 2010. Abstract published in J Cardiovasc Nursing 2010; 25(5):369. See the abstract book. See the poster presented.
Hormone measures in finger-prick blood spot samples: new field methods for reproductive endocrinology. Worthman CM, Stallings JF. Am J Phys Anthropol 1997; 104:1-21.
Measurement of gonadal hormones in dried blood spots versus serum: verification of menstrual cycle phase. Shirtcliff EA, Reavis R, Overman WH, Granger DA. Horm Behav 2001; 39:258-66.
Assessing estradiol in biobehavioral studies using saliva and blood spots: simple radioimmunoassay protocols, reliability, and comparative validity. Shirtcliff EA, Granger DA, Schwartz EB, Curran MJ, Booth A, Overman WH. Horm Behav 2000; 38:137-47.
What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. McDade TW, Williams S, Snodgrass JJ. Demography. 2007;44:899-925.

Useful Resources

Provider Data Sheet: The Science of Dried Blood Spot Testing

Testing Thyroid Hormones

Testing Elements

Testing Hormonal Imbalances in Menopause

How to Collect Samples at Home

Topical Progesterone, Not Synthetic Progestins or Oral Progesterone, Should Be Considered as a Companion for Estradiol Replacement Therapy

Fri, 17 Jan 2020 14:44:48 -0800

In 2002 several large-scale clinical studies were published on the risks of breast cancer in postmenopausal women using conventional FDA-approved hormone therapy. These were the Women’s Health Initiative (WHI) and Million Women’s studies of women using FDA-approved estrogens and progestogens in the United States and Great Britain, respectively [1,2]. Both studies came to the same conclusion – that estrogen therapy, mostly in the form of oral conjugated equine estrogens, by itself did not significantly increase the risk of breast cancer and, to the surprise of many, was associated with a lower risk. However, when estrogen was combined with a synthetic progestin to prevent uterine cancer, the breast cancer risk increased 1.5 to 2-fold. Virtually all forms, of which there are many, of synthetic progestins increased risk to about the same extent. Smaller studies suggested that FDA-approved oral progesterone, which was not as widely used, did not increase risk in combination with estrogen therapy. These results led to widescale panic among postmenopausal women using conventional estrogen and progestogen (both synthetic progestins and natural progesterone) therapies and a precipitous drop in prescriptions for these forms of HRT [1]. Many women stopped cold turkey all forms of hormone replacement therapy (HRT), which significantly diminished their quality of life. Adverse estrogen deficiency symptoms that were effectively suppressed with estrogen therapy (e.g., hot flashes and night sweats, sleep disturbances, memory issues, incontinence, vaginal dryness, depression, weight gain, etc.) resurfaced with a vengeance in many women, as did risk for diseases of advanced aging (e.g., bone loss and osteoporosis, cardiovascular disease, stroke, diabetes, senile dementia, Alzheimer’s disease). Women were left frightened between “damned if you do and damned if you don’t,” and a large majority chose not to continue HRT. Many health care providers refused to prescribe hormone therapy until more information about risks were made available through clinical trials.

Natural Progesterone Reduces Breast Cancer Risk

It was clear to me from the research literature and smaller clinical studies that the natural hormone progesterone, when delivered topically at a physiological dose (25 mg) protects normal breast tissue from the growth promoting actions of estrogens.

When these and other studies emerged in 2002 John Lee, MD, and I along with Virginia Hopkins had just published our book entitled, “What Your Doctor May Not Tell You About Breast Cancer: How Hormone Balance Can Help Save Your Life” [3]. The short of the book is that if your hormones are out of balance and you are suffering from symptoms and conditions of advancing age, meaning the transition into menopause and beyond, then you should replenish your hormones back to physiological levels with the same hormones that your body made when you were healthy early middle age. This meant if you were to use estrogen replacement therapy (ERT) you would replace with estradiol and/or estriol, not a synthetic estrogen like ethinyl estradiol (synthetic estrogen found in birth control pills) or a conjugated horse estrogen (Premarin). And if you were taking a progestogen to help balance the estrogen and protect against its proliferative effects, use bioidentical progesterone, not a synthetic progestin like medroxyprogesterone acetate (Provera) or other synthetic “fake” progesterone. We already knew from earlier studies published several years before WHI that the synthetic progestins were increasing the incidence of breast cancer [3, Chapter 11]. As a research scientist at that time, who had spent 20+ years researching and publishing on the role of estrogen and progestogen binding to their receptors and regulating breast cell proliferation and differentiation, it was clear to me from the research literature and smaller clinical studies that the natural hormone progesterone, when delivered topically at a physiological dose (25 mg) protects normal breast tissue from the growth promoting actions of estrogens [4]. In the breast cancer book [3] we delved into a lot more of what the scientific literature says causes breast cancer and what can be done to help prevent it, like avoid bad foods and environmental chemicals, make sure you’re eating good nutritious foods with plenty of colored vegetables, exercise in moderation, get adequate sleep in the dark, reduce stress as much as possible to lower cortisol-induced estrogen, and take nutritional supplements that bolster the army of antioxidants that protect against environmental toxins that convert good estrogens to bad ones [5,6]. And if your hormones are out of balance, based on testing their levels in body fluids that represent the bioavailable fraction that enters cells, use physiological amounts of bioidentical hormones to adjust them to levels when you were younger and healthy. What has been crystal clear is that bioidentical hormone therapy, if used correctly and in physiological amounts, will significantly reduce risk but is no guarantee you will never develop breast cancer.

Synthetic Progestins Raise Breast Cancer Risk

Fast forward about 20 years to present; several more recent meta-analyses of the risk of breast cancer with FDA-approved, or equivalent for non-US countries, estrogen and progestogen therapies, shed some new light on risk of HRT for breast cancer. These studies (US, British) mostly just reiterated what we already knew: that estrogen therapy only increases breast cancer risk slightly, but significantly when combined with synthetic progestins [1]. The meta-analysis of prospective studies of slightly over 100,000 women using estrogen alone or estrogen combined with progestogens (mostly synthetic), but also oral progesterone [1], revealed that estrogen therapy alone was only associated with a slight increase in risk, but when combined with a synthetic progestin the risk was approximately doubled after 5 years and doubled again at 10 years of use. No question, synthetic progestins were bad for the breasts and should not be used, especially by women who want to reap the many benefits of hormone therapies their entire lives, who should avoid synthetic progestins. Most surprising was that this study also reported that FDA-approved oral bioidentical progesterone, combined with estrogen, carried the same approximately 2-fold higher risk at 5-10 years, but strangely had no increased risk less or greater than that [1]. No data were available for topical progesterone combined with estrogens, perhaps because very few, if any, clinical studies have been carried out in the US or Great Britain and surrounding European countries using natural progesterone delivered topically.

Topical versus Oral Progesterone

Oral progesterone, while it protects the endometrial lining, may not raise progesterone to a level high enough to counter the growth-promoting actions of estrogens in the breast tissue.

Despite research and small clinical studies showing that natural progesterone delivered topically directly to the breasts of humans [4] and primates protects against estrogen-stimulated cell proliferation of normal mammary epithelial cells, a risk factor for breast cancer, surprisingly no large prospective randomized clinical studies have been carried out to investigate if topical progesterone therapy is associated with reduced risk for developing breast cancer. The reason for this is not based on science, but economics. Progesterone cannot be patented; however, progesterone combined with a special delivery system can be, as seen with several forms of FDA-approved oral progesterone (Prometrium and Bijuva). Oral progesterone, however, while it protects the endometrial lining, may not raise progesterone to a level high enough to counter the growth-promoting actions of estrogens in the breast tissue [7].

Factors Influencing Estrogen Metabolism to Mitigate Risk

What is clear is that anything that reduces excessive estrogen burden, be it progesterone, natural or synthetic aromatase inhibitors, diet with more fiber and phytochemicals with colored vegetables, exercise, stress reduction, better sleep habits, etc., translates to lower breast cancer risk. That is what progesterone does. It lowers cell proliferation by down-regulating estrogen receptors (ER), preventing further estrogen-mediated stimulation of cell proliferation and redirects differentiation through progesterone interaction with estrogen-regulated cellular progesterone receptors (PR). Progesterone, via its activation of 17β-hydroxysteroid dehydrogenase type 2 (see diagram below), also increases the conversion of estradiol, a potent estrogen with high affinity for cellular ERs, to estrone, a weak inert estrogen [3]. Progesterone also enhances synthesis of sulfotransferase, an enzyme that then sulfates estrone to estrone sulfate, which is unable to enter cells and serves as a circulating estrogen precursor that must be converted back through two enzymatic steps, sulfatase and 17β-hydroxysteroid dehydrogenase type 1, to estradiol.

Diagram depicts the progesterone-regulated metabolism of E2 to E1 and then to E1-SO4, and then Cyp1A1 and Cyp1B1 conversion, respectively, to 2- and 4-hydroxy estrogens and their further oxidation to 2- and 4-quinones. For details see reference [5].

Progesterone’s Effects on Estrogen-Stimulated Cell Proliferation

What is poorly understood in the scientific/clinical community is that progesterone has dual actions in its effects on estrogen-stimulated cell proliferation. At the lower luteal levels (about 1-10 ng/mL) progesterone synergizes with estrogen to promote cell proliferation with little differentiation. If insufficient progesterone is produced in the presence of high estradiol, as often occurs at perimenopause with compromised luteal function, proliferation may be higher than with estradiol alone [7]. However, at higher luteal levels (10-30 ng/mL) progesterone counters the growth-promoting actions of estrogen by down-regulating ER, and through induction of cellular PR redirects the cellular machinery to drive quiescence and differentiation, causing proliferation to come to a halt. If inadequate progesterone is present, as often happens at the transition to menopause (perimenopause) when the corpus luteum fails to produce adequate progesterone but abundant estrogen (low progesterone/estradiol ratio), then the combination of higher physiological estrogen and lower luteal progesterone (in range, but low-normal) will often result in persistent proliferation and the clinical manifestation of fibrocystic and painful breasts. Excessive proliferation in the absence of progesterone increases risk for gene mutations that have the potential to lead to increased breast cancer risk.

Risks Greatest During the Menopausal Transition

Retrospective clinical studies have shown that the higher the luteal progesterone level 5 years prior to breast cancer diagnosis, the lower the risk of developing breast cancer.

As shown in the diagram above, estrogens in excess, in the absence of progesterone and with exposure to environmental toxins, induce the cytochrome enzyme Cyp1B1 that converts beneficial estradiol to potentially toxic and mutagenic 4-catechol estradiol [5,6]. During this perimenopausal transition is when the rate of increase of breast cancer is greatest. Retrospective clinical studies have shown that the higher the luteal progesterone level 5 years prior to breast cancer diagnosis, the lower the risk of developing breast cancer [8], so it is important to keep progesterone balanced with estradiol, especially during the menopause transition when estrogen metabolite damage is most likely to occur, as shown in a recent study on estrogen metabolite formation and breast cancer risk [6].

Topical Progesterone Achieves Optimal Tissue Levels

As mentioned, oral progesterone, because it is mostly degraded (90-95%) in the GI tract and liver to metabolites with no capacity to bind and activate PR, may not reach sustained luteal levels to counter the growth-promoting actions of estrogens. Based on saliva, capillary blood, and tissue levels of progesterone, only topically applied progesterone [9] can achieve capillary blood and tissue levels of progesterone high enough to counter the proliferative actions of estrogens. While small clinical studies have shown that topical progesterone used at physiological dosing (25-50 mg) effectively reduces estrogen-induced proliferation of the mammary epithelium in humans [4], this form of therapy has not been tested in a large scale prospective case-control randomized study as with FDA-approved conjugated equine estrogens in combination with synthetic progestins [1,2].

What might we learn from what we know about the dual actions of progesterone as it relates to bioidentical estrogen and progesterone therapies for menopausal women and their risk for breast cancer? If we let mother nature be our guide, then we should strive to keep estrogen within the physiological ranges seen throughout a full monthly cycle (about 50-150 pg/mL in serum or about 2% of that seen in saliva, 1-3 pg/mL). For at least half of the month serum or capillary blood (Dried Blood Spot, DBS) progesterone should be about 10-30 ng/mL and saliva 200-600 pg/mL. This results in a progesterone/estradiol ratio of about 100-300, which is consistent with the ratio seen at the peak of the luteal phase and shown to be protective of the breasts exposed to estradiol [4]. Excessive estradiol or too little progesterone will result eventually in estrogen dominance and symptoms of estrogen excess (e.g., weight gain in the hips and thighs, fibrocystic and painful breasts, mood swings, etc.) Those are warning signs. High levels of 4-catechol estrogens ratchet that up to extreme warning [5] as we have found that 4-catechol estrogens are much higher in women harboring breast cancers than healthy women [6].

Many providers believe that topical progesterone is ineffective because it doesn’t raise serum levels. To the contrary, we [9] and others [4,10] have shown that physiological dosing with topically delivered progesterone raises the progesterone to physiological levels in tissues of the body such as the salivary gland, and capillary beds of the finger tips. High dose progesterone (> 50 mg), in an attempt to achieve physiological concentrations in serum never does this, regardless of the topical progesterone dose [9]. What may seem paradoxical based on serum levels is that excessive progesterone and too little estrogen will eventually keep ER persistently down-regulated and may precipitate symptoms of estrogen deficiency with weight gain, vasomotor symptoms, excessive sleepiness, etc. Keep in mind that estradiol and progesterone are produced in a rhythmic pattern each month. Estradiol rises slowly throughout the first half of the cycle and peaks about midway with ovulation. This stimulates growth and proliferation of the uterus and breasts. Progesterone will not work if there is no cellular PR, which requires adequate ER. Excessive progesterone down-regulates ER, stopping the cycle.

In summary, maintaining estradiol levels in balance with natural progesterone is necessary to achieve optimal clinical benefits of these hormones. It is the opinion of this author that these goals for optimal progesterone therapy as a breast cancer preventive can best be achieved with topical progesterone, and not oral progesterone, or a synthetic progestin. However, clinical studies with topical progesterone are needed to confirm this hypothesis.

While you go through the stages of menopause, it is important to get your hormone levels checked to ensure that you are within physiological levels. If you are going into, in the middle of, or at the end of your menopause journey, ZRT's saliva and blood spot testing can give you the information necessary to get your hormones balanced and get you back to optimal health.

This article was first published in the American Academy of Anti-Aging Medicine (A4M) Winter 2019 Issue.

Related Resources

References

[1] Collaborative Group on Hormonal Factors in Breast Cancer. Type and timing of menopausal hormone therapy and breast cancer risk: individual participant meta-analysis of the worldwide epidemiological evidence. Lancet. 2019;394:1159-68.

[2] Manson JE, WHI Investigators, et al. Menopausal Estrogen-Alone Therapy and Health Outcomes in Women With and Without Bilateral Oophorectomy: A Randomized Trial. Ann Intern Med. 2019;171:406-414.

[3] Lee JR, Zava D, Hopkins V. What your doctor may not tell you about breast cancer – how hormone balance can help save your life. Warner Books, 2002.

[4] Chang KJ, et al. Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil Steril. 1995;63:785-91.

[5] Cavalieri EL, et al. Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc Natl Acad Sci USA. 1997;94:10937-42.

[6] Miao S, et al. 4-Hydroxy estrogen metabolite, causing genomic instability by attenuating the function of spindle-assembly checkpoint, can serve as a biomarker for breast cancer. Am J Transl Res. 2019;11:4992-5007.

[7] Sitruk-Ware LR, et al. Inadequate corpus luteum function in women with benign breast diseases. J Clin Endocrinol Metab. 1977;44:771-4.

[8] Micheli A, et al. Endogenous sex hormones and subsequent breast cancer in premenopausal women. Int J Cancer. 2004;112:312-8.

[9] Du JY et.al. Percutaneous progesterone delivery via cream or gel application in postmenopausal women: a randomized cross-over study of progesterone levels in serum, whole blood, saliva, and capillary blood. Menopause 2013;20:1169-75.

[10] Leonetti HB, et al. Topical progesterone cream has an antiproliferative effect on estrogen-stimulated endometrium. Fertil Steril. 2003;79:221-2.

Topical Delivery of Sex Steroid Hormones and Distribution in Different Body Fluids

Thu, 12 Sep 2019 12:50:16 -0700

Delivery of the bioidentical hormones estradiol (E2), progesterone (Pg), and testosterone (T) through the skin as a cream or gel (topically) has become a mainstay of bioidentical hormone replacement therapy (BHRT) for women and men. A plethora of FDA-approved pharmaceuticals, compounded, and even over-the-counter topical BHRT products are available. The popularity of these products lies not only in their ease of use, but in proven clinical efficacy [1-5] in treating hormonal deficiencies mostly brought on by aging of the female (menopause) and male (andropause) reproductive systems.

Dosing for Optimal Clinical Benefit

Over the past 30 or so years, higher FDA-approved topical BHRT dosing has dominated allopathic acceptance. However, more practitioners are opening their thinking to alternative views of much lower dosing as being optimal, based on clinical observations coupled with scientific discovery. It also is becoming more evident that while serum and urine testing work fine for detecting endogenously produced hormones, or hormones supplemented by nearly all other forms of delivery (e.g., transdermal patch, oral, vaginal, troche/sublingual, injections and pellets) these body fluids grossly underestimate hormone delivery to capillary beds and hence, tissues, relative to topical delivery [6]. Attempts to achieve physiological levels of E2, Pg, and T in serum with topical BHRT has met with little success, resulting in use of pharmacological dosing to raise serum levels to physiological ranges [6,7].

Despite the clear clinical efficacy of topical natural estrogens (mostly E2 and estriol -E3) and Pg in women and T in men, controversy still encircles the dose needed to achieve optimal clinical benefit. This controversy has arisen out of attempts to explain why topical BHRT results in very little increase in venipuncture serum or urine levels of the hormone delivered through the skin [6-8] despite use of topical hormone doses that usually are more than 10x higher than daily endogenous production of these hormones in women and men (see Table 1).

How Does Testing in the Wrong Body Fluid Lead to Overdosing?

Justification for higher dosing of both FDA-approved pharmaceutical and compounded products comes from pharmacokinetic studies showing that pharmacological dosing is necessary to increase serum and urine levels of the dosed hormones to physiological levels. But even with supra-high pharmacological BHRT dosing serum and urine levels of some of the hormones rarely increase to even within low physiological ranges. This is most notable for Pg [6,7,9], and less so for E2 and T [5,10,11] which only increase to physiological levels in serum/plasma and urine with pharmacological dosing.

Daily ovarian production of E2 during peak luteal phase of the menstrual cycle is about 100-300 µg [12] when serum and capillary blood levels of E2 are quantitatively equivalent at about 80-200 pg/mL, saliva ranges from about 1.5-4 pg/mL (approximately 2% of serum/plasma levels), and urine levels range from about 0.8-2.0 µg/g Cr (see Tables 2-4). For postmenopausal women to achieve physiological ranges seen in mid-luteal phase of premenopausal women, topical E2 therapy only requires physiological dosing to achieve physiological levels in saliva and capillary whole blood (about 100-300 µg E2), but at least 10x higher dosing (1000-3000 µg = 1-3 mg) to achieve physiological levels in venipuncture serum/plasma and urine (Table 2).

A similar distribution of Pg is seen during peak of the luteal phase of the menstrual cycle where daily Pg production by the ovaries is about 10-40 mg, and the Pg in serum and capillary blood are quantitatively equivalent at about 10-30 ng/mL, saliva about 100-300 pg/mL (approximately 1% of serum/plasma levels), and urine pregnanediol, a surrogate for Pg, about 500-2000 µg/g Cr (Table 3). To achieve premenopausal luteal ranges in postmenopausal women with topical Pg therapy only requires physiological dosing when measuring saliva and capillary whole blood (about 10-50 mg topical Pg) [6,7], but at least 10x higher dosing (100-300 mg topical Pg) when measuring venipuncture serum/plasma. Urine pregnanediol increases very little with increasing doses of Pg [8].

In men, topical T distributes into the same body fluids as topical E2 and Pg in women. In healthy young males, the testes produce about 5-7 mg of T daily and the morning T levels range from about 500-1200 ng/dL in serum/plasma and capillary blood, 100-250 pg/mL in saliva, and 1-4 µg/g Cr in urine (Table 4). To achieve physiological ranges of a healthy young male in the body fluids of hypogonadal men with topical T therapy only requires physiological dosing for saliva and capillary whole blood (about 5-10 mg topical T), but at least 10x higher dosing (50-200 mg topical T) for venipuncture serum/plasma or urine levels.

The lack of increase in serum and urine levels of topically delivered hormones at physiological dosing (50-200 µg E2 and 10-30 mg Pg in women and 5-10 mg T in men) (Tables 2-4) has resulted in justification of dose escalation of FDA-approved and compounded products to amounts that are 10 to 20-fold higher (500-5000 µg E2 and 100-300 mg Pg in women and 50-200 mg T in men) than levels of these hormones that are produced by the gonads of healthy young women and men over a 24-hour period (Table 1). Justification for these 10x pharmacological topical doses is based more on serum and urine levels than clinical response. Only a handful of studies have evaluated the clinical efficacy of lower physiological topical hormone dosing relative to the more allopathic acceptance of pharmacological dosing. However, some studies have clearly shown that more physiological dosing with topical E2 and progesterone are clinically effective for increasing bone mass [3] and protecting the breasts [13] and endometrium [4] at physiological dosing of E2 and Pg applied topically.

Saliva and Capillary Whole Blood Testing More Closely Reflect Tissue Levels

The advent of monitoring steroid hormones in body fluids that more closely reflect tissue levels, namely saliva and capillary whole blood derived from the fingertip (Dried Blood Spot, DBS), has serendipitously shed light on why such high pharmacological dosing is required to achieve even suboptimal physiological levels of hormones in serum and urine. Studies over the past 20 years have revealed that while salivary and capillary blood levels of endogenously-produced steroids quantitatively parallel levels of parent hormones (E2, Pg, T) seen in serum and their metabolites in urine, these equivalencies no longer hold when these steroid hormones are delivered percutaneously as gels or creams. Instead, salivary and DBS levels rise in a more linear fashion with dosing and achieve physiological levels in these body fluids at 10x lower physiological dosing (Tables 2-4); in stark contrast serum and urine levels increase very little at physiological doses.

This has raised the question of why salivary and DBS levels, which are equivalent to free serum levels with endogenously produced hormones, are so much higher (10-100x) than levels seen in serum and urine when hormones are delivered topically. Which one of these categories of body fluids (serum and urine vs. saliva and capillary whole blood) more closely reflect tissue levels of the hormone and the optimal dose that is needed for optimal physiological response?

As a lab that tests all four of these body fluids and is involved in many IRB-approved clinical research projects related to topical BHRT, we have had the opportunity to evaluate levels of endogenous steroids in women and men at different stages of life and supplementing with different hormone delivery systems (oral, occluded patch, topical, vaginal, injections and pellets, troche, etc.) and doses at different time points following hormone application. From these tests and evaluation of symptom relief, we have come to the conclusion that the bioavailable fractions of topically delivered hormones are not accurately quantified by testing serum or urine and that saliva and DBS levels of the topically delivered steroids more closely reflect tissue levels of E2 and Pg in women, and T in men.

Tables 2 and 3 show the median levels of E2 and Pg in saliva, DBS, serum, and urine of postmenopausal women before and after increasing doses of topical E2 and Pg therapies. Table 4 shows the median levels of T in these same body fluids in men over the age of 40 before and after increasing doses of topical T.

Insufficient information was available from our database for serum E2 and Pg (women) and T (men) with different topical doses of these hormones as we test much fewer serum and urine samples than saliva and DBS,; therefore, we used reported median values from several well designed clinical studies that investigated different topical doses and reported serum/plasma levels of each hormone category [1,3,14].

As seen in Tables 2 and 3, physiological luteal levels of E2 and Pg are seen in saliva and capillary blood with physiological topical dosing of E2 (100-300 µg) and Pg (10-30 mg). E2 and Pg in saliva and DBS continue to increase in a somewhat linear fashion with increasing doses. In sharp contrast, the data clearly show that to achieve luteal premenopausal serum levels of estradiol (range 80-200 pg/mL) requires pharmacological dosing (1000-3000 µg) with topical E2, as reported by others [3,14]. Further, physiological luteal levels of Pg in serum and urine are not achieved even with pharmacological (100-300 mg) topical Pg dosing, as reported by others [7]. Dosing at this pharmacological range results in salivary and capillary blood levels of E2 and Pg that are 10-20x higher than physiological range.

As shown in Table 4, topical T in men follows the same pattern as seen with topical E2 and Pg in women in that the physiological levels of T found in young healthy males (Table 1) are seen in the saliva and capillary blood of older hypogonadal men (> 40 y/o) with topical T dosing in a physiological range (5-10 mg). Very little increase in serum and urine T occurs at a physiological topical dose. At least 10-20x higher dosing (50-200 mg) is required to achieve even mid-physiological levels, as reported by others [5]. Dosing at this pharmacological range results in salivary and capillary blood levels of T 10-20x higher than physiological range.

Results from Tables 2-4 clearly show that pharmacological topical dosing with E2 (1000-3000 µg) and Pg (100-300 mg) in women, and T (50-200 mg) in men are required to achieve near physiological mid-luteal levels of serum E2 seen in premenopausal women, and T in healthy young men. In sharp contrast, much lower physiological topical dosing (10-20x lower than pharmacological) is required to achieve optimal physiological levels of these hormones based on saliva and capillary blood levels of E2 and Pg in women, and T in men.

Accurate Testing Leads to Better Dosing Decisions

These data suggest that at least for topical E2 and Pg in women, and topical T in men, serum and urine underestimate by at least 10-fold the amount of topically delivered hormone distributed to several tissues of the body (salivary gland and capillary beds of the finger tip). Similar results have been seen in tissues such as breast biopsies following physiological dosing with topical estradiol (1 mg) and progesterone (25 mg) [13] where mammary tissue levels of hormones increased 100-fold and Pg suppressed E2-stimulated mammary cell proliferation, but serum levels of E2 and Pg showed no significant change from patients treated only with placebo topical cream. These doses of E2 and Pg significantly raise salivary and DBS levels of E2 and Pg to physiological levels but have little impact on serum or urine levels of these hormones. Similar results have been reported for bone response to physiological (250 µg) levels of E2 [3] and Pg (30 mg) to protect the uterus from overstimulation by E2 [4].

Our data show that serum and urine are not appropriate body fluids to monitor topical hormone therapy but are fine for measuring endogenous hormones, or BHRT delivered by other methods.

Saliva or DBS testing provide a more accurate picture of topical doses needed to achieve physiological levels of these hormones in tissues, thus avoiding the potential for overdosing. Continued use of serum or urine for establishing dosing regimens of topically delivered hormones could potentially lead to excessive tissue exposure, suboptimal response, and eventual symptoms/conditions of hormone excess as well as pseudo deficiency caused by tachyphylaxis (down-regulation of tissue receptors and lack of response due to excessive prolonged exposure).

More research is needed to clarify these issues regarding the optimal topical hormone dose necessary to achieve optimal physiological levels of and clinical response to BHRT.

Related Resources

References

[1] Basdevant A and de Lignieres B. Treatment of menopause by topical administration of oestradiol. In: Percutaneous Absorption of Steroids, Mauvais-Jarvis P, Vickers, CFH., and Wepierre J, Eds., Academic Press (London), 1980; pp. 249-258.

[2] Egras AM, Umland EM. The role of transdermal estrogen sprays and estradiol topical emulsion in the management of menopause-associated vasomotor symptoms. Int J Gen Med. 2010;3:147-51.

[3] Prestwood KM, Kenny AM, Kleppinger A, Kulldorff M. Ultralow-dose micronized 17beta-estradiol and bone density and bone metabolism in older women: a randomized controlled trial. JAMA. 2003;290:1042-8.

[4] Leonetti HB, Wilson KJ, Anasti JN. Topical progesterone cream has an antiproliferative effect on estrogen-stimulated endometrium. Fertil Steril. 2003;79:221-2.

[5] Bhasin S, Woodhouse L, Casaburi R, Singh AB, Mac RP, Lee M, Yarasheski KE, Sinha-Hikim I, Dzekov C, Dzekov J, Magliano L, Storer TW. Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on the skeletal muscle. J Clin Endocrinol Metab. 2005;90:678-88.

[6] Zava DT, Groves MN, Stanczyk FZ. Percutaneous absorption of progesterone. Maturitas. 2014;77:91-2.

[7] Du JY, Sanchez P, Kim L, Azen CG, Zava DT, Stanczyk FZ. Percutaneous progesterone delivery via cream or gel application in postmenopausal women: a randomized cross-over study of progesterone levels in serum, whole blood, saliva, and capillary blood. Menopause. 2013;20:1169-75.

[8] O’Leary P, Feddema P, Chan K, Taranto M, Smith M, Evans S. Salivary, but not serum or urinary, levels of progesterone are elevated after topical application of progesterone cream to pre- and post-menopausal women. Clin Endocrinol 2000;53:615-620.

[9] Burry KA, Patton PE, Hermsmeyer K. Percutaneous absorption of progesterone in postmenopausal women treated with transdermal estrogen. Am J Obstet Gynecol. 1999;
180:1504-11.

[10] Place VA, Powers M, Darley PE, Schenkel L, Good WR. A double-blind comparative study of Estraderm and Premarin in the amelioration of postmenopausal symptoms. Am J Obstet Gynecol.1985;152:1092-9.

[11] Powers MS, Schenkel L, Darley PE, Good WR, Balestra JC, Place VA. Pharmacokinetics and pharmacodynamics of transdermal dosage forms of 17 beta-estradiol: comparison with conventional oral estrogens used for hormone replacement. Am J Obstet Gynecol. 1985;152:1099-106.

[12] Williams Textbook of Endocrinology, Larsen PR, Kronenberg HM, Melmed S, and Polonsky KS, Eds., 10th Ed., Elsevier Health Services (Saunders), Philadelphia, 2003.

[13] Chang KU, Lee TT, Linares-Cruz G, Fournier S, de Lignieres B. Influence of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil Steril 1995;63:785-91.

[14] Brennan JJ, Lu Z, Whitman M, Stafiniak P, van der Hoop RG. Serum concentrations of 17beta-estradiol and estrone after multiple-dose administration of percutaneous estradiol gel in symptomatic menopausal women. Ther Drug Monit. 2001;23:134-8.

[15] Padwick ML, Endacott J, Whitehead MI. Efficacy, acceptability, and metabolic effects of transdermal estradiol in the management of postmenopausal women. Am J Obstet Gynecol. 1985;152:1085-91.

Monoamine Metabolites – An Essential Factor In Understanding Neurotransmitters

Fri, 13 Oct 2017 10:52:00 -0700

After an extensive and careful review of test results over the past year, the scientists and clinical consultants at ZRT have concluded that the monoamine metabolites ZRT includes as part of its panel of 14 neurotransmitters are essential for the best and most comprehensive interpretation of test results.

We've determined that while looking at a more limited range of seven to nine neurotransmitters is helpful in assessing precursor availability, interpreting results based on those levels alone without their downstream metabolites can result in undertreatment, treatment of the wrong part of the system, or overtreatment with direct precursors.

It is only in looking at a complete set of parent neurotransmitters together with their metabolites that you glean the most precise information about systemic patterns – leading to a provider's ability to develop the most effective treatment plan.

Following are several examples that have led us to these conclusions.

Serotonin Assessment Incomplete without 5-HIAA

It is only in looking at a complete set of parent neurotransmitters together with their metabolites that you glean the most precise information about systemic patterns.

When serotonin is low and its downstream metabolite 5-HIAA is normal or high, this usually indicates high activity of monoamine oxidase A (MAO-A), an enzyme that requires copper and vitamin B2. MAO-A, the gene of which resides on the X chromosome, is sensitive to estrogens. Higher estrogen inhibits MAO-A, allowing more serotonin in the synapse, and a happier you. When estrogen drops at menopause MAO-A activity is increased, resulting in a higher rate of serotonin conversion to its inert metabolite 5-HIAA, and an associated increase in low estrogen symptoms such as hot flashes, night sweats, and depression – an unhappy you. This is why drugs that treat serotonin deficiency, such as SSRIs, are often successfully used for treating symptoms of menopause like hot flashes and depression. Thus, knowledge of the levels of serotonin relative to 5-HIAA can be very valuable for postmenopausal women who suffer from symptoms of estrogen deficiency that are related to low serotonin.

The opposite (high serotonin and low 5-HIAA) can be observed in women who have higher estrogens; however, a high urinary serotonin may also be associated with just simply eating a handful of walnuts (high serotonin content [1]), or taking 5-hydroxytryptophan (5-HTP), which is readily available OTC. Combining 5-HIAA with serotonin provides a much clearer picture of what’s happening to serotonin and determines if, clinically, the problem is real (low estrogens) or an artefact of the patient’s diet or supplement regime.

Dopamine Results Only Actionable with DOPAC & HVA

The downstream metabolites of dopamine, DOPAC and HVA, provide better insight into interpretation of dopamine results. Many of you have probably heard about the “warrior gene” and its relationship to the reward/pleasure neurotransmitter dopamine [2]. When you have an abundance of dopamine you feel on top of the world; however, as we know with most things, too much of anything isn’t good for you – and too much dopamine makes for an aggressive personality and increased likelihood you’re going to end up in trouble [3].

The warrior gene results from a single nucleotide polymorphism in the MAOA gene, which sits on the X chromosome. Men are more susceptible than women to MAO-A deficiency because they only have one X chromosome. A single nucleotide change in the MAOA gene reduces the activity of the MAO-A enzyme, meaning that monoamines (serotonin, dopamine, norepinephrine) formed in the body are more likely to accumulate and overstimulate the brain because they are less likely to be converted to their inactive metabolites. The same thing happens with neurological dysfunctions such as schizophrenia, mania, and ADHD. This is easier to identify when you know the levels of dopamine, DOPAC (MAO-A metabolite), and HVA (methylation end-product of COMT).

A genetic defect in MAOA will generally show an increase only in the monoamines, but not their metabolites. It is these subtleties that allow proper interpretation of the monoamine neurotransmitter results.

However, just because you have high dopamine doesn’t make you a “warrior.” Dopamine might also be elevated with supplementation of OTC extracts of plants such as the velvet bean (e.g., Mucuna Pruriens) that contain very high levels of the dopamine precursor DOPA. These products, used widely in the supplement industry, have profound effects on levels not only of dopamine, but its downstream metabolites DOPAC and HVA. When all three are elevated, and we know the patient is supplementing with dopamine precursors, this has an entirely different clinical interpretation than when some, or all, of the monoamines (e.g., dopamine, serotonin and norepinephrine) and their metabolites are elevated. Mucuna only elevates dopamine and its metabolites. A genetic defect in MAOA (i.e., warrior gene) will have a different profile and generally show an increase only in the monoamines (serotonin, dopamine, norepinephrine), but not their metabolites. It is these subtleties that allow proper interpretation of the monoamine neurotransmitter results.

Dopamine and its metabolites might also be elevated in individuals with tumors [4][5]. While this is rare, we have seen a few cases, which is why the clinical history should include use of OTC herbal extracts that might raise dopamine and its metabolites [6][7]. ZRT is meticulous about collecting information on herbal products that we know profoundly affect urinary neurotransmitter levels. This information is recorded in our database and used in concert with symptoms to create individualized comments about the neurotransmitters. Without this information physicians may go searching for a tumor that doesn’t exist.

High Levels of Normetanephrine May Indicate a Tumor

We also felt compelled to include information on downstream metabolites of the two stress hormones/neurotransmitters, norepinephrine and epinephrine. The metabolites include normetanephrine (NMN) and vanillylmandelic acid (VMA), respectively. Very high levels of NMN are diagnostic of pheochromocytomas, tumors of the adrenal medulla [8][9]. We have seen two of these very rare tumors in the past year, both of which were confirmed by independent serum testing and imaging, and were successfully removed by surgery.

Science Shows Dried Urine has Better Accuracy

Some neurotransmitters and their downstream metabolites are unstable and degrade in the liquid form . . . We have found that drying urine on filter cards immediately after collection is superior to collecting liquid urine in acid.

Another technical problem that is not well appreciated, but well documented in the scientific literature, is that some of the neurotransmitters and their downstream metabolites are unstable and degrade in the liquid form even when stabilized by acid. One example is serotonin and 5-HIAA, which can be unstable in liquid urine and result in false-low levels [10]. The collection vessel for liquid urine contains an acid as a preservative which helps prevent the neurotransmitters from degrading during shipment. The acid (e.g., hydrochloric acid) reduces the urine pH, which stabilizes some neurotransmitters that would otherwise degrade rapidly. However, if the acid is too strong, which can occur with low volume or very dilute urine, it will degrade some neurotransmitters if maintained in the liquid state. This degradation is accelerated by temperature, which makes shipment during the hot months of the summer much more problematic if the sample is sitting in the back of the transporting vehicle for hours.

What we confirmed is that serotonin and its metabolite 5-HIAA are very susceptible to acid degradation [10]. As reported by others, a very low pH of 2-3 is problematic, but in the range of 3-4 it is not. Drying urine on the filter cards affords much better stabilization of all neurotransmitters vs. keeping them in liquid state during transport and storage prior to testing. We have found that drying urine on the filter cards immediately after collection is superior to collecting liquid urine in acid and allowing it to be exposed for a prolonged time in this state. Drying urine, or other body fluids such as blood, shortly after collection has proved to be an excellent way to stabilize the analytes (neurotransmitters, steroids, peptide hormones, etc.) to be tested as we and others have shown and published [11][12][13].

If serotonin and 5-HIAA are allowed to degrade because the acid conditions and time from collection to testing are not optimal, the clinical interpretation would be low serotonin synthesis. Thus, if the levels of both serotonin and 5-HIAA are found to be low in liquid urine in the absence of any other neurotransmitter deficiencies and symptoms of low serotonin (e.g., depression, low energy, sleep disturbances, vasomotor symptoms) it is quite likely that the results are a urine collection problem and not a serotonin deficiency.

Only ZRT Offers the Added Insight of Monoamine Metabolites

Given the importance of these monoamine metabolite levels to correct interpretation of neurotransmitter results and development of subsequent treatment plans, ZRT Laboratory will begin including all 14 tests it offers in all neurotransmitter reports this month.

Going forward, too, ZRT will offer only its NeuroAdvanced Profile, which includes all 14 of these critical tests. This collection is only available from ZRT, and when combined with ZRT’s customized comments, delivers the most insightful report available from any lab.

Learn more about the scientific importance of these tests by joining us for webinar on Case Reviews on the Importance of Testing Neurotransmitters with their Metabolites.

To ask about kits and pricing, please contact customer service at 866.600.1636 or info@zrtlab.com.

Related Resources

References

[1] Strasser, B, et al. Mood, food, and cognition: role of tryptophan and serotonin. Curr. Opin. Clin. Nutr. Metab Care. 2016;19(1): 55-61.

[2] Harro, J and Oreland, L. The role of MAO in personality and drug use. Prog. Neuropsychopharmacol. Biol. Psychiatry 2016;69: 101-11.

[3] Godar, SC, et al. The role of monoamine oxidase A in aggression: Current translational developments and future challenges. Prog. Neuropsychopharmacol. Biol. Psychiatry 2016;69: 90-100.

[4] Foo, SH, et al. Dopamine-secreting phaeochromocytomas and paragangliomas: clinical features and management. Singapore Med. J. 2010;51(5): e89-e93.

[5] Verly, IR, et al. Catecholamines profiles at diagnosis: Increased diagnostic sensitivity and correlation with biological and clinical features in neuroblastoma patients. Eur. J. Cancer 2017;72: 235-43.

[6] Pathak-Gandhi, N and Vaidya, AD. Management of Parkinson's disease in Ayurveda: Medicinal plants and adjuvant measures. J. Ethnopharmacol. 2017;197: 46-51.

[7] Weldin, J, et al. Quercetin, an over-the-counter supplement, causes neuroblastoma-like elevation of plasma homovanillic acid. Pediatr. Dev. Pathol. 2003;6(6): 547-51.

[8] Eisenhofer, G and Peitzsch, M. Laboratory evaluation of pheochromocytoma and paraganglioma. Clin. Chem. 2014;60(12): 1486-99.

[9] Crona, J, et al. New perspectives on pheochromocytoma and paraganglioma: towards a molecular classification. Endocr. Rev. 2017; Aug 4 [Epub ahead of print].

[10] Corcuff, JB, et al. Urinary sampling for 5HIAA and metanephrines determination: revisiting the recommendations. Endocr. Connect. 2017;6(6): R87-R98.

[11] Du JY, et al. Percutaneous progesterone delivery via cream or gel application in postmenopausal women: a randomized cross-over study of progesterone levels in serum, whole blood, saliva, and capillary blood. Menopause. 2013;20(11): 1169-75.

[12] Drolet J, et al. Integrated Metabolomics Assessment of Human Dried Blood Spots and Urine Strips. Metabolites. 2017;7(3).

[13] Zava TT, et al. Iodine and creatinine testing in urine dried on filter paper. Anal Chim Acta. 2013;764: 64-9.

Cortisol Testing in Saliva, Blood & Urine

Fri, 29 May 2015 10:29:00 -0700

Practice Takeaway: Providers should be aware that three different body fluids – saliva, blood or urine – can be used to assess adrenal gland function, and should know each method’s advantages and disadvantages, when deciding how to test patients.

Inadequate or excessive production or disrupted circadian patterns of cortisol synthesis by the adrenal glands in response to stressors can eventually lead to imbalances in blood glucose levels, impaired immune response, as well as a host of different hormonal imbalances, all of which are associated with multiple adverse conditions and symptoms.

Saliva

Cortisol is synthesized by the adrenal glands and released into the bloodstream in response to stress signals from the brain. In the bloodstream cortisol is mostly bound up by Cortisol Binding Globulin (CBG) and albumin, leaving only about 1-3% bioavailable to enter tissues and invoke a biological response. The bioavailability of cortisol can vary considerably due to different levels of CBG; liver synthesis of CBG is controlled by various hormones, such as estrogens, thyroid, and cortisol itself. Salivary cortisol reflects the amount of cortisol that escapes binding proteins, and enters the tissues throughout the body, including the salivary glands and saliva.

As such, saliva is representative of the bioavailability of cortisol to target tissues throughout the body. Saliva collection at four time points throughout the day (morning, noon, evening, and night) provides a simple and convenient means for assessing not only the bioavailability of cortisol but also its circadian pattern of synthesis (i.e. the diurnal cortisol curve).

Blood

Blood serum and plasma derived from venipuncture, as well as whole capillary blood derived from the fingertip and dried on filter paper (dried blood spot, DBS) have been used to determine total cortisol levels in the bloodstream. Cortisol levels in plasma, serum, and DBS are quantitatively equivalent.

DBS testing for cortisol does have the advantage over conventional venipuncture serum/plasma methods in that the former is more convenient for the patient since blood can be collected at any time and place, and allows for morning and night collections outside the clinical setting.

While cortisol testing in blood provides a good estimate of the adrenal glands’ capacity for total cortisol synthesis and release into the circulation, this test is not useful for determining the bioavailable fraction of cortisol without additional testing for CBG, which is not commercially available.

Urine

Urine represents another easily accessible body fluid for measuring cortisol. Urine cortisol is tested commonly in most laboratories by liquid chromatography-mass spectrometry (LC-MS) using urine collected in a large plastic container over a 24 hr time course. More recently, simple and convenient methods have been developed to measure cortisol from urine collected at four times during the day (first morning, late morning, evening, and night before bed) and dried on filter paper.

Steroid hormones like cortisol, whether from urine or blood, are extremely stable at room temperature for up to a month when dried on filter paper, allowing for great latitude in the dried urine assay. The 4x dried urine method has the advantages of ease of sample collection (urination directly on a filter card and hang to dry) and shipment (dry filter card vs liquid urine).

. . . the 4x dried urine cortisol curve, like the 4x saliva cortisol curve, conveys important clinical insight into the adrenal glands' response to stressors throughout the day.

Moreover, by collecting urine 4x from morning to night it is possible to evaluate the circadian rhythm of cortisol synthesis, as opposed to total cortisol synthesis over a 24 hr period by the liquid urine method. The circadian patterns of cortisol synthesis can vary considerably among individuals and thus the 4x dried urine cortisol pattern, like the 4x salivary cortisol assay, will convey important clinical insight into the adrenal glands' response to stressors throughout the day.

Cortisol is present in urine at about equal concentrations in both a conjugated (mostly glucuronidated) and free form. The free form, designated Urinary Free Cortisol (UFC), enters urine in a way similar to salivary cortisol and is representative of the bioavailable fraction.

Interpretation of the 4x circadian UFC is somewhat different from saliva, since the first urine void is representative of cortisol synthesis throughout the night and is expected to be lower than the second urine void, which, like the first saliva collection, should represent the peak of adrenal cortisol synthesis in the early morning. In a healthy individual cortisol in both saliva and urine tapers as the day progresses and reaches a nadir before bed at night. Circadian patterns of adrenal cortisol synthesis (i.e. diurnal cortisol curves) are usually very similar when measured in saliva or urine.

The diagrams below depict typical circadian patterns of salivary and urinary free cortisol synthesis in an individual showing adrenal fatigue as the day progresses. As seen in the diagrams, both the salivary and UFC show normal circadian patterns, but low cortisol synthesis in the afternoon and at night before bed, indicating an inability of the adrenal glands to maintain normal cortisol synthesis by day's end.

While the 24 hr total or free cortisol levels are usually within normal range in these individuals, their circadian patterns reveal abnormally low late evening/night free urinary cortisol levels which commonly parallel their symptom profile of fatigue occurring more at night than in the morning.

In summary, providers should be aware that three different body fluids can be used to assess adrenal gland function, and should know each method’s advantages and disadvantages, when deciding how to test patients. Working with a lab that offers the clinical expertise to interpret the differences between these methods also helps ensure patients get the most meaningful results.

Related Resources

Flaws in the Iodine Loading Dose Urine Test

Thu, 18 Dec 2014 22:57:00 -0800

I began the study of iodine because I believe it has benefits to human health. As a breast cancer researcher I believe that iodine protects the breasts and uterus against toxic and mutagenic estrogen metabolites that form in some people more than others. Thus, choosing the most reliable test to a) determine iodine status, and, b) supplementation to maintain levels seen in populations with lowest rates of breast cancer (ie. the Japanese), is a key health consideration with or without breast cancer risk factors.

That leads us to the all-important discussion of iodine testing methods. Have you ever had your levels evaluated with a 24-hour iodine loading test and been found to be deficient? If so, you are not alone. This is a test where 98-99% of people who take it will be deemed “whole body iodine deficient”. On the basis of this test result, you may be advised to take iodine supplements at a dose that makes some people ill, and raises iodine to hundreds of times the levels recommended by health organizations around the world. In the most serious cases, the dosing recommended to get you to “whole body iodine sufficiency” may cause your thyroid to stop working normally, producing either too much (hyperthyroid) or too little (hypothyroid) thyroid hormone.

Considering that this test is aimed at improving health, how could it potentially result in health problems? It started with...

Related Resources

Testosterone: Elixir or Dangerous Drug?

Wed, 19 Sep 2012 03:42:00 -0700

An FDA-approved testosterone gel was shown to significantly increase risk for adverse cardiovascular, respiratory, and dermatological events in men 65 or older who had impaired mobility and increased health risks.

This is according to a recent study published in the prestigious New England Journal of Medicine (NEJM) (1) that casts a shadow on the common use of testosterone therapy in older men for boosting their energy and vitality.

These unexpected negative results fly in the face of decades of good research on testosterone therapy in older men showing it to be beneficial to the cardiovascular system, and improve sexual function, mood, energy level, and muscle and bone mass and strength (2,3,4,5).

What went wrong? Why were the results so different from hundreds of other publications painting testosterone as the elixir of youth and vitality? Was it the dose of testosterone these men were using, or the way it was delivered as a gel through the skin?

In the NEJM study the testosterone treated men had mean venous serum testosterone levels of 574+/- 403 ng/dL, compared with 292+/- 160 ng/dL in the placebo group. The Endocrine Society Clinical Practice Guidelines suggests that testosterone should be dosed to achieve serum levels about mid-range for a healthy young male (5). The 50-150 mg topical testosterone gel put the testosterone-treated men well within the expected mid-physiological range seen in healthy young men (about 500-1000 ng/dL). So based on conventional serum testing one would conclude that overdosing would not likely be responsible for the adverse reactions these men had to testosterone. Or was it?

Let’s look at some assumptions made about using conventional venous serum to monitor how much testosterone is entering the body following topical testosterone therapy. It is “assumed” that when testosterone is delivered as a topical gel only 10% is absorbed and utilized by the body. This is based on measuring the total amount of testosterone that can be detected in venous serum over a specific time frame, usually about 12-24 hours, after the testosterone has been applied to the skin. With this conventional wisdom of 10% absorption, topical testosterone gel has been FDA-approved at a pharmacological dose about 10 times higher (50-100 mg) than the amount a healthy young male’s testes will manufacture in a day, which is about 5-10 mg of testosterone.

The NEJM study (1), and many others preceding it (4,5), confirmed that the daily topical dosage of 50-150 mg testosterone results in a level of serum testosterone (mean 574 ng/dL) that would be considered healthy in young men. So why was this dose of topical testosterone not healthy for the older men?

It is “assumed” that when testosterone is delivered into the body using a topical gel that serum testosterone measurements, derived from a venous blood draw, actually reflect how much testosterone is entering tissues throughout the body.

What evidence do we have that the pharmacological dose (50-150 mg) of topical testosterone used in the NEJM study was actually “delivering” a physiological dose of testosterone into the tissues of these older men? The correct answer is none. This would require measuring the levels of testosterone in different tissue biopsies, which for obvious reasons is not practical and such studies would not likely find many volunteers.

However, there are other means to look a little closer at “tissue” exposure to testosterone, which is to measure the level in the fluid of tissues bathed by capillary beds (blood flowing to tissues and delivering nutrients and hormones) as opposed to blood flowing away from tissues (i.e. venous blood). Capillary blood and saliva provide the next-best alternative as they are easily accessible body fluids that are more representative of the direct interaction of capillary beds with tissues (6,7).

Over the past 10 years ZRT Laboratory has measured the levels of steroid hormones (estradiol, progesterone, testosterone, DHEAS, cortisol) in saliva and capillary blood (finger stick) from men and women who have used a plethora of different hormone doses and delivery systems (topical creams and alcoholic gels, oral, im-injections, sc-pellets, sublingual drops, and troches). Information on hormone type, dosage, delivery, and timing on well over a million people has been recorded in our database.

In mining this extensive database, we find that when steroid hormones are produced endogenously by the ovaries and testes and released into the bloodstream the levels in venous blood serum are quantitatively equivalent to whole capillary blood drawn from the finger (7). Under the same conditions, salivary hormones are about 2-3% of venous serum or capillary blood hormone levels, as reported extensively in the literature (6).

In contrast to what we see with endogenously produced hormones, when testosterone, or any other steroid hormone, is applied topically as a cream, gel, or spray, levels in capillary blood (finger) are remarkably higher, by 10-20 fold, than venous blood levels. Moreover, salivary hormones are often >100% of venous serum levels, which ordinarily are only 2-3%. This remarkable increase in capillary blood and salivary testosterone is NOT seen when it, or other hormones, are delivered orally, or as im-injections or sc-pellet implants. With these delivery systems capillary blood and venous serum levels are about the same.

This discrepancy in venous serum vs capillary blood and salivary hormone levels following topical hormone delivery has created enormous confusion among physicians attempting to interpret hormone test results. Those using venous serum are often frustrated that pharmacological dosing, as seen in the NEJM article, only leads to lower to mid-physiological serum testosterone levels, but side effects such as excessive buildup of red blood cells associated with pharmacological dosing. On the other side, those using saliva and capillary blood testing are shocked to see that pharmacological dosing leads to pharmacological levels much higher than expected.

Specifically, with reference to the use of topical testosterone gels in men at the 50-150 mg dosage, we find mean capillary (finger tip) blood levels of testosterone to be about 5000 ng/dL, approximately 10 x higher than the venipuncture serum blood levels reported in the NEJM study (574 +/- 403 ng/dL).

Of further relevance to the NEJM study, we find that men using the higher pharmacological topical testosterone gel have capillary blood levels of estradiol that usually exceed 100 pg/ml, which is very high for males. While physiological levels of estrogens are beneficial to the male cardiovascular system, higher levels can be harmful (8). Unfortunately, estrogens were not monitored in the NEJM study so it is not possible to know if levels, even in venous serum, were higher than physiological range. It is very possible that some of the cardiovascular events observed in the older high risk men participating in the NEJM study were precipitated by excess estrogens derived from excess testosterone.

We believe these differences in conventional venipuncture serum vs capillary blood and saliva testosterone provide a rational explanation for the unexpected adverse clinical events seen in the NEJM study.

Based on the exceptionally high testosterone and estradiol levels we have documented in capillary blood and saliva following use of topical testosterone gels in the 50-150 mg range, we suggest that dosage for topical delivery of testosterone, or any steroid hormone, should not be determined by venipuncture serum levels as this will result in over dosage and potential for toxicity, as seen in the men participating in the NEJM study.

Perhaps a lesson is learned from The Swiss alchemist, Paracelsus, who noted some 500 years ago: "All things are poison and nothing is without poison. Only the dose permits something not to be poisonous."

Hormones are no different. A physiological level of testosterone is unquestionably life-enhancing in men of all ages and health conditions who suffer from testosterone deficiency. However, at a high enough dose testosterone, like all hormones, has the potential to transition from an elixir to a dangerous drug.

Related Resources:

References:

Basaria S et.al. N Eng J Med June 30, 2010. Adverse Events Associated with Testosterone Administration (downloaded on line)
Bhasin S, et. al. J Clin Endocrinol Metab 90: 678-688, 2005. Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on skeletal muscle.
Wu FCW et. al. N Engl J Med 363: 123-35, 2010. Identification of late-onset hypogonadism in middle-aged and elderly men.
Fernandez-Balsells et. al. J Clin Endocrinol Metab 95: 2560-2010, 2010. Adverse effects of testosterone therapy in adult men: a systematic review and meta-analysis.
Bhasin S et.al. J Clin Endocrinol Metab 95: 2536-2559, 2010. Testosterone therapy in men with androgen deficiency syndromes: an endocrine society clinical practice guideline.
Vining RF, McGinley RA. J Steroid Biochem 27: 81-94, 1987. The measurement of hormones in saliva: possibilities and pitfalls.
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Natural Progesterone as a Preventive for Breast Cancer

Wed, 19 Sep 2012 03:35:00 -0700

Editor’s Note: After the publication of Dr. David Zava’s last Health Watchers News and Views post, Hormone and Breast Cancer: the Latest Findings from the WHI, he received an attacking comment from a doctor who has been making claims for years that natural progesterone has the same effects as the synthetic progestins.

Although, as an editor, I do not publish comments that attack, Dr. Zava wrote an eloquent, if technical response which adds to the dialogue about progestins, progesterone and breast cancer. We share it here for the reading pleasure of hormone biochemistry geeks everywhere!

I very much disagree with the comment that “Progestins cause more breast cancer than estrogens because they act like progesterone.” Yes, synthetic progestins increase breast cancer, but there is no evidence that progesterone does the same. Quite the contrary. Below is my argument in favor of natural progesterone as a preventive for breast cancer, with reference to the Fournier studies that address this issue.

Fournier and colleagues (Breast Cancer Res Treat 107: 103-111, 2008) originally published their clinical study looking at the relationship of different progestogens, in combination with estrogen, on the risk of developing breast cancer. First, I think it important to define what I mean by a progestogen; it is any molecule with a structure like progesterone that binds to and activates intracellular progesterone receptors, which would include all forms of synthetic progestins in addition to natural progesterone. Overall, what Fournier and colleagues found is that of all the progestogens studied, natural progesterone had the lowest risk, which was lower than no treatment.

Fournier et.al. then did a second study in the same patient population as above (80,391 postmenopausal women), but looked not only at the different types of hormones that were used (estrogens and progestogens), but risk in relationship to the type of tumor (ductal or lobular). They also looked at whether or not the tumors contained receptors for estrogen or progestogens. There are four categories for tumors with estrogen and progestogen receptors: ER+/PR+, ER+/PR-, ER-/PR+, and ER-/PR-. Most breast cancers (60-70%) present with receptors for both estrogens and progestogens (ER+/PR+), and about 20-30% fall into the other categories (about 10-15 % are ER-/PR-). For the record, I helped develop this technology in the 1970s, ran three different breast cancer testing laboratories that did this type of testing for many years (mid 1970s to mid 1990s), and published in this area extensively using breast cancer cell lines and human clinical samples post surgery for breast cancer.

What Fournier and group found in the second study (J Clin Oncol 26 (8): 1260-1268, 2008) is that the combination of estrogens and progestogens for more than 5 years is associated with increased risk of breast cancer. When this is broken down into ductal vs lobular cancers, the use of estrogens plus progestogen (including natural progesterone) is associated with an increased risk for breast cancer. What Dr. Grant fails to mention is that the risk with estrogen plus progesterone is actually less than the risk of estrogen alone (1.7 vs 2.1, respectively), and that of all the progestogens, natural progesterone has the lowest risk. So there is a different way of looking at this data. It is important to keep in mind that this study did not look at natural progesterone by itself, only estrogen plus progesterone. Another way of interpreting this data is that natural progesterone decreased the risk of breast cancer caused by long term use of estrogens (i.e. risk 2.1 to 1.7).

It is also important to keep in mind that the Fournier studies did not compare the effects of oral vs topical progesterone. Based on what we have seen in hundreds of thousands of salivary and capillary blood progesterone levels following different routes of progesterone administration, the topical route of administration delivers much more progesterone to tissues than does the oral route. Luteal tissue levels of progesterone need to be achieved for progesterone to act as an estrogen antagonist to prevent estrogen from excessively stimulating breast cell proliferation, which can increase breast cancer risk. Women who have an excess of estrogen relative to progesterone (low progesterone/estradiol ratio), are more likely going to have atypical benign breast disease that is at increased risk of developing into breast cancer (Sitruk-Ware et.al. J clin Endocrinol Metab 44, 771, 1977). Low endogenous luteal progesterone levels in premenopausal women (much more prevalent in peri-menopausal women) have also been associated with increased breast cancer risk (Micheli et.al. Int J Cancer 112, 312-318, 2004).

Oral progesterone is mostly destroyed in the gastrointestinal tract before it enters the blood circulation and delivered to tissues. Based on saliva and capillary blood progesterone levels, topically delivered progesterone is 20-100 times more efficiently delivered to tissues. Studies in humans have shown that physiological amounts of progesterone (20-50 mg) administered topically deliver physiological amounts of progesterone to the breast tissue and suppress estrogen-stimulated cell proliferation (Chang KJ et.al. Fert Sterility 63: 785-791, 1995 and De Boever J et.al. Endocrinol of Cystic Breast Disease, 1983). My concern is that women using estrogens and oral progesterone as hormone replacement may not be getting enough progesterone to tissues to counter the growth-promoting effects of the estrogens. And the actual estrogen levels relative to progesterone are not evaluated in these studies. The study by Fournier mentioned above looks at oral, not topical, progesterone. Based on saliva, capillary blood, and tissue progesterone levels from the studies mentioned above, topical progesterone is far more effective in delivering a physiological amount of progesterone to the breast and controlling estrogen-stimulated cell proliferation.

It is unfortunate that in spite of all the science and clinical studies behind it, only one small study (Plu-Bureau G et.al. Cancer Detect Prev 23(4), 290-296, 1999) that I am aware of looked at the risk of breast cancer with topical progesterone in a manner similar to what Dr. John Lee recommended for progesterone use (10-30 mg topical progesterone daily). In that study they showed the risk to be reduced by half (0.5) in those using topical progesterone for 3 years or more. This is entirely consistent with the biology behind progesterone delivered topically (protective).

When I talk to very high level people about why they should at least study the relationship of topically delivered progesterone to breast cancer risk I am told they don’t think it could possibly be effective because it doesn’t increase serum levels. This is true, as it is for all topically delivered hormones, but it does increase saliva, capillary blood, and tissue levels of progesterone. With this data in hand, it doesn’t take a genius to figure out something is wrong with serum testing when hormones are delivered topically.

Hopefully one day the medical establishment will finally wake up and listen to what women have been telling them for years—topical progesterone works. Then they might discover what has been right under their noses (the scientific articles supporting the utility and effectiveness of topical progesterone) for too many years.

Continue the discussion:

Learn more about progesterone and breast cancer by viewing ZRT's webinar on Progesterone Metabolites and Breast Cancer Risk.