Reliable Free Steroid Hormone Testing in Saliva
By Dr. Wolfgang Ziemann: 3.10.06 in Endocrinology
1. Introduction: The exact measurement of hormone concentrations is important for the correct estimation of the hormone balance and endocrine function. This measurement traditionally is performed in blood/serum/plasma samples. Several hormones also can be analyzed in saliva. This technique offers several advantages. The sampling is non-invasive and can be done anytime, anywhere. Especially in the case of steroid hormones such as Cortisol, Testosterone, Progesterone, Estradiol, Estriol, and DHEA reliable saliva testing offers significant additional advantages. The diagnostic relevance is much higher than compared to serum/plasma analytics.
2. The hormone activity: Steroid hormones are almost completely bound to their specific binding globulins. This bound fraction is biologically inactive and is considered to be just a kind of hormone reservoir. Only less than 5% of the total hormones are free and represent the active fraction. This free fraction is therefore considered to be responsible for the hormone action. In serum or plasma the steroid measurement will result mainly in a number representing the concentration of the total inactive hormone. This total concentration at best gives only an approximate indication of the hormonal balance in cases of completely healthy and normal patients. In any case it would be very advantageous to just measure the free fraction of the hormones. This is exactly what we can achieve by testing salivary samples.
3. Episodic hormone secretion: Due to the episodic secretion pattern of steroid hormones any result based on a single sample is completely arbitrary. We only can expect reproducible and reliable results in cases of multiple sampling. Therefore we always recommend taking 5 samples within at least a 2 hour period. This is valid for serum/ plasma as well as for saliva. In contrast to blood this sampling strategy easily can be done in cases of salivary testing. Please note that there should not be any food intake several hours prior to saliva sampling. Optimally saliva should be collected while morning-fasting. Intake of water will not affect test results. Other liquids are not recommended.
4. Sampling device: As saliva contains only tiny amounts of protein non-polar analytes such as steroids tend to absorb to various plastic surfaces. Glass would tend to be the best material for saliva sampling and for the most part Polyethylene, although it is still in use in many laboratories worldwide, should be avoided. However there is an inexpensive polypropylene device available from Demeditec Diagnostics or other suppliers. It does not show any interference in saliva even in Progesterone testing, which is the most critical steroid.
5. Blood in saliva: Traces of blood might interfere with the results of saliva testing. It is therefore important that the salivary samples should not show any red colour. In cases of visible blood traces in saliva the sample should be discarded. After rinsing the device several times with tap water a new saliva sample can be taken but only after waiting for approximately 15 minutes.
6. Serum testing: Testing of steroids in serum or plasma in general is questionable. The result represents mainly the inactive hormone fraction and is arbitrary in cases of a single sample. In a relatively short time, usually less than half an hour, the concentration of the hormone in serum can easily change by the factor of 2 or 3. Due to the limited specificity of current (fully automated) routine methods serum testing is unreliable in low concentration ranges. This has been recently published in scientific literature. In obvious contrast to this saliva testing is the most reliable alternative in any concentration range of steroids such as Cortisol, Testosterone, Progesterone, 17OH-Progesterone, Estradiol, Estriol, and DHEA.
7. Summary: Saliva testing for steroid hormones by far is the most reliable and convenient method for measuring the hormone activity in endocrine disorders and in checking the hormonal balance. Routine testkits in ELISA technology now are available for an increasing number of steroids.
Are you aware that most ELISA tests for salivary estradiol are unreliable unless the sample is first extracted by liquid extraction (with ether or ethyl acetate) or by solid phase extraction? Even FDA-cleard kits for testing estradiol in saliva are inaccurate and imprecise for estradiol because it is in such low concentrations. Saliva testing has been largely considered unreliable, and the misunderstanding and overuse of estradiol assays without a prior extraction is the main reason.
Mark Newman
Lab Director
ZRT Laboratory, Beaverton, OR
Comment by Mark Newman — 9. June 2007 @ 18:06
Dear Mark,
thank you for your interesting comments. I was the author of the article “Reliable Free Steroid Hormone Testing in Saliva”, which I wrote for the web site of SMS Gruppen some time ago and I am pleased to have feedback from you. I always try to stay current on literature about steroid testing in serum and saliva, and as a result I can only agree to a certain extent with your comments. I fully agree that serum assays for Estradiol measurements require extraction. This has been proven in recent publications such as:
1. Frank Z. Stanczyk, Michael M.Cho, David B. Endres et.al.
Title: Limitations of direct estradiol and testosterone immunoassay kits
Published in: Steroids 2003 (68) pages 1173 – 1178
2. Jennifer S. Lee, Bruce Ettinger, Frank Z. Stanczyk et al:
Title: Comparison of Methods to Measure Low Serum Estradiol Levels in Postmeopausal Women.
Published in: The J. of Clin. Endocrinol. & Metabol. 2006 (91:10), pages 3791-3797
I am not aware of any publication showing the need for an extraction step for salivary measurement (e.g. comparing extraction with non-extraction methods). Are you aware of any journal articles that have been published on this subject? This would be very interesting for me. there are, however, several recent publications showing that direct salivary Estradiol measurements yield useful clinical information. Below is an extract from our literature data base about the usefulness of salivary measurements:
1. L.J. Bond, E.T. Vella, Y. Kiparissis, K.E. Wynne-Edwards
Title: Anthropometry and body composition do not predict bioavailable Androgen or Progesterone concentration in adolescent girls.
Published in: Am J Hum Biol 2006, Vol.18, pages 639-653
Summary: In this study 3 different groups of girls have been investigated for salivary Estradiol, Testosterone, Progesterone, and DHEA. The mean age of the 3 groups were 10.9 years, 13.7 years, and 16.7 years respectively. There was no evidence for an association between the salivary hormone levels and BMI, % body fat, or WHR.
2. G. Jasienska, A. Ziomkiewitz, I. Thune, S.F. Lipson, P.T. Ellison
Title: Habitual physical activity and levels in women of reproductive age.
Published in: Eur J Cancer Prev 2006, Vol.15, pages 439-445
Summary: The Authors investigated the concentration of salivary Estradiol in 139 young healthy women (age 24-37 years). They found a significant negative correlation between the concentration of salivary Estradiol and regular (daily) physical activities. On the other side it is known that the risk for breast cancer positively correlates with the Estradiol level. This has led the Authors to the assumption that regular (daily) physical exercise might reduce the risk for breast cancer.
3. G. Jasienska, A. Ziomkiewitz, I. Thune, S.F. Lipson, P.T. Ellison
Title: High ponderal index at birth predicts high Estradiol levels in adult women.
Published in: Am J Hum Biol 2006, Vol.18, pages 133-140
Summary: 145 young women were investigated for salivary Estradiol and the results compared birth weights and lengths (taken from their old medical records). The study showed a moderate positive correlation between the ponderal index at birth and the salivary Estradiol in those later adults. This also fits with an earlier published observation that high birth weight is one of the risk factors for the later development of breast cancer.
4. M. Kapiszewska, M. Miskiewicz, P.T. Ellison, I. Thune, G. Jasienska
Title: High tea consumption diminishes salivary Estradiol concentration in Polish women
Published in: Br J Nutr 2006, Vol.95, pages 989-995
Summary: Major constituents of tea, catechins and theaflavines, inhibit aromatase, an enzyme which catalyzes the conversion of androgens to estrogens. This study showed that the secretion of salivary Estradiol was significantly suppressed during the menstrual cycle simply by drinking average or high volumes of black tea during the day. This relatively simple form of diet may have a significant impact of reducing the risk of breast cancer in women.
5. K.L. Klump, K.L. Gobrogge, P.S. Perkins, D. Thorne, S.M. Breedlove
Title: Preliminary evidence that gonadal hormones organize and activate disordering eating.
Published in: Psychol Med 2006, Vol.36, pages 539-546
Summary: Finger-length ratios are known to be somatic markers of prenatal Testosterone exposure. Salivary Estradiol has been measured in 113 adult female males. The findings of this study suggest that lower levels of prenatal Testosterone exposure (as estimated by finger-length ratios) and higher adult levels of adult salivary Estradiol are associated with increasing eating disorder symptoms.
6. Yu-cai Lu, M.D. et al
Title: Salivary estradiol and progesterone levels in conception and nonconception cycles in women: evaluation of a new assay for salivary Estradiol
Published in Fertility and Sterility Vol. 71, No. 5, May 1999, pages 863-868.
Summary from your web site: This study attempted to replicate earlier work by Lipson and Ellison. Daily salivary estradiol and progesterone levels were measured in consecutive menstrual cycles of 11 women, until conception occurred. The authors report that mean estradiol level in the preovulatory period in conception cycles was not statistically different from non-conception cycles.
In the last publication the authors have used a non-extraction E2 assay as well and I note that this publication is cited on your web site.
If you would be interested in the full version of any of the publications I have cited please let me know and I will send it to you.
These positive publications seem to confirm that there is a potential in the market for commercial salivary Estradiol testing. As a result, we offer a direct ELISA for the measurement of salivary Estradiol with a sensitivity of 0.4 pg/ml. This assay has been cleared by the FDA.
I just visited your web site using http://www.zrtlab.com/Page.aspx?nid=426 and I find it curious that you are giving reference ranges for DHEA-S in saliva. Usually the labs here in Europe measure salivary DHEA instead of DHEA-S. In fact there is some evidence that DHEA-S enters saliva mainly by micro bleedings. As a result, some labs are using salivary DHEA-S for measuring blood contamination. This is supported by literature as well:
1. G. Lac, N. Lac, A. Robert
Title: Steroid assays in saliva: A method to detect plasmatic contaminations
Published in: Archives Internationales de Physiologie, de Biochemie et de Biophysique 1993 (101) pages 257-262
2. Ross F Vining, Robynne A McGingley, Richard G. Symons
Title: Hormones in Saliva: Mode of Entry and Consequent Implications for Clinical Interpretation
Published in: Clinical Chemistry 1983, Vol 20 (10), pages 1752 - 1756
A summary of both publications is on your own web site. Both of the papers you cite on your website state that salivary DHEA should be determined instead of DHEA-S in saliva. I agree with the statement on your website that the publication from Vining “…. is a landmark paper for salivary hormone analysis”
I also agree with your statement that “Saliva testing has been largely considered unreliable”. This statement was confirmed in a very impressive way by the publication of Hagen et al in 2003 (see attachment). Having read this publication I became convinced that the main reason for the lack of reliability of salivary testing can be attributed to pre-analytic procedures used by many labs. Taking into consideration the high biological CV you cannot expect reproducible results based on taking just one single saliva sample. We have performed detailed investigations regarding the secretion of salivary testosterone in men. When testing single samples you have to expect an average deviation (from the mean value) of 22% and a maximal deviation of 94% (in either direction). Consequently we highly recommend collecting 5 samples within a period of 2 hours. The lab should pool equal aliquots from all 5 samples and determine the mean value. This simple strategy yields a mean deviation of just 7% from the average value. As far as I have seen there is no such sample collection recommendation on your web site. Can I assume that you still are using the strategy of taking just a single sample? Please correct me if you have changed your sampling strategy. In my opinion the strategy of collecting and testing only a single sample for many of the steroid hormones creates by far the most important weakness of current saliva testing. If you are interested, I can send you detailed secretion profiles of salivary steroids showing obvious and strong episodic secretion patterns.
By the way, I know your lab personally. In fact I have visited your facility some years ago (in 2002 if I remember correctly) and I also talked to David Zava several times. Unfortunately I don’t recall having met you during my visit, and I apologize for that. I am also a colleague and consultant to KMI Diagnostic Laboratories in Minneapolis, and their Director of Laboratory Services, Mike Foley, knows you and speaks highly of you. I believe he is working with you as a participant in your prototype proficiency testing service? Maybe we will have an opportunity to meet at an Anti-Aging conference in the US. Usually I attend the AACC and some of the A4M Anti-Aging shows as well. It would be nice to get your comments and feedback and to continue discussing the subject personally.
Best regards,
Wolfgang
Demeditec Diagnostics
Dr. Wolfgang Ziemann
Comment by Dr. Wolfgang Ziemann — 12. June 2007 @ 08:26