Your aging patients will appreciate noninvasive assessment tools.
As a healthcare professional, you are often asked to clinically evaluate the health of your patients and the ongoing effects of physiological aging. The baby boomers in particular, who are wealthy and following a healthier lifestyle, place emphasis on feeling better, living longer, and looking younger. Consequently, they are seeking help from physicians worldwide.
As people age, however, their hormone levels gradually and steadily decline. The endocrine and sexual glands produce fewer hormones than they once did, changing vital, active individuals to slower, weaker ones. In fact, every 10 years after the age of 20, people lose 15 percent of their hormones. A 70-year-old woman, for example, may have lost up to 75 percent of her natural hormones.1 For the next 14 years, baby boomers will be turning over 65 at the rate of 10,000 per day in the U.S.2 As a result, hormone deficiencies may influence health more than previously thought.
Different types of hormones
More than 95 percent of the hormones in the bloodstream are bound and therefore biologically inactive. These are glycoproteins that are loosely bound to albumin and tightly bound to cortisol-binding globulins (CBG), sex hormone-binding globulins (SHBG), testosterone, and all forms of estrogen.
The remaining hormones in the bloodstream—a tiny fraction—are called unbound or free hormones, which are readily available for the receptors of organs and tissues. They are the only hormones the body can use.
Physiology of saliva production
Saliva is produced by three major glands: The parotid, the sublingual, and the submandibular. The parotid gland is in front of the ears on the lateral portion of the mandible and it normally produces 30 to 40 percent of a person’s saliva (up to 70 percent when stimulated).
The sublingual gland is located in the oral cavity beneath the tongue, and it produces 25 percent of a person’s saliva (50 to 70 percent when stimulated with food). The submandibular gland is beneath the jaw and it produces about 5 percent of a person’s saliva. Normal saliva production is about 1.5 liters per day, but with age, saliva production decreases to less than 0.5 liters per day, possibly due to salivary gland atrophy and hormonal imbalance.
Saliva is produced when blood, possessing both bound and free hormones, and lymphatic components traverse membranes on their way to the glands. According to two researchers, “Unconjugated plasma steroids, such as estradiol, testosterone, progesterone and other related steroids, are able to cross the salivary gland wall and diffuse to saliva regardless of the salivary flow rate.”3
You may wonder how hormones can cross the salivary gland wall. In fact, any hormone with a molecular weight (MW) of less than 1,900 can penetrate the filtration membrane. The hormones testosterone, DHEA, cortisol, and melatonin are generally about 300 MW.
On the other hand, a CBG is approximately 50,000 MW, albumin is typically 66,000 MW, and an SHBG sex hormone is about 115,000 MW. These are all too heavy to cross the membrane barriers, such that only the free hormones can cross over and enter the saliva. As a result, eight free hormones are readily present in saliva: testosterone, estradiol, cortisol, estrone, estriol, DHEA, progesterone, and melatonin.
Saliva testing technology
Key reasons for performing saliva testing are that it is noninvasive, inexpensive, painless, and convenient (for you and the patient). It offers accuracy by directly measuring biologically active or free hormones. As a general protocol, patients should take five samples of their saliva throughout the same day.
Published studies have shown that, when comparing saliva and serum methods, saliva is a more accurate sample.4 It provides precise readings for testosterone bioavailability, especially for low levels.5 (Serum tests are often unreliable in this area.) Saliva testing also can measure hormones, such as testosterone in women or children, and estrogen in men.6
For example, in one study, 50 men, 55 women, and 11 children were assessed for serum testosterone via immunoassays and isotope-dilution gas chromatography spectrometry. None of the serum samples from the women and children were deemed reliable, as these populations have low (< 1.7 nmol/L) and very low (0.17 nmol/L) levels of testosterone.7
8 principles for accurate saliva hormone testing
Principle 1: Hormone cycles. Be aware of natural ebbs and flows of hormone production, such as during the menstrual cycle. For example: High estrogen levels occur during ovulation, and high progesterone levels circulate through the body around day 21 of the menstrual cycle. When you measure hormones, take exercise, medication, illness, and other factors affecting changes in hormone levels into consideration.
Principle 2: Multiple samples. Hormones are produced in high levels of pulsatility and squirts on regular intervals.
Clinical experience shows that five test samples give an average of about 80 percent accuracy, while single sample collections at random reflect only a 20 percent or lower rate of accuracy. The first sample should be collected in the morning, exactly one hour after waking, with the remaining four samples collected every three hours after the initial sample is collected. For the greatest hormone testing accuracy, gather all five samples three hours apart.
Principle 3: Proper sample gathering utensils. Cotton swabs should never be used when collecting a sample. Cotton salivettes, which are balls of cotton with cotton strings attached, have been shown to result in lower cortisol values. There is a high chance that cotton contains substances that interact with cortisol and bind hormones. This makes the cortisol unavailable for measuring. Spitting directly into a container using a small straw is preferred.
Principle 4: Purity of saliva samples. Before collecting saliva samples, patients must remember to wash their hands thoroughly with soap and water and use disposable gloves. Possible contamination will affect the accuracy of the test.
Principle 5: Mouth hygiene. To ensure this, patients should rinse their mouths with clear water right after eating or drinking, and should not eat nor drink one hour before a saliva sample collection. Rinsing the mouth with clear water 15 minutes before a saliva sample is acceptable.
Principle 6: Blood sanitation. Blood in the saliva sample, from bleeding gums to mouth sores, can contaminate the sample. The rejection point of contamination is when blood leaves a red tinge in the saliva specimen. To avoid blood in the saliva, patients should not brush or floss their teeth before collecting a sample.
Principle 7: Proper sample collection containers. A saliva sample can be soiled by improper collection containers—the most common of all contamination mistakes. Because saliva hormones are small molecules that have a static electric charge, they tend to stick to plastic materials that have the opposite electric charge, such as polyethylene.
The best plastic materials seem to be ultra-pure polypropylene and poly- styrene, which do not bind the salivary hormones, as opposed to recycled plastic.
Polyethylene tubes and stoppers readily absorb free hormones, especially progesterone. Therefore, they can absorb up to 87 percent of progesterone on the walls of the container. Although glass is the best container option because it is completely absorption free, it’s not always practical due to the risk of containers breaking during transport.
Principle 8: Exclusion of cortisol level studies. Using blood tests for cortisol levels may give inaccurate and false reports on stress, chronic fatigue, and obesity; the venipuncture itself is a severe stress factor to many patients and may result in false laboratory readings. It is difficult to get a correlation of blood sample cortisol levels to a physical or psychological stress event because no one can predict when or where these events are going to happen, nor will someone be available to draw blood samples during that time.
Saliva testing is easy and convenient. Multiple samples are gathered, which improves the chances of accuracy. Furthermore, saliva testing directly measures free hormone levels. It’s also more accurate when measuring very low levels of free hormones.8
Accurately measuring free hormone levels is essential to diagnosing, planning, and maintaining proper treatment, as well as balancing hormones for total health.
Paul Ling Tai, DPM, FACFS, ABPS, ABAARM, DACBN, is board certified in anti-aging medicine and a professor of clinical nutrition, chairman and president of the Brasil American Academy of Aging and Regenerative Medicine (BARM), and a double-board certified foot and ankle surgeon. He has been featured in renowned medical newsletters, television, and radio health talk shows worldwide. He can be contacted at firstname.lastname@example.org.
1 Reiss U. (2001). Natural Hormone Balance. New York, NY: Atria Books.
2 AARP. “Baby Boomers Turning 65.” http://www.aarp.org/personal-growth/transitions/boomers_65. Published Jan. 4, 2011. Accessed June 8, 2015.
3 Vining RF, McGinley RA. (1984). Transport of steroids from blood to saliva, in Immunoassays of Steroids in Saliva. GF Reid, D Riad-Fahmy, RF Walker, K Griffiths (eds.), pp. 56-63. Cardiff: Alpha Omega Ltd.
4 Khan-Dawood FS, Choe JK, Dawood MY. Salivary and plasma bout and “free” testosterone in men and women. Am J Obstet Gynecol. 1984;148:441.
5 Arregger AL. Salivary testosterone: A Reliable Approach to the Diagnosis of Male Hypo- gonadism. Clin Endocrinol. 2007;67(5):656-62.
6 The Endocrinology Society. J. Clinical Endo & Metab. 2001;86(6).
7 Taieb J, Mathian B, Millot F, Patricot M-C, et al. Testosterone measured by 10 immunoassays and by isotopedilution gas chromatography–mass spectrometry in sera from 116 men, women, and children. Clin Chem. 2003;49:1381-95.
8 Foley M; in communication, KMI Lab, Germany.