Saliva hormone testing

Saliva hormone testing is a non-invasive collection method where patients collect their saliva in plastic tubes in order to measure hormones like cortisol, estrogens, progesterone, and androgens. Saliva hormone testing is a non-invasive saliva collection is ideal for patients because it allows them to collect their sample in the privacy of their home or office. Patients are sent a test kit with easy to follow instructions for saliva collection.

Hormone is a chemical substance produced and secreted by endocrine (ductless) glands that travels through the bloodstream and controls or regulates the activity of another organ or group of cells – its target organ. (For example, growth hormone released by the pituitary gland controls the growth of long bones of the body.) There are two main types of hormones – steroids (e.g., estrogen, testosterone, aldosterone, cortisol) and nonsteroidal. Secretion of hormones is regulated by feedback mechanisms and neurotransmitters.

As a biological medium saliva is a variable and complex fluid and is mostly produced by three pairs of salivary glands (parotid, submandibular and sublingual) with a small contribution from the buccal glands which line the mouth ¹. In addition, saliva contains variable amounts of gingival crevicular fluid, which leaks from the tooth-gum margin, as well as plasma exudates, or blood, from oral abrasions or lesions. Hormones can enter saliva by a variety of mechanisms but for the neutral steroids the most common route is rapid diffusion through the acinar cells and as such their concentration is independent of the rate of saliva flow ². For charged steroids, like DHEAS, the mode of entry is by diffusion between the tight junctions of the acinar cells and its concentration is inversely related to saliva flow rate ³. Saliva pH also alters with saliva flow rate and hence affects the partitioning of charged steroids. Steroids can also enter saliva from blood or plasma via oral abrasions or directly from foodstuffs by contamination with exogenous steroids ².

As hormones play an important role in maintaining health, the assessment of hormone levels can help identify the cause of many health related conditions. In both men and women adequate levels of a number of hormones are necessary for optimal health and wellbeing.

The family of sex hormones, which includes the oestrogens – oestrone (E1), oestradiol (E2), oestriol (E3), progesterone, testosterone, DHEAS and cortisol, support a wide range of essential physiological functions. Variation in these hormones plays a large role in the changes seen in life cycle events such as pregnancy, menopause and ageing.

Baseline measurement of hormone levels in saliva provides an accurate assessment of menstrual irregularities for younger women, climacteric changes for the peri and postmenopausal women and andropause changes in men.

The connection between sex hormones and thyroid function, adrenal activity and liver detoxification may also be evaluated with appropriate testing and clinical history. Research shows that salivary hormone testing provides an accurate assessment of hormone status in both men and women of all ages.

Many factors contribute to altered hormone levels. These include environmental factors, such as chemical exposure and pesticides, lifestyle factors including smoking, alcohol, lack of exercise, poor diet and specific conditions such as infertility, endometriosis and enlarged prostate.

Saliva hormone testing advantages and disadvantages

The prime advantage of saliva testing for hormone is that it offers non-invasive, stress-free and real-time repeated sampling where blood collection is either undesirable or difficult. Saliva hormone testing is well suited for pediatric, time-shift and psychobiological studies. In addition, no special training or equipment is needed and subjects can conveniently collect samples themselves, if required. Salivary steroid levels can reflect the circulating level of free steroid rather than total circulating levels, which are confounded by the presence of circulating high affinity binding proteins ³. There are, however, disadvantages in the use of saliva for hormone levels testing. It can be a difficult matrix to deal with experimentally and may require physical or chemical disruption. Freeze/thaw cycles and centrifugation are often used to break up mucins and dithiothreitol treatment can facilitate filtration ⁴. The analysis of steroids in saliva can present analytical problems since they are present at far lower levels than in circulation. Existing assays may need to be adapted to improve sensitivity, although kits are available for measuring some steroids in saliva. Some use manual methods or automated platforms, designed for plasma, and care is required to address standardisation issues as well as the differing matrices of plasma and saliva ⁵. The possibility of blood contamination and its likely interference can be quantified but the presence of both sex hormone binding globulin (SHBG) and corticosteroid-binding globulin in uncontaminated saliva casts doubt on the reliability of salivary steroids to accurately reflect circulating free steroid levels ⁶. The presence of 11β-hydroxysteroid dehydrogenase type 2 and 17-hydroxysteroid dehydrogenase in salivary glands also complicates the relationship between salivary and plasma free steroids ⁷. During saliva sampling there is also the possibility of oral contamination by exogenously administered steroids. Another point, often overlooked, is that during the course of clinical investigation the physician is likely to investigate other analytes, besides salivary steroids, and more often than not a blood sample is likely to be taken.

Saliva hormone testing accuracy

Salivary androgens

Salivary androstenedione correlates well with free plasma concentrations (r = 0.92) and has been used in paediatric studies along with DHEA and testosterone ⁸. However, in the clinical setting, it has been deemed necessary that salivary androstenedione determination, along with progesterone and 17α-OH progesterone determinations, are performed following extraction and chromatography. This requirement is due to the presence of unidentified cross-reacting material in unpurified extracts ⁹. Salivary DHEA levels, following organic extraction, are reportedly lower in adult depression but salivary DHEAS analysis is probably not as useful as its concentration is dependent on salivary flow rate ¹⁰. This suggests an alternative transport mechanism into saliva. In addition, the high concentration of DHEAS in plasma could contaminate saliva levels via gingival fluid or oral abrasions ³. There are numerous reports of testosterone analysis in saliva and in psychobiological studies it appears to correlate with psychological parameters ¹¹. Salivary testosterone has also been used for monitoring i.v. replacement therapy with testosterone buciclate ¹². However, in view of the current controversies surrounding testosterone measurement by immunoassay at low concentrations in plasma, it would seem that the even lower testosterone concentrations in saliva would likely present an analytical challenge, although it is suggested that its reliability is acceptable for research purposes ¹³. There are, however, real concerns regarding storage and interference ¹⁴. In males, salivary testosterone declines with age and reflects testicular production although neither salivary testosterone nor plasma testosterone appears clearly superior to the other as a measure of bioavailability ¹⁵. Extraction-based assays for salivary testosterone may mitigate some problems and improve sensitivity as well as display acceptable correlation between either total or free testosterone in male plasma and saliva levels (r = 0.71 and 0.67, respectively). However, the correlation between plasma free testosterone and saliva testosterone in females is poor (r = 0.37) ¹⁴. This is in agreement with laboratory findings using extraction-based immunoassays for testosterone. The sex-related correlation between calculated free testosterone in plasma and salivary testosterone is shown in Figure 1 ¹⁶. Scientists conclude that mathematically derived indicators of bioavailable androgen status, using plasma total testosterone and sex hormone-binding globulin, are preferable as they overcome the limitations imposed by saliva.

Figure 1. Testosterone saliva test

Footnote: Correlation of calculated plasma free testosterone with salivary testosterone for females (a) and males (b). Testosterone analysis was by ELISA.

[Source ¹⁶ ]

Salivary progestins and estrogens

Congenital Adrenal Hyperplasia or CAH is an inherited genetic disorder that affects the adrenal glands. Those with Congenital Adrenal Hyperplasia lack one of the enzymes that the adrenal glands use to produce hormones that regulate metabolism, the immune system, blood pressure, and other essential functions. This can cause a number of symptoms, including accelerated puberty, infertility, and low blood pressure.

Congenital Adrenal Hyperplasia has been diagnosed by the immunoassay of 17α-OH progesterone in both plasma and saliva and an excellent correlation reported ¹⁷. An liquid chromatography/mass spectrometry method for saliva steroids has also been reported to effectively diagnose congenital adrenal hyperplasia in children ¹⁸. Latterly, commercial immunoassays for 17α-OH progesterone have been adapted and used to establish reference ranges in children. However, the use of oral collection devices for newborns induced stress responses, jeopardising both 17α-OH progesterone and cortisol determinations ¹⁹. A recent study examined the use of three different saliva collection systems, plain tube and either cotton or polyester Salivette®. Here, all saliva samples were centrifuged prior to assay, using a modified commercial assay, and no differences detected ²⁰. However, a shortcoming was the absence of comparisons with intact extracted saliva.

Progesterone is metabolically stable in saliva and has been used in a variety of applications ²¹. It has been used as an index of ovulation but there was a high degree of discrepant classifications between the follicular and luteal phases ²². Similar findings were reported recently where salivary progesterone in the mid-luteal phase yielded a sensitivity and specificity of 78% and 77%, respectively ²³. While suited for research and long-term clinical observation salivary progesterone determination may have a limited application for individual diagnosis ²⁴. The parallel changes of salivary progesterone and 17α-OH progesterone over the menstrual cycle were suggested as an additional parameter to assess ovarian function ²⁵. Similarly, paired determinations of progesterone and estradiol in saliva may be useful for fertility monitoring although wide variations in oestradiol were noted ²⁶. Probably, the most reliable way of assessing corpus luteal function is via plasma progesterone and if noninvasive testing is required the best option is the determination of urinary pregnanediol excretion indexed to creatinine ²⁷. Salivary progesterone determinations were used to monitor the absorption of progesterone following topical application of progesterone cream to pre- and postmenopausal women. Paradoxically, salivary progesterone levels were elevated whereas plasma and urinary progesterone and its metabolites remained low suggesting minimal benefit to target tissues using this alternative replacement therapy ²⁸.

Salivary cortisol

There is little information on 11-deoxycortisol in saliva, presumably due to its very low level, although it can be detected following metyrapone ²⁹. On the other hand, salivary cortisol determinations are more promising. Since the earliest reports, there is evidence that saliva levels reflect unbound concentrations in plasma ³⁰. Direct measurement in saliva may be comparable to extraction but extraction has the advantage of allowing the analysis of low volume saliva samples. This can avoid the use of stimulants and the loss of data due to insufficient sample volume ³¹. Salivary cortisol determinations have proved popular in psychobiology, stress and sports medicine studies ³². Their use is based on the assumption that salivary cortisol is a reasonable reflection of hypothalamic-pituitary- adrenal (HPA) axis function. In the diagnostic setting, salivary cortisol levels parallel those in plasma following ACTH and CRH stimulation, and following exercise induced-stress ³³. However, the correlation of salivary cortisol levels with total plasma cortisol is confounded by the presence of corticosteroid-binding globulin in plasma which is largely saturated up to 500–600 nmol/L of cortisol ³⁴. Salivary cortisol correlates better with measured plasma free cortisol than total plasma cortisol. However, it appears to be subject-specific as considerably variability is found between individuals for daily paired samples (Figure 2). Salivary cortisol determinations were used as markers of metabolic disturbances in obese and diabetic patients and used to investigate changes in glucocorticoid control of the hypothalamic-pituitary- adrenal axis following oral prednisolone ³⁵. Conversely, salivary cortisol measurement is not a useful tool in determining dose adequacy in subjects on oral glucocorticoid replacement therapy ³⁶. Blood spots or serum is preferable, due to contamination of saliva by oral hydrocortisone. The diurnal variation of plasma cortisol is reflected by similar changes in salivary cortisol and hence timed salivary cortisols have been used in a diagnostic setting. Early morning salivary cortisols are useful as a screening tool for adrenal suppression and salivary cortisol is helpful to investigate the control of diurnal cortisol secretion following exposure to darkness and light ³⁷.

Recently, there has been considerable interest in the use of night time salivary cortisols for the initial screening for Cushing’s syndrome. However, despite the optimism, an element of caution is required. Reported cut-off values differ considerably. Papanicolaou et al. ³⁸ determined a normal night time (2330–2400 hour) saliva cut-off of 15 nmol/L with values above this threshold suggestive of Cushing’s. They reported 100% specificity and 91% sensitivity for saliva, similar to a late night serum cortisol measurement cut-off value exceeding 240 nmol/L ³⁸. Both were reportedly clearly superior to urinary free cortisol excretion, contrasting with other recent reports. Yaneva et al. ³⁹ determined a lower saliva cut-off value of 5.7 nmol/L, with 100% sensitivity and 96% specificity which effectively separated Cushing’s from obese subjects ⁴⁰. The corresponding excretion of urinary free cortisol determined a cut-off value of 248 nmol/day, with similar sensitivity and specificity to saliva. Viardot et al. ⁴⁰. reported a similar night time saliva cut-off value of 6.1 nmol/L and view salivary cortisol as a reliable first line alternative to urinary free cortisol, the urinary free cortisol:creatinine ratio, and the 1mg overnight dexamethasone suppression test. However, their cut-off value for urinary free cortisol was considerably higher at 504 nmol/day compared to the 248 nmol/day reported by the Yaneva group ³⁹. Both groups used extraction based assays for urinary free cortisol which, as an aside, highlights the importance of well characterised methods and reagents for the determination of urinary free cortisol.

Late night salivary cortisol measurement is nevertheless very promising for the diagnosis of Cushing’s although elevation above threshold values can occur in the elderly, diabetics and women in late pregnancy ⁴¹. The variations in reported late night salivary cortisol cut-off values could result from the relatively small numbers in each of the study control groups, which may have varying degrees of obesity, non-adrenal disorders and pseudo-Cushing states, which themselves may be influenced by periodic hypercortisolism ³⁸. Methodological and standardisation issues are also likely contributors to differences in reported cut-off values but a major factor is probably differing specificities of cortisol antibodies towards cortisone. The salivary gland has abundant 11 β-hydroxysteroid dehydrogenase type 2 activity and as a consequence, saliva, unlike plasma, has up to three times the level of cortisone compared to cortisol ⁴². Depending on the relative cross-reactivity of cortisol antibodies towards cortisone, there could be quite different values of salivary cortisol measured by different immunoassays. Conversely, differences in plasma would be expected to be minimal as cortisone levels are normally only 10% of circulating cortisol levels ⁴². It is therefore desirable that laboratories establish their own method-specific reference ranges before using salivary cortisol for diagnostic purposes.

Figure 2. Cortisol saliva test

Footnote: Correlation of salivary cortisol and plasma free cortisol in paired samples from two normal individuals (a and b). Salivary cortisol was measured by ELISA and plasma free cortisol by ligand binding/ultrafiltration.

Summary

Salivary steroid testing has a recognised place in research and diagnostic medicine although its limitations must be acknowledged. It is clearly not desirable for androgen assays as well as assays to assess ovarian function and the monitoring of absorption of steroids from transdermal creams. Caution must be exercised for these applications. The use of salivary cortisol for measuring endogenous cortisol is the most encouraging. It can be successfully applied to research studies, adrenal stimulation tests, investigating diurnal variation as well as night time samples as a screening test for Cushing’s syndrome. However, there is a need to establish methodspecific reference ranges and sample stimulation, collection and storage should remain consistent across study groups with a preference for the collection of whole unstimulated saliva, if possible.

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