Article by Michel Y. Farhat, Ph.D and Elias F. Ilyia, Ph.D

The use of saliva as a vehicle for determination of plasma steroid hormone levels has increased dramatically in recent years. Since 1983, more than 2500 papers and research articles dealing with salivary diagnostic tests have been published1. Both clinicians and investigators have used saliva to assess numerous clinical problems including, digitalis toxicity, celiac disease, liver function and immunodeficiency. Saliva has also been used for pharmakicokinetic studies and therapeutic drug monitoring in a variety of clinical situations. The value of saliva as a monitoring medium resides in the fact that it is easy to collect, store and ship, and is non-invasive, thus convenient for multiple sampling.

Plasma-saliva transfer

The reliability of saliva testing depends on establishing a direct correlation between saliva and plasma concentration of a particular substance. The transfer of substances from plasma into the saliva is dependent on their physiochemical properties1. A small molecular weight and a great lipid solubility are normally associated with a faster transfer rate. A good correlation has also been established between saliva/plasma ratio of substances, their pKa and salivary pH. Salivary flow rate and the existing pathophysiology of the oral cavity have also been shown to affect salivary distribution of substances.

Saliva concentration of a particular hormone is dependent on the affinity and total binding capacity of various binding proteins in plasma. As blood passes through salivary glands, free "unbound" and weakly bound (low affinity binding protein) forms of hormones will diffuse through the salivary gland epithelium into the saliva. As in other clearing organs, membrane transfer occurs in both directions, is passive for most substances and equilibrium is governed by the transmembrane concentration gradient. Thus, saliva levels reflect the free concentration of hormones in plasma, and in the absence of high affinity, high capacity-binding proteins, these levels correlate with plasma concentrations.

Salivary steroid hormone analysis

Monitoring plasma steroid levels is essential for the clinical assessment of a patient's endocrine function. Saliva becomes an important diagnostic tool, since in many instances, the standard plasma and urine sampling techniques may not provide the optimum sampling conditions. Some of the problems that diagnostic laboratories had to overcome in establishing the validity of salivary steroid assays, were to determine whether steroid concentration in saliva can be measured with accuracy and whether these small values are meaningful and correlate with plasma levels or any other physiological parameter. ,Salivary steroid levels tend to be much lower than those in plasma, because they reflect the level of unbound steroid, which
represents about 2-5% of total plasma concentration. Salivary glands may also transform certain steroids during their passage across the salivary epithelium3. Highly polar molecules, such as sugars and conjugated steroids do not cross the lipid lining of cells and can only get into whole saliva via blood contamination from small cuts and ulcers or from gingival fluid. Here we review the pharmacodynamics and partitioning of few hormones, that have been characterized in saliva and evaluate the correlations between their saliva and plasma concentrations.

Progesterone

Progesterone was one of the first steroids to be reliably assayed in human saliva. Once saliva samples are collected, salivary progesterone concentrations remain stable under a wide range of handling conditions. Because of its high circulating blood levels (ng/ml during luteal phase), saliva concentrations of the hormone usually remain within the limits of sensitivity of most conventional assays. Average salivary progesterone concentrations vary from 20-100 pg/ml during the follicular phase to 100-500 pg/ml during the luteal phase (Figure 1). Progesterone levels are also affected by women's age, degree of activity, nutrition and race4,5. A high correlation coefficient (between 0.8 and 1.0) has been established between plasma and salivary levels of the hormone6,7, making saliva a useful diagnostic tool. Serial measurement of salivary progesterone has been used to assess ovarian function, and in particular for diagnosis of defective or inadequate luteal function, as well as to monitor response to hormone therapy. Other clinical applications may include monitoring placental function by repetitive measurement of salivary progesterone during pregnancy9.

Estradiol

Salivary estradiol (E2) is about 1-2% of total plasma values, Earlier studies, using assays with variable sensitivities and specificities, have described a wide range of salivary estradiol levels. Three independent studies using radioimmunoassay10-12, enzyme immunoassay13 and chemi-luminescence immunoassay14 have reported comparable salivary E2 levels during normal nonstimulated menstrual cycles ranging from 5-15, 10-30 and 7-20 pmol/L during the follicular, periovulatory and luteal phase, respectively (Figure 2). These levels in stimulated cycles ranged from 10 to 120 pmol/L15. Moreover, Wong et al.10 showed a mid-cycle salivary E2 peak, corresponding to the mid-cycle LH surge, followed by a midluteal rise. In these women, the changes in salivary E2 were similar to the E2 pattern in serum, with a high degree of correlation (r=0.93). A similar correlation (r=0.96) was observed between salivary E2 + estrone and free plasma E2 in FSH-stimulated cycles13. Testosterone Under physiological conditions, a very good correlation exists between salivary and serum testosterone (T) in men16,17. Wang et al. have demonstrated that following exogenous T

Salivary cortisol exhibits clear diurnal variation and circadian rythmicity both in normal and depressed individuals. Salivary cortisol is highest in the morning and varies between 13 and 23 nmoles/L These levels decrease significantly during the day and reach their lowest value at night (1-3 nmoles/L) (Figure 3).

Dehydroepiandrostenedione (DHEA)

DHEA ia an adrenal steroid produced in abundant amounts and is conjugated to sulfate to form DHEAS before its release into the
circulation32. DHEA and DHEAS are interconvertable and exist in dynamic equilibrium with each other33. Salivary DHEAS is found in saliva at about 0.1% of its plasma concentration. Serum fluctuations in DHEA(S) concentrations are accurately and rapidly reflected in salivary levels.34Other hormones

Salivary estriol levels are also high, easy to measure and correlate well with plasma unbound unconjugated estriol35,36. Androstenedione can also be measured with sufficient accuracy in saliva. The absence of specific high affinity binding proteins for androstenedione, results in a linear relationship between plasma total and plasma free fraction, and hence an excellent correlation between plasma and saliva levels of the hormone37.

Conclusion

The above studies clearly demonstrate that saliva is a useful diagnostic tool for measurement of steroid hormones. Salivary concentration represents the free form of a particular hormone, and thus is a true reflection of its bioactivity. Moreover, the non-invasive nature of saliva collection and the convenience of multiple sample facilitate the design of functional assays for the assessment of various endocrine functions. In a recent review, Mandel38 has likened saliva to a mirror reflecting the emotional, hormonal, immunological as well as nutritional and metabolic status of the body. The broad spectrum of interactions and relationships among these factors opens a whole field of diagnostic possibilities worth exploring and evaluating.


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