All Things Saliva
03.15.08
Saliva and Its Use as a Diagnostic Fluid
Saliva is the familiar fluid present in the mouths of humans and some animals, which serves principally to moisten and lubricate food. In addition, it contains enzymes that begin the process of digestion, it aids our sense of taste, and it helps cleanse and protect the teeth, gums, and other tissues inside the mouth. (1,2)
The Salivary Glands
Human saliva is produced by glands in various locations in and around the mouth. Three primary glands occur in pairs located symmetrically on both sides of the head: the parotids, the submandibulars (also known as the submaxillarys), and the sublinguals. In addition to the primary glands, there are also hundreds of smaller glands located in the lips, cheeks, tongue, and palate. Although the parotid glands are the largest in size, they produce only about 25% of the total saliva in the unstimulated rest state, and the minor glands and the sublinguals together contribute only about an additional 5% . The submandibulars are by far the most active glands in the unstimulated state, and they are estimated to produce about 70% of the total rest volume. (3)
Figure 1. Locations of primary salivary glands.
Salivary Gland Structure
The primary salivary glands are composed of numerous clusters of 15 to 100 secretory cells arranged in globular or tubular configurations. These clusters are called acini (singular acinus.) The acini open into ducts, which merge to carry the saliva towards the mouth. Duct cells also transport electrolytes in and out of saliva, and they can participate in secretory activity to a limited degree. The acini and ducts are surrounded by myoepithelial cells, which can contract to help accelerate saliva flow. (4)
Figure 2. Cross section of a non-realistic salivary gland, showing three types of acini.
Acini are composed principally of two types of secretory cells, serous and mucous, which are both specialized for the production of large quantities of proteins. Serous cells produce a thin, watery saliva containing the digestive enzyme a-amylase. Mucous cells produce a thicker saliva rich in mucins–a type of glycoprotein–which help lubricate food for swallowing.
The proportions of serous and mucous cells are different in the various salivary glands, and each gland secretes a saliva that reflects the cellular makeup of its acini. (5,6) (See Table 1.)
| Table 1—Salivary Gland Secretions | |
|---|---|
| Gland Type | Saliva Type |
| Parotid, and Von Ebner’s (on the tongue) | Serous |
| Submandibular | Mixed, more serous than mucous |
| Sublingual | Mixed, but mostly mucous |
| Most minor | Mucous |
The Composition of Saliva
Saliva is principally a mixture of water and electrolytes; both pass into the acini from a dense network of capillaries that surround the salivary glands. The initial product secreted by the cells in the acini has concentrations of sodium, potassium, chloride, and bicarbonate ions similar to plasma. As the saliva passes through the ductal regions of the glands sodium and chloride ions are absorbed, and additional potassium and bicarbonate ions are secreted. (See figure 3.) The total ionic concentration of the final product is lower than that of plasma. (6,7)
Figure 3. Ion exchanges during saliva production.
Ionic concentrations change as saliva production is stimulated, however, and concentrations of sodium, chloride, and bicarbonate ions all increase with accelerated flow. As bicarbonate levels increase the pH of saliva changes from slightly acidic (6-7) to slightly basic (around 8). (6,7) Changes in the pH of the saliva can be a concern for saliva testing because pH can have an effect on the amount of ionic charge present on certain drugs or other compounds. The presence of these charges can affect the ability of the compound to diffuse through neutral lipid membranes, and be present in saliva. (8)
Saliva also contains organic compounds that are synthesized primarily in the cells of the acini, and also to a lesser extent by some ductal cells. These organic products are mostly proteins or peptides, including enzymes, mucins, lactoferrin, cystatins, and histatins. (9) Nutrients needed for the synthesis of these compounds pass from the capillaries surrounding the glands into the cells, either by simple diffusion, or by active transport mechanisms.(6) Immunoglobulins released from nearby B-lymphocytes are also transported through cell membranes of the salivary cells and secreted into the saliva. (9)
The many components of saliva serve a wide range of functions. Some of the more important of these are summarized in the Table 2. (10)
| Table 2-Functions of Saliva Components | |
|---|---|
| Mucins | Lubricate food; Protect teeth against acid; Help protect against bacteria, viruses, fungi |
| Digestive Enzymes | a-Amylase – digests starches, Lipase – digests fats, Protease – digests proteins |
| Lysozyme, Peroxidases, Lactoferrin, Histatins, Cystatins | Anti-bacterial agents |
| Secretory Immunoglobulin A, Histatins, Cystatins | Anti-fungal, anti-viral agents |
| Bicarbonate ions, Phosphate ions, Proteins | Help protect teeth and soft tissues against acidic conditions |
| Calcium ions, Phosphate ions, Proline-rich proteins | Help maintain mineral content of tooth enamel |
Whole Saliva
The whole saliva that pools on the floor of the mouth is a mixture of the fluids secreted by all of the various saliva glands, and it may also contain the following components in varying degrees:
- Bronchial and nasal secretions
- Fluid that comes from the junctions between gums and teeth (gingival crevice fluid or GCF)
- Blood and serum from wounds in the mouth, including the gums if they are not healthy
- Micro-organisms (bacteria, viruses, fungi) and products derived from them, including enzymes
- Assorted cellular components and food debris
The Control of Saliva Secretion and Composition
Saliva production changes throughout the course of the day. It is greatest during the waking hours, and diminishes greatly during sleep. (11) Various stimuli including taste, smell, and chewing motions of the jaw greatly increase saliva flow. (2,11,12) Control over saliva production is shared by the sympathetic and parasympathetic branches of the autonomic nervous system, which work together in a complex relationship. The parasympathetic system is largely responsible for increases in fluid secretion by the salivary glands, but the sympathetic system also plays a smaller role. Both systems can signal the myoepithelial cells in the salivary glands to contract, increasing the flow of saliva. (13)
Concentrations of some components in whole saliva can be altered because of differing flow rates from the principal glands. While in the unstimulated rest state, the parotid glands contribute only a relatively small proportion of the total mix, and the viscous, mucin-rich saliva from the sub-maxillary and sub-mandibular glands predominates. When stimulated, however, the parotid glands disproportionately increase their output of watery saliva, effectively lowering the concentration of mucins in the mixed saliva. (10)
Activity in the sympathetic and parasympathetic systems also stimulates secretory cells in the salivary glands to increase the secretion rates of specific compounds that are synthesized in the cells. These compounds are stored in small granules within the cell, and they can be quickly released into the saliva when the signal is given. (13) An interesting example of this ability involves the body’s reaction to stress through the sympathetic system. Recent research has confirmed that stressful situations cause the cells to increase a-amylase levels in saliva, and that these increases are independent of salivary flow rates. (14) This relationship is useful to researchers, since it provides a simple, indirect way to measure activation of the sympathetic nervous system.
The Movement of Substances from Blood into Saliva
In addition to the organic compounds that are produced locally in the saliva glands, there are some that pass into saliva from blood. These compounds include drugs, drug by-products, hormones, and some proteins. The presence of these compounds in saliva has spurred research into its use as a diagnostic fluid, especially in view of the relative ease and safety of collection it offers when compared to more traditional diagnostic fluids such as blood and urine. (8)
Figure 4. Movement of compounds through membrane into saliva.
The most common way for substances to migrate from blood to saliva is believed to be by unaided, or passive, diffusion. As described above, the capillaries surrounding the salivary glands are quite porous for many substances. Materials can pass from the blood system into the space surrounding the glands, and then make their way directly through the membranes of acinus or duct cells. The ability of a molecule to diffuse passively through cell membranes depends partly on its size, and partly on how much electrical charge it carries. If a molecule is polar in nature, or if it separates into charged ions while in solution, it will have a hard time passing through the membranes, which are made out of neutral fatty compounds called phospholipids. Steroid hormones are relatively small in size, and most of them are fatty, non-polar compounds, so they tend to pass relatively easily by diffusion. Other molecules such as the large protein hormones, or hormones or drugs that are bound to large carrier proteins while in the bloodstream, are too big to enter by this route. (15)
A second pathway used by molecules to enter saliva is by filtering through the tight spaces between acinus or duct cells. In order to do this they must be relatively small. Sulfated steroids such as dehydro-epiandrosterone sulfate (DHEA-S) and estriol sulfate, which are not able to pass through the fatty cell walls because of their electrical charges, are believed to enter principally by the filtration route. Compounds such as DHEA-S are slower to migrate into saliva than the neutral steroid hormones, and when saliva output is stimulated they may move too slowly to keep up with the accelerated flow rates, causing concentrations in saliva to drop. (15)
Blood components can also gain entry into saliva from the outflow of the serum-like gingival crevicular fluid (GCF) from the gums, or from small injuries or burns in the mouth. GCF is believed to be a major route by which certain molecules, which would ordinarily be too large to pass by either diffusion or filtration, can find their way from serum into saliva. (15,16)
A fourth pathway for the entry of a substance into saliva is by active transport through the secretory cells of the glands, which is the route used by secretory immunoglobulin A (SIgA). Polymeric IgA is secreted by B-lymphocyte cells close to the salivary cells, then bound and transported across the cells by a Polymeric Immunoglobulin Receptor, and finally released into salivary secretions. (17) It has been shown that secretion of SIgA is increased by nervous stimulation of the saliva glands, but the exact manner in which the transport is accelerated is not yet understood. (18) Saliva flow rates are also affected by stimulation, and this effect is greater than the increase in the secretion rate. SIgA concentrations in saliva are known to decrease as saliva flow is stimulated. (19)
The Use of Saliva Testing for Hormones
Due to the ease with which saliva can be collected, it is an appealing medium for hormone studies that require multiple samples to be taken over the course of the day. In addition to simply being more convenient, saliva testing can actually be preferable to serum testing in several ways. First, for hormones such as cortisol that reflect stress levels, the collection of a saliva sample is much less invasive and stress-inducing than blood collection. Using saliva as a testing medium should therefore help avoid measurement of reaction to the collection process itself. Secondly, measurement of steroid hormone levels by salivary testing is actually preferable to serum measurement because the presence of specific and non-specific binding proteins in serum complicates attempts to measure the levels of active hormones. In the bound form, the hormone is not biologically active, and it is also too large to pass into saliva. Only a small, unbound fraction of the hormone is available to diffuse into the saliva, and for this reason salivary steroid hormone levels are consistently lower than in serum. The low level of a hormone measured in saliva is believed to be a direct measure of the biologically active, free fraction in serum. (20)
One of the most-studied steroid hormones is cortisol, and it has been demonstrated that salivary cortisol levels have a steady and predictable relation to the free, unbound cortisol levels in serum. It has also been shown that the rate of equilibrium of cortisol between blood and saliva is rapid, which helps insure that cortisol levels in saliva do accurately reflect the free-serum levels regardless of the degree of stimulation of the saliva glands. (21) Commercials kits for assaying cortisol levels in saliva are widely used to identify patients with Cushing’s syndrome and other diseases, as well as in research to investigate the role cortisol plays in the body’s response to stress.
Other steroid hormones have been studied in saliva, and a number, including progesterone, testosterone, the various estrogenic compounds, and common precursor molecules, have also been shown to have stable relationships between free-serum and saliva levels, and sufficiently rapid migration rates. (22,23) Like cortisol, the levels of these hormones measured in saliva are lower than in serum, and for some like estradiol and testosterone the saliva levels can be very low, requiring assay methods with very high sensitivity.
The Growing Use of Salivary Testing
Given the ease with which saliva samples may be collected, it is an ideal medium for monitoring the use of illegal drugs or other harmful substances, such as tobacco, and numerous test devices are being introduced to serve this need. Another notable development is the recent introduction of the first saliva test kit to check for the presence of antibodies to the HIV virus.
It is also becoming increasingly clear that saliva contains many more substances than had been previously realized. The UCLA Human Salivary Proteome Project, funded by the National Center for Dental and Craniofacial Research, has already identified more than 1000 proteins in the saliva of healthy individuals, and its participants are now studying the saliva of individuals with various diseases, looking for substances that could be used for screening and diagnostic purposes. Studies are already beginning to report results, such as the identification of RNA molecules that are associated with oral cancer, which could lead to practical tests for the disease in the near future. As additional substances of interest are discovered in saliva, methods to assay their presence quickly and efficiently will need to be developed, and made commercially available.
References
1. Mese, H. and Matsuo, R. (2007). Salivary secretion, taste and hyposalivation. J Oral Rehabil, 34(10),711-23.
2. Spielman, A.I. (1990). Interaction of saliva and taste. J Dent Res, 69(3), 838-843.
3. Medical College of GA, Section VI: Gastrointestinal Physiology, p. 4. Retrieved from the internet, 9/11/07 at http://www.lib.mcg.edu/edu/eshuphysio/program/section6/6cch4/s6ch4_4.htm.
4. Boron, W.F. and Boulpaep, E.L. Medical Physiology: A cellular and molecular approach. Saunders (Elsevier Science), Philadelphia, 2003, pp. 908-29.
5. Seeley, R.R. and Stephens, T.D. Anatomy and Physiology, 2nd ed. St. Louis, Mosby Year Book, 1992, pp. 774-780.
6. Guyton, A.C. and Hall, J.E. Textbook of Medical Physiology, 10th ed. Philadelphia, Saunders, 2000, pp. 739-42.
7. Johnson, L.R., ed. Essential Medical Physiology, 3rd ed. San Diego, Elsevier Academic Press, 2003, pp. 499-502.
8. Kaufman , E. and Lamster, I.B. (2002). The diagnostic applications of saliva–A review. Critical Reviews in Oral Biology & Medicine, 13(2), 197-212.
9. Nieuw Amerongen, A., Bolscher, J.G.M., and Veerman, E.C.I. (2004). Salivary proteins: Protective and diagnostic value in cariology? Caries Research, 38, 247-253.
10. Adapted from Nieuw Amerongen, A., Ligtenberg, A.J.M., and Veerman, E.C.I. (2007). Implications for diagnostics in the biochemistry and physiology of saliva. Ann. N.Y. Acad. Sci, 1098, 1-6.
11. Thie, N.M., Kato, T., Bader, G., Montplaisir, J.Y., and Lavigne, G.J. (2002). The significance of saliva during sleep and the relevance of oromotor movements. Sleep Med Rev, 6(3), 213-27.
12. Lee, V.M. and Linden, R.W. (1992). An olfactory-sumandibular salivary reflex in humans. Exp Physiol, 77, 221-224.
13. Garrett, J.R. (1987). The proper role of nerves in salivary secretion: A review. J Dent Res, 66(2), 387-397.
14. Rohleder, N., Wolf, J.M. Maldonado, E.F., and Kirschbaum, C. (2006). The psychosocial stress-induced increase in salivary alpha-amylase is independent of saliva flow rate. Psychophysiology, 43 (6), 645-52.
15. Vining, R.F., McGinley, R.A. and Symons, R.G. (1983). Hormones in saliva: Mode of entry and consequent implications for clinical interpretation. Clin Chem, 29(10), 1752-56.
16. Embery, G. and Waddington, R. (1994). Gigival crevicular fluid: Biomarkers of periodontal tissue activity. Adv Dent Res, 8(2), 329-36.
17. Mostov, K.E. (1994). Transepithelial transport of immunoglobulins. Annu Rev Immunol, 12, 63-84.
18. Proctor, G.B., Carpenter, G.H., Anderson, L.C. and Garrett, J.R. (2000). Nerve-evoked secretion of immunoglobulin A in relation to other proteins by parotid glands in anaesthetized rat. Experimental Physiology, 85(5), 511-518.
19. Kugler, J., Hess, M., and Haake, D. (1992). Secretion of salivary immunoglobulin A in relation to age, saliva flow, mood states, secretion of albumin, cortisol, and catecholamines in saliva. J Clin Immunol, 12(1), 45-9.
20. Gozansky, W.S., Lynn, J.S., Laudenslager, M.L., and Kohrt, W.M. (2005). Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity. Clin Endocrinol (Oxf), 63(3), 336-41.
21. Vining, R.F., McGinley, R.A., Maksvytis, J.J., and Ho, K.Y. (1983). Salivary Cortisol: a better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem , 20(6), 329-35.
22. Read, G.F. (1993). Status report on measurement of salivary estrogens and androgens. Saliva as a diagnostic fluid, ed. Daniel Malamud and Lawrence Tabak. Annals of the New York Academy of Sciences, 694, 146-160.
23. Ellison, P. (1993) . Measurements of salivary progesterone. Saliva as a diagnostic fluid, ed. Daniel Malamud and Lawrence Tabak. Annals of the New York Academy of Sciences, 694, 161-76.
