The diagnostic uses of saliva
Irwin D. Mandel Columbia University, School of Dental and Oral Surgery, New York, USA
Mandel ID: The diagnostic uses of saliva. J Oral Pathol Med 1990; 19: 119-25. It is becoming increasingly apparent to investigators and clinicians in a variety of disciplines that saliva has many diagnostic uses and is especially valuable in the young, the old and infirm and in large scale screening and epidemiologic studies. The highly sensitive test procedures that are now commonplace makes it practical to quantitate, despite very low concentrations, a large number of hormones and drugs in saliva. Indeed, all steroids of diagnostic significance in routine clinical endocrinology can now be readily measured in saliva. Drug monitoring can include abusive as well as therapeutic agents. The concordance between anti HIV antibodies in saliva and serum has stimulated application to various other antiviral antibodies as well as to viral antigens per se. Saliva has found use as a diagnostic aid in an increasing number of clinical situations and in systemic diseases that can affect salivary gland function and composition such as Sjogren's syndrome, cystic fibrosis and diseases of the adrenal cortex. The list keeps growing.
SaHva is not one of the popular bodily fluids. It lacks the drama of blood, the sincerity of sweat and the emotional appeal of tears. Despite the absence of charisma, however, a growing number of internists, pediatricians, pharmacologists, clinical and forensic pathologists, psychologists and dentists are finding that saliva provides an easily available, noninvasive diagnostic medium for a rapidly widening range of diseases and clinical situations (1, 2). The limited utilization of saliva for diagnostic purposes in the past may be a reflection of the ambivalence that has characterized both the public and professional view of the nature of saliva over the ages. Prior to the 17th century and the anatomic demonstrations by STENSON and WHARTON of the ducts that bear their name, salivary glands were thought to be accessory excretory organs, emunctories, that strained off the evil spirits of the brain (3). With the realization that the glands could generate an external secretion, physicians who practiced medicine based on humoral pathology, the need to balance the body humors (phlegm, blood, yellow bile and black bile), bled, blistered, purged and stimulated salivation. It was not uncommon to prescribe massive doses of bichloride of mercury to cleanse the system by causing saliva to literally pour from the mouth. It was the fortunate patient who survived both the disease and the treatment.
This negative image of saliva, however, was not uniform. In the cosmologies of ancient Egypt, Thoth the wise is said to have spat into the empty eye socket of Horus, the sun god, to restore his vision (4). The new testament (Mark 8: 23-25) tells us that Jesus took the blind man by the hand and led him out of town; and when he had spit on his eyes, and put his hands upon him, he asked him if he saw ought; and he was restored and saw every man clearly. The people who really appreciate the "miracle" of saliva, however, are not the blind, but those who suffer from xerostomia (dry mouth) from a variety of causes (5). They recognize belatedly that saliva is a natural resource with many functional capabilities that include food preparation, digestion, lubrication and protection of the teeth and mucous membranes (6). One function not bestowed by nature is the use of saliva for diagnostic purposes. Historically this diagnostic value may have been recognized first by the ancient judicial community who employed salivary flow (or its absence) as the basis for a primitive lie detector test. The accused was given a handful of dry rice. If anxiety (and presumably guilt) so inhibited salivation that he or she could not form an adequate bolus to chew and swallow, than off with their head. In more recent times, where the vagaries of the secretory-motor system have been replaced by those of the court
Key words: drugs in saliva; hormones in saliva: saliva: salivary chemistry; salivary diagnosis: salivary nnonitoring: siaiochemistry. irwin D. Mandei, Coiumbia University, School of Dental and Oral Surgery, 630 West 168th Street, New York, New York 10032, LISA. Accepted for publication December 22, 1989.
system, saliva found its widest use at the race track where the saliva test for drugs became the determinant of a "fixed" horse race. It is interesting to note that both the ancient and modern use of saliva are different forms of lie detection. Apparently ';";; saliva veritas'. Sample collection
The fiuid most frequently employed for salivary diagnostic purposes is expectorated whole saliva, a mix composed largely of the secretions from the major salivary glands, the right and left parotid, submandibular and sublingual glands. There are also modest contributions from the minor salivary glands (which are distributed inside the lips and cheeks, on the palate and under the tongue) and the gingival crevicular fluid from the spaces between the teeth and the gingiva. This latter Ouid is primarily a serum transudate but can also contain cells and products of the inflammatory process when gingivitis or periodontitis is present. Due to the presence of large numbers of bacteria continually loosed from tooth and soft tissue surfaces, as well as shed epithelial cells, whole saliva usually requires centrifugation to provide a clear sample. In some situations, however, the bacteria or cells have diagnostic value. Unstimulated or resting saliva is usually collected by passive drooling into a graduated tube or preweighed
vial so that fiow rate per unit time can be measured. The concentration of some salivary constituents is fiow rate dependent (1). When volume measurement is not required the saliva can be collected on cotton swabs, cotton rolls, gauze or filter paper strips, then eluted or centrifuged, or, aspirated directly from the fioor of the mouth with plastic pipettes. For evaluating salivary gland function or when large volumes of saliva are required for analytic purposes, saliva is stimulated by a masticatory or gustatory stimulus, expectorated and handled in a similar manner as the unstimulated fluid. Softened paraffin wax or a washed rubber band are the usual masticatory stimuli and 2% citric acid applied directly to the tongue the standard gustatory stimulus (1, 5). In many situations separately collected secretions from individual glands are preferable and this can be accomplished in a non-invasive manner with suitable collecting devices (1, 5). Parotid saliva is best collected with plastic modifications of a simple cup first introduced by CARLSON & CRITTENDEN in 1910
Disposable and individualized collectors have also been introduced (8, 9). Submandibular-sublingual saliva can be collected by customization of a basic plastic collector (10, 11) or by aspiration from the duct openings with a micropipette (5). Oral diseases
For many years dental investigators have been exploring changes in salivary flow rate and composition as a means of diagnosing and monitoring a number of oral diseases (1, 2). The examination of secretions collected from individual glands has been especially valuable in the differential diagnosis of local diseases of the salivary glands such as infiammatory and autoimmune diseases and in evaluating the effects of pharmacologic agents and therapeutic regimens which impact on salivary function (1,
5). Since individual gland secretions contain some locally shed cells attempts have been made to use saliva for cytologic examination as an aid in diagnosis of tumors, but so far cytology is being restricted to fine needle biopsy (12). Shed buccal mucosal cells, however, are being used as a simple, non-invasive method for obtaining DNA for gene analysis in cystic fibrosis and is applicable to other disease as well (13). Saliva collected by expectoration contains a fair representation of the bacterial and fungal species that coat the teeth, tongue and mucous membranes. By culturing a known volume of saliva (in various dilutions) on selective media a quantitative determination can be made of specific organisms. Counts of Streptococcus mutans and lactobacilli are being employed for identifying children at high risk for enamel caries (14, 15) and older adults susceptible to root surface caries (16). Oral candidiasis is frequently found in people wearing complete dentures, in people with a marked diminution in fiow rate due to a variety of therapies and medications, and in HIV infection. Several recent studies have shown that quantitation of Candida (usually albicans) from whole saliva can be used as an indicator of infection (17-19). System diseases affecting saliva
With the increase in investigator interest it is becoming apparent that many systemic diseases affect salivary gland function and/or salivary composition (Table 1) (1). Although the study of these effects have been valuable in the understanding of the pathogenesis of the diseases, their use as diagnostic markers have been more limited, but none the less helpful in some situations. In Sjogren's syndrome, an autoimmune disease which affects several million people, one of the main diagnostic procedures is biopsy of the minor salivary glands of the lip (the labial glands) to assess the presence of and the extent
Table 1. Systemic diseases affecting salivary glands and saliva Sjogren's syndrome Rheumatoid diseases Graft VS host disea.se Sarcoidosis Cystic fibrosis Hypertension Hyperlipidemia Alcoholic cirrhosis Malnutrition
Hromonal dysfunction Diabetes Pancreatitis Adrenal-cortical diseases Thyroiditis Acromegaly Neurological diseases Parkinsonism Bell's palsy Cerebral palsy
of the lymphocytic infiltration characteristic of this disease (20). Siaiochemistry provides a helpful screening procedure to determine whether the biopsy is indicated. A number of studies have shown that if the disease is developing in the salivary glands the periductal infiltrate and its products (cytokines) can have a profound effect on the resorptive, transport and synthetic function of the striated duct cells which results in: a) elevated sodium and chloride concentration and a decreased phosphate concentration despite reduced fiow rate (1, 21); b) elevation in lactoferrin (21-24); c) elevation in beta 2-microglobulin (25, 26) and in d) kaUikrein (27). Parotid lysozyme was found to be elevated in patients with primary Sjogren's syndrome (dry eyes, dry mouth but no rheumatoid disease) but not in secondary Sjogren's syndrome (with an accompanying rheumatoid disease) (28). The alteration in glandular structure produced by the disease resulted in a marked impact on the lipid content of saliva with a 20 fold elevation in the concentration of phospholipids (29). If confirmed in a larger group of subjects this could be the basis of a valuable diagnostic test. Salivary gland chemistry in Sjogren's syndrome is not only potentially useful for diagnostic purposes but for following disease development and monitoring therapy (1, 26). Cystic fibrosis affects all of the exocrine glands to varying degrees. The impact on salivary gland function at a clinical level is minimal, but there are very definite effects on the composition of saliva (1, 30, 31). The most dramatic changes reported have been an elevation in calcium and proteins, especially apparent in the submandibular-sublingual glands and minor salivary glands. In the former these elevations result in a very apparent turbidity in the fluid secreted due to formation of a calcium-protein complex (32) and possibly of hydroxyapatite as well (33). In the minor salivary glands the precipitate physically obstructs the narrow excretory duct and markedly reduces the rate of secretion to virtually zero. This phenomenon can be used as a diagnostic test by measuring the fiow from the readily accessible labial glands on the lower lip with a capillary tube (34). Although many hormones influence the composition of saliva, the most dramatic changes have been noted in diseases of the adrenal cortex. The sodium and potassium concentration is markedly affected by corticosteroids, especially
The diagnostic uses of saliva 121 ical depression. In more than a dozen studies the data supported the view that patients with affective disorders secrete significantly less saliva than normal. A more recent study noted a similar trend (43). However, another study found no difference between depressed patients and controls (44). Apparently widescale use of psychoactive drugs with xerostomia as a side effect makes quantitation of flow rate in these patients more unreliable than in the past (1). An alternative, showing great promise, is the measurement of sahvary prostaglandins (PGD2, PGEj and PGF2a). In the saliva of patients with major depressive disorder the concentrations of immunoreactive PG's were significantly higher than cortex (36, 37). WOTMAN et al. (38) those of healthy controls (45). In pashowed that the ratio had value pre- tients with minor depressive or neurotic and post-surgically as an early index of disorders the values were comparable to prognosis and recovery as well as a those of controls. Salivary PG levels means of differentiating the adenoma may be a good indicator of major defrom "pseudoprimary aldosteronism", a pressive disorders. hyperplastic disease of the cortex (39). One of the unhappy consequences of cancer chemotherapy with such agents as high-dose methotrexate and cycloDiagnostic aids for ciinical phosphamide is the induction of an problems acute, severe mucositis with severe disIn several diverse clinical situations comfort and the high risk of fatal infec(Table 2) salivary analysis has provided tion. IzuTSU et al. (46) found that the valuable information for both the clini- loss of epithelial barrier function and cian and the investigator. The common- increased vascular penneability results ly used cardiac glycosides have a rela- in a marked increase in the albumin tively narrow margin of safety and de- concentration in the whole expectorated termining if a patient is manifesting saliva. The parotid secretion is not aftoxic effects has critical clinical implica- fected, hence the elevation is purely lotions. WOTMAN et al. (40) demonstrated cal in origin. The increase in albumin in 1971 that both potassium and calci- always preceeded the stomatitis and um concentration in whole saliva was could be a useful predictor of the clinmarkedly elevated in toxic patients and ical problem. Monitoring whole saliva that the calcium-potassium product albumin is "useful in establishing treatprovided a very easy and sensitive ment schedules for chemotherapy promeans of identifying these patients. tocols that have stomatitis as the limitThere have been a number of affirm- ing factor in treatment" (46). ations of these findings (1, 2). The curChronic respiratory infection, esperent view is that cardiac glycosides not cially in children, is often associated only affect monovalent cation transport with specific secretory IgA deficiency in cardiac cells, but also modify cation (47). Secretory IgA is the major immuATPase systems in erythrocytes and sal- noglobulin of exocrine gland secretions ivary gland cells (41). (48) and detennination of complete or There is an extensive literature (re- near-cotnplete IgA deficiency can readiviewed by BROWN) (42) on the relation- ly be made with a whole saliva sample, ship between salivary flow rate and clin- aspirated from the floor of the mouth in young children or expectorated in older children. With a cooperative child a parTable 2. Clinical problems in which saliva otid saliva sample is preferable and flow contributes to diagnosis rate should be determined for the most Digitalis toxicity precise measure of IgA level since saliAffective disorders vary IgA concentration varies inversely Stomatitis in cancer chemotherapy with fiow rate (49). Immunodeficiency of secretory IgA Cigarette usage The thiocyanate concentration in Dietary nitrates, nitrites and gastric cancer saliva is appreciably higher in smokers Ovulation time than non-smokers (50). Advantage has
aldosterone, via their impact on the NaK/ATPase in the striated duct cells where resorption of the primary secretion occurs. FRAWLEY & THORN (35) were the first to demonstrate the value of the sodium to potassium ratio of paraffin - stimulated whole saliva in diagnosing and monitoring Cushing's syndrome and Addison's disease. The mean Na to K ratio of Addisonian patients was 5.0 and decreased to 1.8 following treatment with corticosteroids. In normal subject the ratio was 1.3; in Cushing's syndrome, 0.5. Several investigators have demonstrated the diagnostic value of salivary Na to K ratio in primary aldosteronism, a hormone-producing adenoma of the
been taken of this observation to confirm or reject self-reporting of cigarette usage among children and adolescents (51). The test is somewhat limited, however, by the impact of exposure to smoke from heavy smokers in the home environment (52). A more sensitive indicator of exposure to tobacco smoking is measurement of salivary cotinine (53). It strongly correlated with urinary levels and with number of cigarettes smoked per day (54). Salivary cotinine is a useful measure in both compliance and epidemiologic studies. There is increasing interest in the relation of dietary factors to various types of cancer. One such association is between ingested nitrate, its conversion to nitrite and nitrosamines and the development of oral and gastric cancer (55). Since the amount of nitrate secreted by the salivary glands is directly related to the amount ingested (56), measuretnent of salivary nitrate can provide a convenient index for epidemiologic studies. Development of methods for determination of time of ovulation and the fertile period has been an active research area for many years (57). A recent multicenter study indicated that measurement of salivary estradiol was a promising method of prediction (58). The method requires the services of a clinical laboratory, however, and is not readily applicable to home testing. Since the composition of human saliva is altered during the menstrual cycle many simpler methods of using saliva have been explored (1). These involved measurement of a variety of enzymes, sialic acid, glucose and electrolytes. None of the methods appear to be sufficiently reliable for routine use. A new approach has recently been introduced which has considerable potential - the measurement of electrical resistance (59). A special device which provides a digital measurement of the electrical resistance of saliva has been shown to predict ovulation on average of 5.3 (± 1.9 SD) days in advance. With further confirmation this could prove to be a very useftjl method. Hormone monitoring
Since the pioneering studies of SHANNON et al. (60) it has generally been recognized that the lipid-soluble unconjugated steroids pass readily into saliva and that their concentrations in saliva are proportional to the concentrations of free, unbound steroids in plasma (2). The conjugated steroids diffuse with
Table 3. Hormones whose salivary levels reflect serum levels Cortisol Aldosterone Dehydroepiandrosterone Testosterone 5a-Dihydrosterone 17p-hydroxyprotesterone
Progesterone I7p-Estradiol Estriol Estrone Insulin Melatonin
great difficulty because of their low lipid-solubility and high molecular weight (62). An exception has recently been noted for corticosteroid binding globulin (CBG) and some modification of the assay may be required (63). A workshop on the immunoassay of steroids in saliva concluded that, "All steroids of diagnostic significance in routine clinical endocrinology can now be measured in saliva" (67). The list of steroid hormones currently being assayed in saliva includes cortisol, aldosterone, dehydroepiandrosterone, testosterone, 5adihydrotestosterone, 17P-hydroxy-progesterone, progesterone, 17P-estradial, estriol and estrone (2, 65) (Table 3). The literature on the clinical utilization of salivary monitoring of steroid hormones is rapidly expanding. According to RIAD-FAHMY et al. (66) salivary
progesterone is being used for: 1) assessing the functional capacity of the corpus luteum in both normal women and those with defects in the hypothalamicpituitary-ovarian axis; 2) studies of subfertile women; 3) studies of pregnant women; 4) examining the effect of contraceptive steroids on ovarian activity and 5) assessing hormonal changes during adolescence. Salivary estriol measurement during pregnancy has been shown to be an excellent means of detecting fetal growth retardation (67) and the estriol to progesterone ratio shows promise as a predictor of preterm labor (68). Some investigators have found that salivary cortisol is a better measure of adrenal cortical function than serum cortisol (69) and is particularly useful in studies with children (70-72). In many instances the children have been taught to collect their own saliva (71). Measurement of salivary cortisol at 11 P.M.
Table 4. Drugs curently monitored in saliva Phenytoin Primidone Ethosuximide Carbamazepine Theophylline Caffeine
Lithium Methadone Cyclosporine Marijuana Cocaine Alcohol
has been reported to be a reliable and practical index of hypothalamic-pituitary-adrenai axis activity in depression, especially in outpatients (73). Recently hormones other than steroids have been found to be refiective of their plasma levels and could be considered for salivary monitoring. MARCHETTi et al. (74) found a positive linear relationship between plasma and salivary insulin during the oral glucose tolerance test in Type 2 diabetic patients, in obese non-diabetic subjects and in normal volunteers. Further study by these investigators in a large group of non-diabetic subjects affirmed the highly significant correlation between sahvary and plasma insulin and indicated the potential of salivary insulin measurement in clinical practice (75). Excellent correlation has also been found between salivary and plasma levels of melatonin (76-78) and several clinical applications have been suggested (78).
ing on this basis can overcome this difficulty. Monitoring for salivary lithium in manic depressive patients is also subject to the problem of individual variation in the ratio and can be dealt with in the same way (88). Salivary monitoring is being used for patients on methadone, (84) for assay of cyclosporine in kidney-transplant patients (85) and for detection of marijuana smoking (86, 87) and cocaine use (88, 89). It also is a very practical way for determining alcohol concentration (90). Salivary caffeine levels can be accurately measured and overnight caffeine clearance appears to be a simple, safe test for measuring liver function (91). There are numerous other examples.
Screening for antivirai antibodies and viral antigens
The proliferation of new technologies and their application to large-scale screening for presence of HIV antibody Drug monitoring has not only stimulated research into Over the past 15 yr there has been a the use of saliva for this specific purburgeoning interest in the use of saliva pose, but into the whole area of viral in pharmacokinetic studies of drugs in diagnosis and screening (92-95). Invesgeneral and in therapeutic drug moni- tigators in both the United States (92) toring in a variety of clinical situations and Great Britain (94) have shown the (Table 4). The salivary/plasma ratio has complete concordance between salivary been established for a long list of drugs and serum findings for HIV positive (79) and the list has been continually people. Indeed with the use of the Ig expanded. As with hormones, lipid solu- capture radioimmunoassay (GACRIA) bility is a determining factor in their the low level of IgA, IgG and IgM antisalivary excretion. For lipid soluble body in whole saliva (relative to serum) acidic or basic substances the diffusibili- is not a limitation, since "the proportion ty is dependent on degree of ionization of specific to total immunoglobulin is in plasma and saliva. Only the un-ion- similar in the saliva and serum of each ized fraction can cross biologic mem- individual and the signals from capture branes and hence the degree of acidity assays on the two sorts of specimen are or basicity of a drug will determine its much the same and almost independent salivary/plasma ratio. Drug levels in of immunoglobulin concentration" (95). saliva, (like hormone levels) reflect the PARRY et al. (93) have shown the applifree, non-protein-bound portion in plas- cation of this method for salivary monima and hence may have a greater thera- toring of hepatitis A and B infection peutic implication than the total blood and rubella as well. Salivary assay of antiviral antibodies has also been used levels. Currently therapeutic drug monitor- as an indicator of rotavirus infection in ing is most effectively used when the neonates (96). saliva to plasma concentration ratio is In addition to measuring antibody, it constant over a wide range. This is espe- is possible to identify a number of specially so with anticonvulsant drugs such cific viral antigens in saliva. This has as phenytoin, primidone, ethosuximide been put into clinical practice as a and carbamazepine, (79, 80) and has screening procedure for feline leukemia special applicability to dose adjustment virus in vetinary medicine (97) and in children (81). Theophylline monitor- could be applied to mumps virus, cytoing for asthmatic children has also megalo-virus and probably several proven helpful, (82) although there are others as well in humans. often differences among individuals in Identification of salivary antibodies the S/P ratio (79). Establishing the ratio and antigens need not be confined to for the individual patient and monitor- viral diseases, although they have re-
The diagnostic uses of saliva 123 ceived the most attention to date. GRANSTROM et al. (98) used saliva for rapid diagnosis of pertussis by measuring the specific immunoglobulin A response to Bordetella pertussis antigens. Because of the common mucosal effect secretory IgA antibodies in saliva could be especially useful in measuring response to other infectious diseases of the nasopharyngeal and tracheo-bronchial surfaces. However, with the antibody capture technique (95) salivary screening could be used for measuring response to any bacterial infection be it IgG, IgM or IgA.
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