Review article Oral medicine

The diagnostic uses of saliva Irwin D. MandelColumbia University, School of Dental andOral Surgery, New York, USAMandel 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 varietyof disciplines that saliva has many diagnostic uses and is especially valuable inthe young, the old and infirm and in large scale screening and epidemiologicstudies. The highly sensitive test procedures that are now commonplace makesit practical to quantitate, despite very low concentrations, a large number ofhormones and drugs in saliva. Indeed, all steroids of diagnostic significance inroutine clinical endocrinology can now be readily measured in saliva. Drugmonitoring can include abusive as well as therapeutic agents. The concordancebetween anti HIV antibodies in saliva and serum has stimulated application tovarious other antiviral antibodies as well as to viral antigens per se. Saliva hasfound use as a diagnostic aid in an increasing number of clinical situations andin systemic diseases that can affect salivary gland function and compositionsuch as Sjogren's syndrome, cystic fibrosis and diseases of the adrenal cortex.The list keeps growing.

Key words: drugs in saliva; hormones insaliva: saliva: salivary chemistry; salivarydiagnosis: salivary nnonitoring:siaiochemistry.

irwin D. Mandei, Coiumbia University, Schoolof Dental and Oral Surgery, 630 West 168thStreet, New York, New York 10032, LISA.

Accepted for publication December 22, 1989.

SaHva is not one of the popular bodilyfluids. It lacks the drama of blood, thesincerity of sweat and the emotional ap-peal of tears. Despite the absence ofcharisma, however, a growing numberof internists, pediatricians, pharmacolo-gists, clinical and forensic pathologists,psychologists and dentists are findingthat saliva provides an easily available,noninvasive diagnostic medium for arapidly widening range of diseases andclinical situations (1, 2).

The limited utilization of saliva fordiagnostic purposes in the past may bea reflection of the ambivalence that hascharacterized both the public and pro-fessional view of the nature of salivaover the ages. Prior to the 17th centuryand the anatomic demonstrations bySTENSON and WHARTON of the ductsthat bear their name, salivary glandswere thought to be accessory excretoryorgans, emunctories, that strained offthe evil spirits of the brain (3). With therealization that the glands could gener-ate an external secretion, physicianswho practiced medicine based on hu-moral pathology, the need to balancethe body humors (phlegm, blood, yel-low bile and black bile), bled, blistered,purged and stimulated salivation. It wasnot uncommon to prescribe massivedoses of bichloride of mercury tocleanse the system by causing saliva toliterally pour from the mouth. It wasthe fortunate patient who survived boththe disease and the treatment.

This negative image of saliva, how-ever, was not uniform. In the cosmologi-es of ancient Egypt, Thoth the wise issaid to have spat into the empty eyesocket of Horus, the sun god, to restorehis vision (4). The new testament (Mark8: 23-25) tells us that Jesus took theblind man by the hand and led him outof town; and when he had spit on hiseyes, and put his hands upon him, heasked him if he saw ought; and he wasrestored and saw every man clearly.

The people who really appreciate the"miracle" of saliva, however, are notthe blind, but those who suffer fromxerostomia (dry mouth) from a varietyof causes (5). They recognize belatedlythat saliva is a natural resource withmany functional capabilities that in-clude food preparation, digestion, lubri-cation and protection of the teeth andmucous membranes (6).

One function not bestowed by natureis the use of saliva for diagnosticpurposes. Historically this diagnosticvalue may have been recognized firstby the ancient judicial community whoemployed salivary flow (or its absence)as the basis for a primitive lie detectortest. The accused was given a handfulof dry rice. If anxiety (and presumablyguilt) so inhibited salivation that he orshe could not form an adequate bolusto chew and swallow, than off with theirhead. In more recent times, where thevagaries of the secretory-motor systemhave been replaced by those of the court

system, saliva found its widest use atthe race track where the saliva test fordrugs became the determinant of a"fixed" horse race. It is interesting tonote that both the ancient and modernuse of saliva are different forms of liedetection. Apparently ';";; saliva veritas'.

Sample collectionThe fiuid most frequently employed forsalivary diagnostic purposes is expecto-rated whole saliva, a mix composedlargely of the secretions from the majorsalivary glands, the right and left par-otid, submandibular and sublingualglands. There are also modest contribu-tions from the minor salivary glands(which are distributed inside the lips andcheeks, on the palate and under thetongue) and the gingival crevicular fluidfrom the spaces between the teeth andthe gingiva. This latter Ouid is primarilya serum transudate but can also containcells and products of the inflammatoryprocess when gingivitis or periodontitisis present. Due to the presence of largenumbers of bacteria continually loosedfrom tooth and soft tissue surfaces, aswell as shed epithelial cells, whole salivausually requires centrifugation to pro-vide a clear sample. In some situations,however, the bacteria or cells have diag-nostic value.

Unstimulated or resting saliva isusually collected by passive droolinginto a graduated tube or preweighed

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vial so that fiow rate per unit time canbe measured. The concentration ofsome salivary constituents is fiow ratedependent (1). When volume measure-ment is not required the saliva can becollected on cotton swabs, cotton rolls,gauze or filter paper strips, then elutedor centrifuged, or, aspirated directlyfrom the fioor of the mouth with plasticpipettes.

For evaluating salivary gland func-tion or when large volumes of saliva arerequired for analytic purposes, saliva isstimulated by a masticatory or gustato-ry stimulus, expectorated and handledin a similar manner as the unstimulatedfluid. Softened paraffin wax or awashed rubber band are the usualmasticatory stimuli and 2% citric acidapplied directly to the tongue the stan-dard gustatory stimulus (1, 5).

In many situations separately collect-ed secretions from individual glands arepreferable and this can be accomplishedin a non-invasive manner with suitablecollecting devices (1, 5). Parotid salivais best collected with plastic modifica-tions of a simple cup first introducedby CARLSON & CRITTENDEN in 1910 (7).Disposable and individualized collec-tors have also been introduced (8, 9).Submandibular-sublingual saliva can becollected by customization of a basicplastic collector (10, 11) or by aspirationfrom the duct openings with a micropi-pette (5).

Oral diseasesFor many years dental investigatorshave been exploring changes in salivaryflow rate and composition as a meansof diagnosing and monitoring a numberof oral diseases (1, 2). The examinationof secretions collected from individualglands has been especially valuable inthe differential diagnosis of local dis-eases of the salivary glands such as in-fiammatory and autoimmune diseasesand in evaluating the effects of pharma-cologic agents and therapeutic regimenswhich impact on salivary function (1,

5). Since individual gland secretionscontain some locally shed cells attemptshave been made to use saliva for cyto-logic examination as an aid in diagnosisof tumors, but so far cytology is beingrestricted to fine needle biopsy (12).Shed buccal mucosal cells, however, arebeing used as a simple, non-invasivemethod for obtaining DNA for geneanalysis in cystic fibrosis and is applica-ble to other disease as well (13).

Saliva collected by expectorationcontains a fair representation of thebacterial and fungal species that coatthe teeth, tongue and mucous mem-branes. By culturing a known volume ofsaliva (in various dilutions) on selectivemedia a quantitative determination canbe made of specific organisms. Countsof Streptococcus mutans and lactobacilliare being employed for identifying chil-dren at high risk for enamel caries (14,15) and older adults susceptible to rootsurface caries (16). Oral candidiasis isfrequently found in people wearingcomplete dentures, in people with amarked diminution in fiow rate due toa variety of therapies and medications,and in HIV infection. Several recentstudies have shown that quantitation ofCandida (usually albicans) from wholesaliva can be used as an indicator ofinfection (17-19).

System diseases affecting salivaWith the increase in investigator interestit is becoming apparent that many sys-temic diseases affect salivary glandfunction and/or salivary composition(Table 1) (1). Although the study ofthese effects have been valuable in theunderstanding of the pathogenesis ofthe diseases, their use as diagnosticmarkers have been more limited, butnone the less helpful in some situations.

In Sjogren's syndrome, an autoim-mune disease which affects several mil-lion people, one of the main diagnosticprocedures is biopsy of the minor sali-vary 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 syndromeRheumatoid diseasesGraft VS host disea.seSarcoidosisCystic fibrosisHypertensionHyperlipidemiaAlcoholic cirrhosisMalnutrition

Hromonal dysfunctionDiabetesPancreatitisAdrenal-cortical diseasesThyroiditisAcromegaly

Neurological diseasesParkinsonismBell's palsyCerebral palsy

of the lymphocytic infiltration char-acteristic of this disease (20). Siaio-chemistry provides a helpful screeningprocedure to determine whether the bi-opsy is indicated. A number of studieshave shown that if the disease is devel-oping in the salivary glands the periduc-tal infiltrate and its products (cytokines)can have a profound effect on the re-sorptive, transport and synthetic func-tion of the striated duct cells which re-sults in: a) elevated sodium and chlorideconcentration and a decreased phos-phate concentration despite reducedfiow rate (1, 21); b) elevation in lactofer-rin (21-24); c) elevation in beta 2-mi-croglobulin (25, 26) and in d) kaUikrein(27). Parotid lysozyme was found to beelevated in patients with primary Sjo-gren's syndrome (dry eyes, dry mouthbut no rheumatoid disease) but not insecondary Sjogren's syndrome (with anaccompanying rheumatoid disease)(28). The alteration in glandular struc-ture produced by the disease resulted ina marked impact on the lipid content ofsaliva with a 20 fold elevation in theconcentration of phospholipids (29). Ifconfirmed in a larger group of subjectsthis could be the basis of a valuablediagnostic test. Salivary gland chemistryin Sjogren's syndrome is not only poten-tially useful for diagnostic purposes butfor following disease development andmonitoring therapy (1, 26).

Cystic fibrosis affects all of the exo-crine glands to varying degrees. The im-pact on salivary gland function at a clin-ical level is minimal, but there are verydefinite effects on the composition ofsaliva (1, 30, 31). The most dramaticchanges reported have been an elevationin calcium and proteins, especially ap-parent in the submandibular-sublingualglands and minor salivary glands. In theformer these elevations result in a veryapparent turbidity in the fluid secreteddue to formation of a calcium-proteincomplex (32) and possibly of hydroxy-apatite as well (33). In the minor sali-vary glands the precipitate physicallyobstructs the narrow excretory duct andmarkedly reduces the rate of secretionto virtually zero. This phenomenon canbe used as a diagnostic test by measur-ing the fiow from the readily accessiblelabial glands on the lower lip with acapillary tube (34).

Although many hormones influencethe composition of saliva, the most dra-matic changes have been noted in dis-eases of the adrenal cortex. The sodiumand potassium concentration is marked-ly affected by corticosteroids, especially

aldosterone, via their impact on theNaK/ATPase in the striated duct cellswhere resorption of the primary secre-tion occurs. FRAWLEY & THORN (35)were the first to demonstrate the valueof the sodium to potassium ratio of par-affin - stimulated whole saliva in diag-nosing and monitoring Cushing's syn-drome and Addison's disease. The meanNa to K ratio of Addisonian patientswas 5.0 and decreased to 1.8 followingtreatment with corticosteroids. In nor-mal subject the ratio was 1.3; in Cush-ing's syndrome, 0.5.

Several investigators have demon-strated the diagnostic value of salivaryNa to K ratio in primary aldosteronism,a hormone-producing adenoma of thecortex (36, 37). WOTMAN et al. (38)showed that the ratio had value pre-and post-surgically as an early index ofprognosis and recovery as well as ameans of differentiating the adenomafrom "pseudoprimary aldosteronism", ahyperplastic disease of the cortex (39).

Diagnostic aids for ciinicalproblemsIn several diverse clinical situations(Table 2) salivary analysis has providedvaluable information for both the clini-cian and the investigator. The common-ly used cardiac glycosides have a rela-tively narrow margin of safety and de-termining if a patient is manifestingtoxic effects has critical clinical implica-tions. WOTMAN et al. (40) demonstratedin 1971 that both potassium and calci-um concentration in whole saliva wasmarkedly elevated in toxic patients andthat the calcium-potassium productprovided a very easy and sensitivemeans of identifying these patients.There have been a number of affirm-ations of these findings (1, 2). The cur-rent view is that cardiac glycosides notonly affect monovalent cation transportin cardiac cells, but also modify cationATPase systems in erythrocytes and sal-ivary gland cells (41).

There is an extensive literature (re-viewed by BROWN) (42) on the relation-ship between salivary flow rate and clin-

Table 2. Clinical problems in which salivacontributes to diagnosisDigitalis toxicityAffective disordersStomatitis in cancer chemotherapyImmunodeficiency of secretory IgACigarette usageDietary nitrates, nitrites and gastric cancerOvulation time

ical depression. In more than a dozenstudies the data supported the view thatpatients with affective disorders secretesignificantly less saliva than normal. Amore recent study noted a similar trend(43). However, another study found nodifference between depressed patientsand controls (44). Apparently widescaleuse of psychoactive drugs with xero-stomia as a side effect makes quantita-tion of flow rate in these patients moreunreliable than in the past (1). An alter-native, showing great promise, is themeasurement of sahvary prostaglandins(PGD2, PGEj and PGF2a). In the salivaof patients with major depressive disor-der the concentrations of immunoreac-tive PG's were significantly higher thanthose of healthy controls (45). In pa-tients with minor depressive or neuroticdisorders the values were comparable tothose of controls. Salivary PG levelsmay be a good indicator of major de-pressive disorders.

One of the unhappy consequences ofcancer chemotherapy with such agentsas high-dose methotrexate and cyclo-phosphamide is the induction of anacute, severe mucositis with severe dis-comfort and the high risk of fatal infec-tion. IzuTSU et al. (46) found that theloss of epithelial barrier function andincreased vascular penneability resultsin a marked increase in the albuminconcentration in the whole expectoratedsaliva. The parotid secretion is not af-fected, hence the elevation is purely lo-cal in origin. The increase in albuminalways preceeded the stomatitis andcould be a useful predictor of the clin-ical problem. Monitoring whole salivaalbumin is "useful in establishing treat-ment schedules for chemotherapy pro-tocols that have stomatitis as the limit-ing factor in treatment" (46).

Chronic respiratory infection, espe-cially in children, is often associatedwith specific secretory IgA deficiency(47). Secretory IgA is the major immu-noglobulin of exocrine gland secretions(48) and detennination of complete ornear-cotnplete IgA deficiency can readi-ly be made with a whole saliva sample,aspirated from the floor of the mouth inyoung children or expectorated in olderchildren. With a cooperative child a par-otid saliva sample is preferable and flowrate should be determined for the mostprecise measure of IgA level since sali-vary IgA concentration varies inverselywith fiow rate (49).

The thiocyanate concentration insaliva is appreciably higher in smokersthan non-smokers (50). Advantage has

The diagnostic uses of saliva 121been taken of this observation to con-firm or reject self-reporting of cigaretteusage among children and adolescents(51). The test is somewhat limited, how-ever, by the impact of exposure tosmoke from heavy smokers in the homeenvironment (52). A more sensitive indi-cator of exposure to tobacco smokingis measurement of salivary cotinine (53).It strongly correlated with urinary levelsand with number of cigarettes smokedper day (54). Salivary cotinine is a usefulmeasure in both compliance and epide-miologic studies.

There is increasing interest in the rela-tion of dietary factors to various typesof cancer. One such association is be-tween ingested nitrate, its conversion tonitrite and nitrosamines and the devel-opment of oral and gastric cancer (55).Since the amount of nitrate secreted bythe salivary glands is directly related tothe amount ingested (56), measuretnentof salivary nitrate can provide a conve-nient index for epidemiologic studies.

Development of methods for deter-mination of time of ovulation and thefertile period has been an active researcharea for many years (57). A recent mul-ticenter study indicated that measure-ment of salivary estradiol was a promis-ing method of prediction (58). Themethod requires the services of a clinicallaboratory, however, and is not readilyapplicable to home testing. Since thecomposition of human saliva is alteredduring the menstrual cycle many sim-pler methods of using saliva have beenexplored (1). These involved measure-ment of a variety of enzymes, sialic acid,glucose and electrolytes. None of themethods appear to be sufficiently reli-able for routine use. A new approachhas recently been introduced which hasconsiderable potential - the measure-ment of electrical resistance (59). A spe-cial device which provides a digitalmeasurement of the electrical resistanceof saliva has been shown to predict ovu-lation on average of 5.3 ( 1.9 SD) daysin advance. With further confirmationthis could prove to be a very useftjlmethod.

Hormone monitoringSince the pioneering studies of SHAN-NON et al. (60) it has generally beenrecognized that the lipid-soluble uncon-jugated steroids pass readily into salivaand that their concentrations in salivaare proportional to the concentrationsof free, unbound steroids in plasma (2).The conjugated steroids diffuse with

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Table 3. Hormones whose salivary levels re-flect serum levels

CortisolAldosteroneDehydroepiandrosteroneTestosterone5a-Dihydrosterone17p-hydroxyprotesterone

ProgesteroneI7p-EstradiolEstriolEstroneInsulinMelatonin

great difficulty because of their low lipi-d-solubility and high molecular weight(62). An exception has recently beennoted for corticosteroid binding globu-lin (CBG) and some modification of theassay may be required (63). A workshopon the immunoassay of steroids in salivaconcluded that, "All steroids of diag-nostic significance in routine clinical en-docrinology can now be measured insaliva" (67). The list of steroid hor-mones currently being assayed in salivaincludes cortisol, aldosterone, dehy-droepiandrosterone, testosterone, 5a-dihydrotestosterone, 17P-hydroxy-pro-gesterone, progesterone, 17P-estradial,estriol and estrone (2, 65) (Table 3).

The literature on the clinical utiliza-tion of salivary monitoring of steroidhormones is rapidly expanding. Accord-ing to RIAD-FAHMY et al. (66) salivaryprogesterone is being used for: 1) assess-ing the functional capacity of the corpusluteum in both normal women andthose with defects in the hypothalamic-pituitary-ovarian axis; 2) studies ofsubfertile women; 3) studies of pregnantwomen; 4) examining the effect of con-traceptive steroids on ovarian activityand 5) assessing hormonal changes dur-ing adolescence. Salivary estriol meas-urement during pregnancy has beenshown to be an excellent means of de-tecting fetal growth retardation (67) andthe estriol to progesterone ratio showspromise as a predictor of preterm labor(68).

Some investigators have found thatsalivary cortisol is a better measure ofadrenal cortical function than serumcortisol (69) and is particularly useful instudies with children (70-72). In manyinstances the children have been taughtto collect their own saliva (71). Meas-urement of salivary cortisol at 11 P.M.

Table 4. Drugs curently monitored in saliva

PhenytoinPrimidoneEthosuximideCarbamazepineTheophyllineCaffeine

LithiumMethadoneCyclosporineMarijuanaCocaineAlcohol

has been reported to be a reliable andpractical index of hypothalamic-pituit-ary-adrenai axis activity in depression,especially in outpatients (73).

Recently hormones other than ste-roids have been found to be refiectiveof their plasma levels and could be con-sidered for salivary monitoring. MAR-CHETTi et al. (74) found a positive linearrelationship between plasma and sali-vary insulin during the oral glucose tol-erance test in Type 2 diabetic patients,in obese non-diabetic subjects and innormal volunteers. Further study bythese investigators in a large group ofnon-diabetic subjects affirmed the high-ly significant correlation between sah-vary and plasma insulin and indicatedthe potential of salivary insulin meas-urement in clinical practice (75). Excel-lent correlation has also been found be-tween salivary and plasma levels of mel-atonin (76-78) and several clinicalapplications have been suggested (78).

Drug monitoringOver the past 15 yr there has been aburgeoning interest in the use of salivain pharmacokinetic studies of drugs ingeneral and in therapeutic drug moni-toring in a variety of clinical situations(Table 4). The salivary/plasma ratio hasbeen established for a long list of drugs(79) and the list has been continuallyexpanded. As with hormones, lipid solu-bility is a determining factor in theirsalivary excretion. For lipid solubleacidic or basic substances the diffusibili-ty is dependent on degree of ionizationin plasma and saliva. Only the un-ion-ized fraction can cross biologic mem-branes and hence the degree of acidityor basicity of a drug will determine itssalivary/plasma ratio. Drug levels insaliva, (like hormone levels) reflect thefree, non-protein-bound portion in plas-ma and hence may have a greater thera-peutic implication than the total bloodlevels.

Currently therapeutic drug monitor-ing is most effectively used when thesaliva to plasma concentration ratio isconstant over a wide range. This is espe-cially so with anticonvulsant drugs suchas phenytoin, primidone, ethosuximideand carbamazepine, (79, 80) and hasspecial applicability to dose adjustmentin children (81). Theophylline monitor-ing for asthmatic children has alsoproven helpful, (82) although there areoften differences among individuals inthe S/P ratio (79). Establishing the ratiofor the individual patient and monitor-

ing on this basis can overcome this diffi-culty. Monitoring for salivary lithium inmanic depressive patients is also subjectto the problem of individual variationin the ratio and can be dealt with in thesame way (88).

Salivary monitoring is being used forpatients on methadone, (84) for assayof cyclosporine in kidney-transplant pa-tients (85) and for detection of marijua-na smoking (86, 87) and cocaine use (88,89). It also is a very practical way fordetermining alcohol concentration (90).Salivary caffeine levels can be accuratelymeasured and overnight caffeine clear-ance appears to be a simple, safe testfor measuring liver function (91). Thereare numerous other examples.

Screening for antivirai antibodiesand viral antigensThe proliferation of new technologiesand their application to large-scalescreening for presence of HIV antibodyhas not only stimulated research intothe use of saliva for this specific pur-pose, but into the whole area of viraldiagnosis and screening (92-95). Inves-tigators in both the United States (92)and Great Britain (94) have shown thecomplete concordance between salivaryand serum findings for HIV positivepeople. Indeed with the use of the Igcapture radioimmunoassay (GACRIA)the low level of IgA, IgG and IgM anti-body in whole saliva (relative to serum)is not a limitation, since "the proportionof specific to total immunoglobulin issimilar in the saliva and serum of eachindividual and the signals from captureassays on the two sorts of specimen aremuch the same and almost independentof immunoglobulin concentration" (95).PARRY et al. (93) have shown the appli-cation of this method for salivary moni-toring of hepatitis A and B infectionand rubella as well. Salivary assay ofantiviral antibodies has also been usedas an indicator of rotavirus infection inneonates (96).

In addition to measuring antibody, itis possible to identify a number of spe-cific viral antigens in saliva. This hasbeen put into clinical practice as ascreening procedure for feline leukemiavirus in vetinary medicine (97) andcould be applied to mumps virus, cyto-megalo-virus and probably severalothers as well in humans.

Identification of salivary antibodiesand antigens need not be confined toviral diseases, although they have re-

ceived the most attention to date.GRANSTROM et al. (98) used saliva forrapid diagnosis of pertussis by measur-ing the specific immunoglobulin A re-sponse to Bordetella pertussis antigens.Because of the common mucosal effectsecretory IgA antibodies in saliva couldbe especially useful in measuring re-sponse to other infectious diseases of thenasopharyngeal and tracheo-bronchialsurfaces. However, with the antibodycapture technique (95) salivary screen-ing could be used for measuring re-sponse to any bacterial infection be itIgG, IgM or IgA.

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