Vol. 34, No. 1INFECTION AND IMMUNITY, Oct. 1981, p. 208-2140019-9567/81/100208-07$02.00/0

Inhibition of Dental Plaque Acid Production by the SalivaryLactoperoxidase Antimicrobial System

JORMA TENOVUO,' BRITTA MANSSON-RAHEMTULLA,l KENNETH M. PRUITT," ANDROLAND ARNOLD2

Division ofMolecular Biology, The Medical Center, University ofAlabama in Birmingham,Birmingham, Alabama 35294'; and Department of Oral Biology, School of Dentistry, University of

Louisville, Louisville, Kentucky 402082

Received 20 February 1981/Accepted 30 June 1981

Resting human whole saliva inhibited acid production by glucose-stimulated,homologous plaque. The degree of inhibition of plaque acid production correlatedpositively with the concentration of hypothiocyanite (OSCN-) ions in saliva.Supplementation of saliva with an appropriate combination of thiocyanate andhydrogen peroxide resulted in a significant increase in the concentration ofOSCN- ions and in more effective inhibition of plaque acid production. In mostcases, the inhibition was complete when the supplements were added directly tosaliva-plaque mixtures. Acid production resumed when the inhibitory effect ofOSCN- was reversed by addition of thiols. Among the oral defense factors, thesalivary lactoperoxidase system seems to play an important role by producinghighly reactive antibacterial products (including OSCN-) which can regulatebacterial metabolism in the human mouth. The concentration of OSCN- innormal human whole saliva seems to be just below the threshold level requiredfor plaque inhibition. Therefore, enhancement of this system in vivo may beeffective in the regulation of plaque acid production.

Oral microorganisms interact with secretoryantibodies as well as with the innate (nonim-munoglobulin) defense factors present in humansaliva. The innate defense factors include lyso-zyme, lactoferrin, and lactoperoxidase. Despiteextensive research in the area of oral defensemechanisms during recent years, the relativecontributions of different salivary antimicrobialfactors to bacterial inhibition and to oral healthhave not been determined. Arnold et al. (2)showed that patients with immune system irreg-ularities consistently demonstrated normal orelevated levels of innate defense factors. Numer-ous studies have shown that many gram-positiveand gram-negative bacteria are inhibited bythese factors in vitro, but clear relationshipsbetween these factors and oral health have notbeen described.

Practical attempts to prevent dental carieswith the aid ofa salivary peroxidase system havebeen made with positive (10, 15) and negativeresults (13). These attempts have been based onthe activation of a salivary peroxidase-thiocya-nate-H202 antimicrobial system by addition ofperoxide, which is known to be a critical factorfor the antibacterial effect of this system (19; H.Hoogendoorn, Ph.D. thesis, Delft, Mouton DenHaag, 1974). Antimicrobial properties have beenreported for the hypothiocyanite ion, OSCN-,

which is formed during the oxidation of SCN-by peroxidase and H202 (4, 9). Hypothiocyaniteis a normal component of human whole saliva(20). Recently, we observed that, in addition toH202, SCN- can be a limiting factor for OSCN-generation in human saliva (K. M. Pruitt, J.Tenovuo, W. Fleming, and M. Adamson, CariesRes., in press). A positive correlation betweenoral health and the generation of OSCN- insaliva sediment has been found (18).The aim of the present study is to describe the

effects of individual saliva samples (containingvarious amounts of OSCN-) on the acid produc-tion of homologous plaque samples. Preliminarystudies have indicated that the variability inplaque response to saliva is related, at least inpart, to the dynamics of OSCN- generation inhuman saliva (3). Detailed knowledge of thissaliva-plaque interaction is essential for our un-derstanding ofthe regulation of bacterial metab-olism in the mouth.

MATERIALS AND METHODSCoilection and treatment of plaque and saliva

samples. Eleven subjects with no apparent abnor-malities in oral health were selected. Subjects wereinstructed to refrain from all measures of oral hygienefor 24 h but to maintain their normal dietary patterns.Plaque was collected at 8 a.m. (before the subjects had

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INHIBITION OF PLAQUE ACID PRODUCTION BY SALIVA 209

eaten) with sterile explorers and curettes from allsupragingival tooth surfaces and pooled in 2 ml ofsterile saline (pH 6.5) at room temperature. Immedi-ately after collection, the plaque suspension was soni-cated for 15 s. The turbidity of the suspension wasestimated by measuring the absorbance at 660 nm(Am0) of 100 id of the sonicated suspension mixed with2 ml of water.

Unstimulated whole saliva samples (5 ml) from eachsubject were collected in an iced water bath. Aliquotswere immediately taken for peroxidase, thiocyanate,and hypothiocyanite assays, and the pH and flow ratewere recorded. Saliva samples were kept in ice beforeacid production experiments. All of the experimentswere completed within 4 h after the collection ofplaque and saliva.Acid production assay. Portions (50 or 100 pl

each) of plaque suspension were mixed with equalvolumes of saliva (test) or phosphate-buffered saline(control). The pH of phosphate-buffered saline wasadjusted to that of the respective saliva sample. After5 min of incubation at 37°C in a titration cell withconstant stirring, 2 ml of saline was added, and the pHwas controlled with a pH-Stat titrator type TI'T2combined with an SBR2 Titragraph and an ABUlAutoburette (Radiometer America, Westlake, Ohio).The mixture was maintained at pH 6.5 by automaticaddition of 1.00 mM NaOH. Acid production wasstimulated by the addition of 100 I of glucose solutionto give a final glucose concentration of 1%. The rate ofacid production was measured as the slope (AV/At) ofthe curve of volume (V) of added base (required tomaintain pH at 6.5) versus time (t) taken from therecorder tracing. The slope was converted into nano-moles of acid produced by multiplying AV/At by theconcentration (C) of the NaOH solution.The acid production in any given experiment de-

pends upon the number (n) of metabolically activebacteria present in the titration cell. To compare re-sults from experiments utilizing different quantities ofbacteria, it is necessary to take the number of cellsinto account. Ideally, the acid production per activecell would be given by the ratio of the total acidproduced to the number of active cells, or:

acid production per active cell =A

.

However, there is no practical way to determine n ona routine daily basis. We have assumed that acidproduction is directly proportional to biomass, andthat biomass, in turn, can be estimated from theabsorbance. Although this assumption may not bestrictly true, we have found that acid production de-fined in the following way is a satisfactory measure ofmetabolic activity for comparative purposes:

AVC 1acid production estimate = *- x

where v is the volume of bacterial suspension added tothe titration cell andAm is the absorbance at 660 nmof that bacterial suspension. These measurements ofacid production are of convenient numerical magni-tude (10 to 100) when the units of acid production areexpressed as nanomoles x minute-' x milliliter-1 xA660-1.

Effect of supplemented saliva on plaque acidproduction. Two types of experiments were carriedout. (i) Supplements of H202 and SCN- sufficient togive final concentrations of 700 uM and 10 mM, re-spectively, were added to saliva, and the mixture wasincubated for 5 min at 37°C. Then a volume of ho-mologous plaque suspension equal to the volume ofsaliva was added. After a further 5 min of incubation,acid production was stimulated by glucose additionand measured as described above. (ii) The supple-ments of H202 and SCN- were added directly to mix-tures of equal volumes of saliva and saline suspensionsof homologous plaque, and the final mixture was in-cubated for 5 min before glucose addition. AU of theincubations of both types of experiments were madein open plastic cups with constant magnetic stirringand with temperature maintained at 37°C.Chemical assays. Peroxidase activity was mea-

sured with ABTS [2,2'-azino-di-(3-ethyl-benzthiazo-line-6-sulfonic acid)] as a substrate. Assay conditionshave been described by Shindler et al. (16). The A412data obtained from a Cary model 219 spectrophotom-eter (Varian Instrument, Houston, Tex.) were digitizedat 1-s intervals by using a model 2200 Datalogger(Fluke Instrument Co., Seattle, Wash.) and processedon an ILSI-11 computer system. Peroxidase activitycalculations were based on the first 15 s ofthe reaction,during which the initial A412 values were linear. Oneenzyme unit is equivalent to a change in A412 of 32.4per min and corresponds to the amount of enzymecatalyzing the oxidation of 1 mmol of substrate permin under the assay conditions. This definition as-sumes a molar absorption coefficient of 32,400 M-1cm ' for ABTS at 412 nm. In the ABTS assay, 1 Ucorresponds to about 17 U in the pyrogallol assay,which was previously used as a substrate in this labo-ratory. The change in substrate was made because ofthe higher sensitivity and reproducibility of the ABTSmethod.

Hypothiocyanite (OSCN-) ion was assayed by re-action with the colored anionic monomer of DTNB[5,5'-dithiobis-(2-nitrobenzoic acid)] as described byAune and Thomas (5). The reaction mixture was 64pM in DTNB and 60 pM in mercaptoethanol in 2.0 mlof 0.1 M tris(hydroxymethyl)aminomethane hydro-chloride buffer, pH 8.0. The concentrations of thiocy-anate were determined by using the ferric nitratemethod (14).

Chemicals. All chemicals were of reagent grade.DTNB was obtained from Aldrich Chemical Co., Mil-waukee, Wis., and ABTS was from Boehringer Mann-heim Corp., New York, N.Y. Hydrogen peroxide waspurchased as a 30% solution (J. T. Baker ChemicalCo., Phillipsburg, N.J.) and stored at 4°C. Potassiumthiocyanate was a product of J. T. Baker ChemicalCo. The water used was deionized with a mixed bedresin and subsequently distilled with a two-stagequartz glass still.

RESUJLTSThe range of values (Table 1) of flow rate, pH,

peroxidase activity, and thiocyanate concentra-tion for this group of subjects was consistentwith normal values reported by others. The av-erage concentration of OSCN- in fresh, unstim-

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TABLE 1. Saliva andplaque analysis of the testsubjectsa

Unstimulated whole saliva

Subject Flow Plaqueb(sex) rate Peroxidase SCN- Ame

(ml/ pH (mU/ml) (mM)min)

1 (i) 0.71 6.40 132.9 0.75 3.682 (f) 0.49 6.62 47.0 1.51 5.463 (f) 0.22 6.00 131.0 3.87 3.054 (m) 0.28 6.65 93.4 1.95 3.475 (m) 0.26 6.60 85.5 1.08 4.626 (m) 1.00 7.05 69.4 1.01 4.737 (f) 0.50 6.70 94.5 0.48 1.898 (m) 0.35 7.10 66.4 1.65 5.259 (f) 0.30 6.30 130.8 1.52 10.1910 (m) 0.41 6.75 59.4 1.11 4.2011 (f) 1.00 6.65 113.7 0.99 2.84

Mean 0.50 6.62 93.1 1.45 4.49SDC 0.27 0.30 29.3 0.87 2.08

aHypothiocyanite (OSCN-) concentrations aregiven in Table 2.

b Plaque was collected into 2 ml of sterile saline (pH6.5) sonicated for 15 s. The turbidity was estimated bymixing 100 id of the sonicated suspension with 2 ml ofwater and measuring the Amso. This result was multi-plied by 21 to obtain the listed values.

'SD, Standard deviation.

ulated whole saliva samples was 57.6 ,tM (Table2). When saliva samples were supplementedwith H202 (700 tiM) and SCN- (10 mM), themean concentration of OSCN- increased to avalue of 217.8 ,uM (Table 3); i.e., almost a four-fold increase in the amount of the inhibitor wasobtained. When saliva was supplemented withthe above-mentioned concentrations of H202and SCN- in the presence of plaque, the concen-trations of OSCN- generated were even higher,averaging 265 !LM (Table 4). The hypothiocya-nite concentrations measured under the condi-tions of these experiments did not show anyobvious correlation with the SCN- concentra-tion, peroxidase concentration, the pH, or theflow rate of the saliva samples (Tables 1 to 3).When plaque samples were exposed to ho-

mologous saliva, the rate of acid productiondecreased as a function of saliva OSCN- concen-tration (Fig. 1), and plaque acid production wasinhibited to a greater extent by supplementedsaliva than by unsupplemented saliva (Tables 2and 3). When saliva was supplemented withH202 and SCN- in the presence of bacteria, acomplete inhibition of acid production was usu-ally achieved (Table 4).When H202 (final concentration, 0.21 mM)

was added to the plaque-saliva mixtures whichwere producing acid, in most cases a completeinhibition of acid production was obtained

TABLE 2. Glucose-stimulated acidproduction ofdental plaque exposed to unsupplemented resting

saliva

Subject OSCN-a Acid pro- Peroxide RecoverdSbet (AtM) duction' effect' eoey1 56.2 22.2 + 11.02 65.9 22.2 + 11.43 76.5 24.0 + 14.14 55.1 11.2 + 7.05 57.2 26.2 06 21.6 78.1 07 85.3 38.5 08 60.9 35.5 + 6.69 37.7 71.1 010 81.6 9.0 + 4.311 35.3 74.2 0

Mean 57.6 37.5 9.1SD' 19.0 24.1 3.4

'The concentration of OSCN- in fresh, unstimu-lated whole saliva.

b The acid production rate (nanomoles x minute'Ix milliliter-1 x Am-1) of the plaque-saliva-saline sus-pension after addition of glucose (final concentration,1%) and before addition of peroxide.

' +, Shutdown (in 2 to 10 min) after addition ofH202 (calculated final concentration, 0.21 mM); 0, noshutdown of acid production.

d The rate of acid production after addition of 2-mercaptoethanol (0.24 mM).

' SD, Standard deviation.

TABLE 3. Acid production of dentalplaque exposedto saliva which was supplemented with H202 (700ILM) and SCN- (10 mM); hypothiocyanite (OSCN-)was generated before saliva was mixed with plaque

Subject OSCN-a Acid pro- Peroxide Recover(pM) duction' effectcRcoey1 336.2 9.1 + 16.52 114.5 13.2 + 15.63 324.2 8.7 + 25.24 253.0 12.2 + 22.75 231.4 23.3 06 93.5 50.3 07 245.3 2.6 * 14.58 135.6 10.0 + 9.29 454.7 40.1 + 11.110 102.8 6.1 + 0.011 103.9 38.4 0

Mean 217.8 19.5 14.4SD' 113.9 15.4 7.4a The concentration of OSCN- in saliva after 5 min

of incubation at 370C in the presence of supplements.b Acid production (nanomoles x minute-' x mnilli-

liter-' x Am601) before second addition of peroxide.' +, Shutdown within 2 min after addition of H202

(0.21 mM); *, spontaneous shutdown in 6 min; 0, noshutdown of acid production.dThe rate of acid production after addition of 2-

mercaptoethanol (0.24 mM).'SD, Standard deviation.

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TABLE 4. Acidproduction of dentalplaque exposedto saliva which was supplemented with H202 (700ILM) and SCN (10 mM) in the presence ofplaque

Subject OSCN-G Acid produc- Recove(pLM) tionlb Reoey

1 191.1 0 12.42 261.0 0 19.33 300.8 0 19.54 352.7 0 16.85 292.3 10.9 28.66 326.0 45.7 20.37 208.1 0 20.58 220.1 1.1 15.59 284.0 0 14.310 344.4 0 14.611 134.6 15.8d 12.8

Mean 265.0 7.6 17.7SD" 65.9 13.3 4.5

The concentration of OSCN- in saliva after 5 minof incubation at 37°C in the presence of supplementsand plaque.

b Nanomoles x minute-' x milliliter-' x A-'.'The rate of acid production after addition of 2-

mercaptoethanol (0.24 mM).d Spontaneous shutdown in 8 to 15 min.'SD, Standard deviation.

within a few minutes (Tables 2 and 3). Subse-quently, when 2-mercaptoethanol (final concen-tration, 0.24 mM) was added, the inhibition byOSCN- was reversed in all cases, and acid pro-duction started again (recovery). The rate ofrecovery seemed to be inversely related to therate of preceding acid production (Tables 2 to4).

In the control experiments (phosphate-buffered saline instead of saliva), the mean rateof acid production was 27.4 (standard deviation,12.0; range, 11.9 to 54.6 nmol x min-I x ml-, xAasO'1). This value is to be compared with themean rate of acid production in glucose-stimu-lated saliva, which was 37.5 (Table 2). The dif-ference between these two means was not statis-tically significant. The addition of peroxidealone, thiocyanate alone, or peroxide and thio-cyanate together to the control plaque suspen-sions did not significantly inhibit acid produc-tion.

DISCUSSIONPrevious work has shown that supplementa-

tion ofhuman whole saliva with the appropriatecombination of H202 and SCN- results in sig-nificant increases in the concentration ofOSCN-ions in saliva (K. M. Pruitt, J. Tenovuo, W.Fleming, and M. Adamson, Caries Res., inpress).

In the present study, supplementation of sa-liva by an H202-SCN- combination also pro-duced a remarkable increase in the concentra-

'50 0

O1000000 3o00

0~~~~~

[oscu-] PoFIG. 1. The rate ofplaque acid production (nano-

moles x milliliter-' x minute-' x A6w-') as a functionof salivary OSCN- concentration (0, unsupple-mented saliva; 0, saliva supplemented with H202 andSCN-). Plaque samples (50 or 1)0 Al of sonicatedplaque) were incubated for 5 min at 370C with anequivalent amount of saliva, followed by stimulationof acid production with 1% glucose. The line is thetheoretical curve calculated from the equation (P -P.,b (S - Sc) = K, where P is the acid production, Sis the OSCN- concentration, and P., So, and K areconstants (the values of the constants [6.24 nmol xml-' x min-' x A66-', 16.43 M, and 865.2 nmol xml-' x min ' x As66&' x 6LM, respectively] were deter-mined by trial and error).

tion of OSCN-. In, most cases, this increase waseven higher when saliva was supplemented inthe presence of plaque. This difference suggeststhat plaque samples produced H202 which, byreacting with lactoperoxidase and SCN-, furtherincreased the concentration of OSCN-. A cor-relation between the accumulation of OSCN-and the production of H202 by saliva sedimenthas been described previously (17, 21). No H202-producing reactions have been detected in salivasupernatant, but H202 production in saliva sed-iment is stimulated by endogenous carbohydratecomponents of saliva (21). Tenovuo and Antto-nen (18) found a positive correlation betweenoral health and OSCN- generation by salivasediment, probably because of higher endoge-nous H202 production by saliva of persons withgood oral health. These observations suggestthat H202-generating bacteria in human salivaare more resistant to OSCN- inhibition than areother microorganisms.Plaque acid production is often reported in

terms ofpH changes. For comparative purposes,the acid production data shown in Tables 2 to 4

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212 TENOVUO ET AL.

may be converted into pH changes as follows.Since pH is defined as the negative of the loga-rithm to base 10 of the hydrogen ion concentra-tion, the relationship between pH change andchange in hydrogen ion concentration is givenby the following equation:

ApH =-log 1 + [H+]}

where ApH is the pH change resulting from theaddition of A[H+] moles of hydrogen ions perliter to a solution whose initial concentration is[H+]i. To make the calculation, we must convertour measurements of acid production into A[H+]or moles of hydrogen ions added per liter. Wemeasured acid production in terms ofnanomolesx minute-' x milliliter-1 x A.-'. If we assumea volume of 1 ml of an unbuffered plaque sus-pension with an A660 of 4.5 (the average fromTable 1), then in 1 min:

A[H+] = acid production

1ml x 4.5 x 1 minx 10-9x lo-3

The factor 10-9 converts nanomoles to moles,and the results must be divided by the volume(1 ml) of the solution expressed in liters (10-3).By using the average acid production rate fromTable 2 (37.5 nmol x min-' x ml-' x A660-o), wefind that [H+] = 169 x 10-6 mol/liter. Substitut-ing this result into the ApH equation togetherwith an [H+]i of 10-65, we obtain ApH = -2.73.Values for the individual plaque samples calcu-lated from the data in Tables 1 and 2 rangedfrom 2.08 to 3.07. These are the pH changeswhich would occur in 1 min in 1 ml of theunbuffered plaque suspensions under the givenconditions. Ofcourse, the pH change in a plaque-saliva suspension would be much less because ofbuffering. A typical value which we have mea-

sured for the actual direct addition of lactic acidto 1 ml of normal saliva at this rate is a ApH of<0.01. Thus, although plaque may produce sig-nificant quantities of acid, the actual pH changeis greatly restricted by the buffering effects ofsaliva.The present study (Fig. 1) clearly shows the

dependence of plaque acid production upon sal-ivary OSCN- concentration. The trend of thedata plotted in Fig. 1 suggested to us that thereis a consistent relationship between these vari-ables. It is clear that as hypothiocyanite concen-

tration (S) increases, there is an initial sharp fallin acid production (P) followed by an apparentlyasymptotic approach to some limiting, minimallevel of acid production (Pii,,,) at very high hy-pothiocyanite concentrations. This minimum

level of acid production may represent the me-tabolism of bacteria which are not inhibited byhypothiocyanite. The data in Table 2 suggestthat normal resting saliva always contains somebasal level of hypothiocyanite. These consider-ations led us to hypothesize that the differencebetween plaque acid production (P) at nominalconcentrations of hypothiocyanite and the acidproduction (Pn,.) at very high concentrationsdecreased inversely in proportion to the differ-ence between the normal basal salivary concen-tration of hypothiocyanite (SO) and higher con-centrations. Quantitatively, this hypothesis canbe written as follows:

(P - Pmin)(S - So) = constant.

The line drawn in Fig. 1 was based on calcula-tions made from this equation. The theoreticalline clearly follows the trend of the data. Thedata presented here are not sufficient to allowbroad generalizations, but it seems reasonable toadopt the suggested relationship between plaqueacid production and salivary hypothiocyaniteconcentration as a working hypothesis. In anyevent, the inhibition of plaque acid productionby saliva seems to vary among different individ-uals according to the efficiency of the lactoper-oxidase system in generating OSCN- ions.The fact that OSCN- inhibition ofplaque acid

production is reversed by addition of thiols sup-ports the suggestions that OSCN- is responsiblefor the inhibition (5, 9, 14). The present studyshows also that dental plaque is more sensitiveto inhibition by the products of the salivaryperoxidase system when the products are gen-erated in the presence of plaque rather thanwhen they are generated separately and subse-quently added. This suggests the formation ofsome additional, short-lived antibacterial oxi-dation product which is more effective thanOSCN-. Bjorck and Claesson (6) have madesimilar observations with Escherichia coli. WithE. coli, the lactoperoxidase system caused irre-versible inhibition or a bactericidal effect whichwas not achieved with nonenzymatically pre-pared OSCN- solutions. In saliva sediment, theoxidation products formed within the sedimentclearly inhibit acid production more thanOSCN- ions applied from outside (17). Theseother oxidation products of the lactoperoxidasesystem may include higher oxyacids of SCN-,such as 02SCN- and 03SCN- (8), as well as(SCN)2 and HOSCN (4). These other oxidationproducts are all more unstable than OSCN-.Unpublished data from this laboratory suggestthat these highly reactive, short-lived interme-diates may be formed when OSCN- is utilizedas a substrate by lactoperoxidase and H202.

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INHIBITION OF PLAQUE ACID PRODUCTION BY SALIVA 213

The importance of the salivary lactoperoxi-dase system in vivo may be understood in termsof the following model (Fig. 2). (i) During andimmediately after eating, food carbohydratesstimulate the metabolism of aerobic bacteria,and some species then produce H202 (11). ThisH202 production is further stimulated by bacte-rial metabolism of the glycoproteins and mono-and disaccharides found in whole saliva (21). (ii)The peroxide generated by the bacteria reactswith SCN- and lactoperoxidase both present insaliva and dental plaque, yielding OSCN- andpossibly other short-lived oxidation products.(iii) The oxidation products cause inhibition ofbacterial metabolism by inhibiting sugar trans-port (12) and by inactivating key glycolytic en-zymes (1; H. Hoogendoorn, Ph.D. thesis, Delft,Mouton Den Haag, 1974). (iv) Inhibition of me-tabolism results in decreased peroxide excretionand decreased generation of OSCN-. At lowerOSCN- levels, there may be either spontaneous(7) or thiol-induced (14) recovery of inhibitedbacteria. Because all of the bacteria are notinhibited by this system and because the lowconcentration ofOSCN- in vivo (20), the rate ofacid production varies considerably among dif-ferent individuals depending upon the bacterialflora and the composition of saliva. Also, wehave observed that plaque collected from thesame individual on different occasions may showvariability in acid production and susceptibilityto homologous salivary OSCN- inhibition, pos-sibly because of variations in plaque or salivacomposition. Furthernore, it should be notedthat the other salivary defense factors may alsocontribute significantly to the regulation of bac-teria in the human mouth.

In conclusion, the data presented here showthat in vitro acid production by mixtures ofhuman dental plaque and homologous saliva is

FMN INCESTIOUUN"STIMIUTEl

ACTERIA carb,drates

RECOVERI STIOULATISIIIIl-*tbglrlollI /

IN ISE ACTIVE NUBACTERIA sacrENA

INIISI91T-10NI

FIG. 2. Proposed regulation ofplaque bacteria bysalivary lactoperoxidase system (details in the text).

inversely proportional to the concentration ofhypothiocyanite in the mixture, that the normalresting level of hypothiocyanite in human wholesaliva is at the threshold concentration requiredfor significant inhibition of plaque acid produc-tion, and that appropriate supplementation ofsaliva with peroxide and thiocyanate can pro-duce nearly total inhibition of plaque acid pro-duction in vitro.

ACKNOWVLEDGMENTISThis study was supported in part by National Institute of

Dental Research contract 54457, Public Health Service Inter-national Fellowship grant IF05 TWO 2903-OI, and PublicHealth Service grant DE 07026-04.

The technical assistance of Bonnie Jenkins is gratefullyacknowledged.

LITERATURE CITED1. Adamson, M., and K. M. Pruitt. 1981. Lactoperoxidase-

catalyzed inactivation of hexokinase. Biochim. Biophys.Acta 658:238-247.

2. Arnold, R. R., K. M. Pruitt, M. F. Cole, J. M. Adam-son, and J. R. McGhee. 1979. Salivary antibacterialmechanisms in immunodeficiency, p. 449-462. In I.Kleinberg, S. A. Ellison, and I. D. Mandel (ed.), Salivaand dental caries. Information Retrieval, Inc., Washing-ton, D.C.

3. Arnold, R. R., K. M. Pruitt, W. Fleming, and M.Adamson. 1981. Variations in salivary inhibition ofplaque metabolism. J. Dent. Res. 60 (Special Issue A):482.

4. Aune, T. M., and E. L. Thomas. 1977. Accumulation ofhypothiocyanite ion during peroxidase-catalyzed oxi-dation of thiocyanate ion. Eur. J. Biochem. 80:209-214.

5. Aune, T. M., and E. L. Thomas. 1978. Oxidation ofprotein sulfhydryls by products of peroxidase-catalyzedoxidation of thiocyanate ion. Biochemistry 17:1005-1010.

6. Bjorck, L, and 0. Claesson. 1980. Correlation betweenconcentration of hypothiocyanate and antibacterial ef-fect of the lactoperoxidase system against Escherichiacoli. J. Dairy Sci. 63:919-922.

7. DeMuth, R., M. Adamson, and K. M. Pruitt. 1981.Streptococcus mutans spontaneous recovery from hy-pothiocyanite inhibition-a theoretical analysis. J.Dent. Res. 60 (Special Issue A):444.

8. Hogg, D. M., and G. R. Jago. 1970. The antibacterialaction of lactoperoxidase. The nature of the bacterialinhibitor. Biochem. J. 117:779-790.

9. Hoogendoorn, H., J. P. Piessens, W. Scholtes, and L.A. Stoddard. 1977. Hypothiocyanite ion: the inhibitorformed by the system lactoperoxidase-thiocyanate-hy-drogen peroxide. Caries Res. 11:77-84.

10. Koch, G., and G. Strand. 1979. Effect of an enzymedentifrice on caries. A two-year clinical pilot study.Swed. Dent. J. 3:9-13.

11. Kraus, F. W., J. F. Nickerson, W. L. Perry, and A. P.Walker. 1957. Peroxide and peroxidogenic bacteria inhuman saliva. J. Bacteriol. 73:727-735.

12. Mickelson, M. N. 1977. Glucose transport in Streptococ-cus agalactiae and its inhibition by lactoperoxidase-thiocyanate-hydrogen peroxide. J. Bacteriol. 132:541-548.

13. Muhlemann, HL R.,R. Schmid, and A. R. Firestone.1981. Effect on rat caries of endogenous and exogenoushydrogen peroxide. Caries Res. 15:46-53.

14. Pruitt, K. M., M. Adamson, and R. Arnold. 1979.Lactoperoxidase binding to streptococci. Infect. Immun.25:304-309.

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15. Rotgans, J., and H. Hoogendoorn. 1979. The effect ofbrushing with a toothpaste containing amyloglucosidaseand glucose oxidase on dental caries in rats. Caries Res.13:150-153.

16. Shindler, J. S., R. E. Childs, and W. G. Bardsley. 1976.Peroxidase of human cervical mucus. Isolation andcharacterization. Eur. J. Biochem. 65:325-331.

17. Tenovuo, J. 1979. Formation of the bacterial inhibitor,hypothiocyanite ion, by cell-bound lactoperoxidase.Caries Res. 13:137-143.

18. Tenovuo, J., and T. Anttonen. 1980. Peroxidase-cata-lyzed hypothiocyanite production in human salivary

INFECT. IMMUN.

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