Evidence for thiocyanate sensitive peroxidase activity in human saliva

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0095-1137/83/111177-06$02.00/0

Evidence for

Thiocyanate-Sensitive

Peroxidase

Activity

in

Human

Saliva

R. A.COWMAN,1,2* S. S. BARON,' S.D.OBENAUF,2ANDJ.J. BYRNES3

Dental Research Unit1 andHematology Section,3Miami VeteransAdministration MedicalCenter, and DepartmentofMicrobiology andImmunology, UniversityofMiamiSchoolof Medicine,2Miami, Florida

33125

Received10June1983/Accepted 22August 1983

Aprocedurewas developedfordetermining the relative levels of

lactoperoxi-dase, leukocyte myeloperoxilactoperoxi-dase, and thiocyanate-sensitive peroxidase in human

saliva. With thisprocedure, most of the peroxidase activity in whole saliva from

normal(those without cancer) subjectswasfoundtobe associated with lactoper-oxidase and thiocyanate-sensitive peroxidase, with only a minor contribution

from leukocyte myeloperoxidase. In contrast,

thiocyanate-sensitive peroxidase

and leukocyte myeloperoxidase were the major peroxidase activities present in theresidualsalivary secretion obtainablefrom two xerostomicpatients examined, and these enzymes were present at concentrations much higher than those normally occurring in human saliva. The occurrence of thiocyanate-sensitive peroxidaseinsaliva hasnotbeenpreviously reportedand may representeitheran

additional peroxidase activity of saliva or a form oflactoperoxidase which is particularly sensitive toinhibition by

thiocyanate.

Radiation damagetothemajor salivary glands in patients receiving ionizing radiation for tu-mors of the head or neck has been shown to

result inqualitative andquantitative changes in

theprotein composition of residual salivary

se-cretion (6). Amongtheearly qualitative changes occurring during radiationtreatment, there was arapid disappearance from the electrophoretic

protein patterns of zones corresponding to sali-varyamylase isozymes andother more cationic

proteins inthepHregion expected for lactoper-oxidase (LPO), lactoferrin, and secretory

immunoglobulinA. These alterationssuggested

that muchof thepotential antimicrobial activity

of saliva

might

be lost early in the radiation

treatmentphase.

However, residual salivary secretion obtained from some patients in the postradiation period exhibited iodide-oxidizing peroxidase activities equivalent to or higher than those seen at

pre-treatment. The reappearance of peroxidase

ac-tivity in these salivas suggested that a selective

partialrecovery ofglandularfunction may have

occurred or that peroxidases ofextraglandular

origin such as myeloperoxidase (MPO), an en-zyme normallyfound in low levels in saliva (12, 19), might be present in substantially increased

amounts. Further attempts to elucidate the

na-ture ofpostradiation salivary peroxidase activi-ty, however, were complicatedby the fact that

iodide,

guaiacol, or thiocyanate, which have

been used to measuresalivaryperoxidase

activi-ty, areoxidized

by

both LPOandMPO

(7-9, 11,

15, 17). Inaddition,eventhough gel permeation chromatography has been usedtoseparate LPO

and MPO (12, 19), such procedures were not

considered

practical

with the limited volumes of

saliva obtainable fromxerostomic individuals.

The purposes of this

investigation

were to

develop a suitable

procedure

for

differentiating

LPO, MPO, or other

iodide-oxidizing

peroxi-dasesinsaliva andusethis

procedure

in

compar-ing the peroxidase activities present in saliva

fromnormal(those without

cancer)

and

xerosto-mic individuals.

MATERIALS

ANDMETHODS

Chemicals and enzymes. Guaiacol and p-phenylene-diamine (PPD) were from Sigma Chemical Co., St. Louis, Mo., 4-aminoantipyrine (4-AP) wasfromJ. T. BakerChemical Co., Phillipsburg, N.J., and all other chemicals or reagents were of the highest quality commercially available.

LPOfrom bovine milk and chloroperoxidase (CPO) from Caldariomyces fumagowerefromSigma Chemi-cal Co. Human leukocytes were obtained by leuko-phoresis from a patient with chronic granulocytic leukemiaat atimewhen the white count was 480,000 and consisted of50%matureforms.MPO was isolated from these leukocytesby the method of Bakkenhist et al.(1)andfurther purifiedby gel permeation chroma-tography on Sephadex G-75 fine (Pharmacia Fine

Chemicals, Piscataway, N.J.) (14) and ion-exchange

chromatography on Bio-Gel CM-2 (Bio-Rad, Rich-mond, Calif.). The purified MPO gave a single zone 1177

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migrating toward the cathode afteranalytical

isoelec-tric focusing in thin-layer polyacrylamidegel

contain-ing ampholines in thepHrangeof3.5to10andastwo

distinctzonesafter sodium dodecyl sulfate-thin-layer

polyacrylamide gel electrophoresis. The latter

obser-vationwasconsistent for thetwosubunitsof MPO (1).

Collection andtreatmentof saliva. Whole salivawas

collected from normal (without cancer) control sub-jects and from two patients with radiation-induced xerostomia (age matched with controls) who had

re-ceived a minimumof 5,000 rads ofionizing radiation over an8-week interval fortreatmentof headorneck tumors. In these xerostomic patients, the major

sali-varyglandshad been bilaterally exposedinthefield of

radiation. Before sampling, each donor rinsed his mouth withdistilledwater toremoveloosely adherent debris, and salivary flowwasstimulated by rinsing for

3-min intervals with three separate2-ml portions ofa

stimulant solution containing 0.8% citric acid. The three rinsings were combined and constituted the

salivasample. Actual volumes of saliva collectedwere

estimated as thedifference between the total volume

of the three rinsesrecovered and thestarting 6 ml of rinse solution. By this method, 6 ml of saliva was

usually obtained from the control subjects, with 0.5 ml obtained from thexerostomic patients.

The saliva specimens werefirsttreated with 0.1 ml

of 0.1 N NaOH to reduce viscosity (necessary with xerostomia salivasamples) and then clarified by

cen-trifugation (15,000 x g, 20min). The resultant

super-natants were neutralized to pH 7.0, transferred to Spectraphor (Spectrum Medical Industries, Los Ange-les, Calif.) selective membrane dialysis sacks (3,500-daltoncutoff) and dialyzed against 2 liters of distilled water at 4°C overnight to remove

small-molecular-weight constituents. These dialyzed protein-contain-ing retentates were used in the studies described

below.

Peroxidase activity assays. All absorbance

measure-mentsweremade inaGilford 240spectrophotometer (GilfordInstrumentLaboratories, Inc., Oberlin, Ohio) with either a 1.5- or 4-ml 1-cm light path quartz cuvette, depending on the specific assay being

per-formed.Enzyme reaction timesweremonitoredtothe nearest0.01 s.

Oxidationofguaiacolwasdeterminedbyamodified

method of ChanceandMaehly (5). Inoursystem, 0.5

ml of 10mMpotassium phosphatebuffer(pH 6.0),0.25

ml of20 mMguaiacol,and 0.25 ml ofdilutedenzyme

preparation were mixed in the cuvette. The

absorb-anceat470 nm was adjustedto zero, after which the

enzymaticreactionwasinitiatedbythe addition of 10

,ul of40 mMhydrogen peroxide. The timerequiredto attain an absorbance increase of 0.05 was recorded,

and the units ofperoxidase activity weredetermined

as described by Makinen etal. (13). Guaiacol

oxida-tion in thepresenceof 0.05mMpotassium thiocyanate

wasdeterminedbyreplacingthe buffer withoneof the samecomposition containing0.1 mM KSCN.

Iodide oxidationwasdetermined inaccordance with

theguaiacolassay,except50 pumolofpotassiumiodide

replaced guaiacol and the enzymatic reaction was

monitoredat353 nm.

The oxidationof4-APwasdeterminedbythe meth-od of Mathesonetal.(14)with thefollowing modifica-tions. Thecuvette contained 0.2 ml of 0.2 M sodium

phosphate buffer(pH 6.1),0.2 ml of 4-AP(2.5mg/ml

stock in 0.17 M phenol, prepared fresh beforeuse),0.4

ml of distilled water, and 0.1 ml ofdiluted enzyme

preparation. The absorbanceat 510nm wasadjusted

tozero, and the reactionwas started by adding0.1ml

of0.85 mM hydrogen peroxide. The increase in absor-bancewasrecorded after 20s,and1 Uof peroxidase

activitywasdefinedasthatamountofenzymecausing

a0.001 absorbance increase in 20s.

For PPD oxidation, the substrate was dissolved in

boiling water, filtered, cooled, stored in adark

con-tainer, and used within 30 min of preparation. Peroxi-dase activity on this substrate was determined as

described previously by Pilz et al. (16) except that hydrogen peroxide was added last. The amount of

enzymecausinga0.001absorbance increaseat485nm

in 30s wasconsidered tobe 1 U.

Salivary peroxidase activity. Peroxidase activity

pres-ent in the dialyzed saliva protein preparations was

assayed using the KI, guaiacol, and guaiacol plus0.05

mM KSCN (SCN- inhibition assay) substrate

sys-tems. From theseactivity measurements, therelative levels of LPO, MPO, and thiocyanate-sensitive

perox-idase (TSP) ina given test sample weredetermined.

For comparing peroxidase activities in saliva from differentdonor subjects, the units of activity attribut-ableto LPO, MPO, orTSP wereconverted to their microgram equivalent after which the microgram amountof eachenzyme permilliliter of saliva actually obtainedwas calculated.

RESULTS

The relativeoxidative activities of milk LPO,

leukocyte MPO, and CPO toward KI, guaiacol,

4-AP, or PPD were compared to establish

whetheranyof these substrates mightserve as a

selective indicator for either LPO or MPO.

These initial tests revealed that whereas LPO

oxidized KI farmore readily thanguaiacol

(Ta-ble 1), both MPO and CPO exhibited agreater

oxidativeactivity toward guaiacolrelativetoKI.

The threeperoxidases displayedcomparable

ox-idative activities toward 4-AP, as did LPO or

MPO towardPPD. The latter substratewasonly

weakly oxidized by CPO.

Although none ofthe substrates tested were

specific for either LPO or MPO, a distinctive

difference did exist between these peroxidases

TABLE 1. Comparisonof theoxidativeactivity of LPO,MPO,and CPOondifferent substrates

Uof enzymeactivity/>lgof enzyme

Enzymesource inassaya

KI Guaiacol 4-AP PPD

LPO 7.75 3.18 24.0 1,600

MPO 1.48 4.90 18.0 1,350

CPO 1.52 5.00 22.5 30

a Units of enzyme activity on each substrate are defined in thetext. Forthesecomparisons 0.4 ,ug of LPOorCPO and 0.26jigof MPOwereused in each assay. Specificactivityvalues represent the meansof threereplications.

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PEROXIDASES OF HUMAN SALIVA 1179

with respect to the oxidation of iodide and

guaiacol. Todetermine if this difference could be

mademoreselective for either LPOorMPO,the

effect of known inhibitors of these peroxidases

on theit oxidative properties toward guaiacol

and iodide was examined. For this purpose, potassium cyanide, sodium azide, or KSCN

were selected and tested at concentrations of

0.05 or 0.1 mM. The oxidation of iodide and

guaiacol by LPO, MPO,orCPOwascompletely

inhibited at either concentration of potassium

cyanide or sodium azide (data not shown).

KSCN similarly abolished the iodide-oxidizing activity associated with the peroxidases, but

pronounced differences were observed with

re-spect to its effect on theguaiacol-oxidizing

activ-ity. Specifically, 0.1 mM KSCN completely

in-hibited guaiacol oxidation by either MPO or

CPO, but in the presence of 0.05 mM of this

inhibitor, activity was reducedonly about 50%.

In contrast, the oxidation ofguaiacol by LPO

wasstronglyenhanced at either concentration of theinhibitor.

Because the foregoing studies had been

car-ried out atonly one enzyme concentration, the

oxidative activities of LPO, MPO, and CPO

toward guaiacol or

guaiacol

plus 0.05 mM

KSCN and toward KI were compared over a

range ofenzyme concentration. In these tests

the oxidation of KI by LPO was linear for

enzyme concentrations up to 0.3 ,g, and a

nearly linearresponse wasobserved forguaiacol oxidation (Fig. 1A). From these data a mean

iodide/guaiacol oxidation ratio of 2.75 + 0.20

was calculated for LPO at enzyme

concentra-tions ranging from 0.05 to 0.3 ,ug. As noted

previously, 0.05 mM KSCN enhancedLPO

ac-tivity toward guaiacol. Conversely, the oxida-tion of eitherguaiacol or KI by either MPO or

CPO was nonlinear (Fig. 1B); nonetheless, a

mean iodide/guaiacol oxidation ratio of0.40 ±

0.03 was calculated from these

activity

curves

for both enzymes. As expected,the addition of

0.05 mMKSCN resulted in a 51 ± 3.8%

inhibi-tion in the oxidation ofguaiacol by either

en-zyme.

On the basis ofthe difference in the iodide/

guaiacol oxidation ratios forLPO andMPO,the

following method was developed for

determin-ingthe relative level of these peroxidase

activi-ties in a given test saliva. Enzymatic activity

assays were carried out withguaiacol, guaiacol

plus 0.05 mM KSCN, or KI. Theproportion of

the enzymatic activity on guaiacol attributable

to LPO or MPO was calculated by using the

relationships that: a + b = activity on guaiacol

and 0.40 a + 2.75 b = activity on KI, where a

represents the units of MPO in the sample, b

represents the units ofLPO, and 0.50 and 2.75

arethe meaniodide/guaiacol oxidationratios for

t

w

N z

Lu

U-0

C,)

I-z

A 0.20 0.40 0.60 B 0.20 0.40 0.60 0.80 ugENZYME IN ASSAY

FIG. 1. Effect of enzyme concentrationonthe oxi-dation ofpotassium iodide, guaiacol,orguaiacol plus

0.05 mM KSCNby LPO,MPO, orCPO. (A) Lacto-peroxidase. (B) Solidlines, CPO;brokenlines,MPO. Symbols indicate the oxidation of: A, guaiacol; 0,

guaiacol plus 0.05 mM KSCN; and 0, potassium

iodide. Barsrepresentthestandard deviation of three replications.

MPO and LPO, respectively. Thefeasibility of

this method was initially tested with samples

which contained differing amounts of LPO and

CPO. The results show that the relativeamounts

ofperoxidase activityrecoveredasLPOorCPO

fromthe sixsamples tested agreed wellwith the

amountsofeach enzymewhichhad been added

to aparticular sample (Table 2). The guaiacol-oxidizing activitiescalculatedforLPO andCPO

were thentransformed to the respective

activi-ties for the oxidation ofKI and fortheeffectof SCN- by referencetothe standardizedactivity

curves (see Fig. 1A and B). In most cases (five

ofsix), these summed expected activity values

were consonantwiththe totalactivity observed

oneach substratesystem.Similartests werenot

carried out with mixtures of LPO and MPO because ofthelimited availability of MPO.

Preliminary tests were next conducted with

salivapreparations. Although the combined

ac-tivities calculated as LPO or MPO in these

salivas were in consistent agreement with the

total activities measured by either iodide or

guaiacol oxidation, the total activity, as

deter-mined by SCN- inhibition assay, was always

much lower than that expected on the basis of

the calculated levels of LPO and MPO in these

salivas. The possibility that this difference was

causedbythepresence of at least one additional

peroxidase activity which could contribute to theoxidative activity toward guaiacol or iodide

butwhich wascompletely inhibited in the

pres-ence of 0.05 mM KSCN was considered. In

testing this possibility, it was assumed that

sali-18,

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TABLE 2. Calculation of LPO andCPO concentration insamples containing different amounts of each peroxidase after assay with guaiacol, guaiacol plus 0.05 mM KSCN, or

potassium iodide

Sample LPO(>Lg) CPO(,g) tested Added Recovered Added Recovered

1 0.20 0.21 0.00 0.00

2 0.15 0.13 0.05 0.06

3 0.10 0.08 0.15 0.14

4 0.10 0.11 0.20 0.21

5 0.05 0.06 0.20 0.19

6 0.05 0.04 0.25 0.25

vary LPOwas not inhibited by SCN- and that

the SCN- inhibitionassayreflected only

peroxi-daseactivity derived from LPOorMPOorboth.

On thisbasis, the proportion of the totalsalivary

peroxidase activity attributabletoLPO could be

determined from the following: 0.49 a + b =

activityonguaiacol plus 0.05 mM KSCN, where

0.49 a represents that fraction of the MPO

activity on guaiacol not inhibited by 0.05 mM

KSCN. This calculationgavemuchlower values

for LPO and indicated that a portion of the

peroxidase activity originally attributed to LPO

wasduetothepresenceof TSP activity.

Correc-tion of the original data for TSP contribution

resulted in excellent agreement between the

combined activities calculated for LPO, MPO,

andTSP and the totalsalivary peroxidase

activi-ty as measured by the SCN- inhibition assay

system.

On the basis of the foregoing findings, the

peroxidase activities present insalivafrom

nor-mal(without cancer) andradiation-induced

do-nor sources were compared after assay with

guaiacol, guaiacol plus 0.05 mM KSCN, andKI.

Theresults indicate that the peroxidase activity

insaliva from normal donorswasderivedmostly

fromacombination of LPO and TSP, with onlya

minor contribution from MPO (Table 3).

Sub-jects D and E, however, appeared to possess

relatively high levels of MPO in their saliva. On

the other hand, TSP and MPO represented the

major peroxidase activitiespresentin the

residu-al salivary secretions from the two xerostomic

patients examined. These latter secretions

con-tained ca. 1.51 ,ug of MPO and 2.35 ,ug of TSP

per mg of saliva proteinas comparedwith0.09

,ug of MPOor0.31 ,ug of TSPper mgof protein

in salivafromthe controlsubjects.

DISCUSSION

Inthispaper arapid procedure for

differenti-ating LPO, leukocyte MPO, andathird

peroxi-dase, TSP, in human saliva is described. The

method is basedondifferences in the enzymatic

properties of the three peroxidases toward

guaiacol and iodide and on a difference in the

sensitivity of MPO and TSPtoinhibition by 0.05

mM KSCN. Although the differentiation

re-quires the use of three substrate systems, the

assays and associated calculations are easily

performed. Also, since only small amounts of

testsampleareneededfor analysis, this method

offers an advantage in situations in which only

limited amounts of saliva may be available for

study.

Using this method, we found thatwhole

sali-vas frommostof the controlsubjects examined

possessedlow levels ofMPO, with LPO

activi-ties ranging between 1.1 to 1.5 U per ml of

TABLE 3. Comparisonof the relative concentrations ofLPO, MPO,andTSP in saliva from normal(without

cancer) andtworadiation-inducedxerostomia donorsources

Enzymeconcn(pg/mlofsaliva)

Salivasource

LPO MPO TSP

Controls

A 0.50 0.05 0.80

B 0.55 0.00 0.70

C 0.70 0.00 0.90

D 0.50 0.90 0.50

E 0.30 0.50 0.90

F 0.50 0.20 1.00

Mean + SD 0.51 + 0.11 0.28 ±0.33 0.97 ± 0.25

Xerostomia

S;6mopostradiation 0.00 7.14 5.20

S;10mopostradiation 0.00 14.% 16.2

T; S mopostradiation 1.78 6.50 16.0

Mean(5to6mopostradiation) ± SD 0.89± 0.89 6.80± 0.30 10.6 ± 5.40

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PEROXIDASES OF HUMAN SALIVA 1181

saliva. Although others have reported similarly low levels of MPO in saliva (19), the LPO activities we measured were much lower than

the 3 to 5 U of LPO per ml (as determined by

guaiacoloxidation) considered normal forsaliva

(19, 20). Using only the guaiacol oxidation

as-say, we obtained LPO activities ranging

be-tween 3 and 8 U/ml, but as indicated by the

SCN- inhibition assay, only a portion of this activity was specifically attributable to LPO.

Theremainderwasderived either fromTSP or a

combination of TSP and MPO. Because these latterperoxidases possesstheability to oxidize either iodide orguaiacol, their

potential

occur-rence in saliva must be considered whenever

iodide orguaiacol is used alone for theassayof salivary LPOactivity. The

lower-than-expected

LPO activities observed inthe saliva from the

donorsexamined in thisstudy,therefore,appear

to be related to the presence inthese salivas of

TSP, inparticular, and, in some cases, MPO as well.

Tenovuo(18)hassuggestedthat humanwhole

salivacontainstwotypes ofperoxidases: oneof salivary glandular origin, namely LPO, and the other of leukocytic

origin.

Evidence obtained here indicates that saliva may also possess a

third

peroxidase

activity, TSP, which is distin-guishable from either MPOorLPOby its partic-ularsensitivitytoSCN- andwhichmay account

for ca.

50%

of the guaiacol or iodide-oxidizing activity associated with saliva. The occurrence

of TSP

activity,

firstnotedby the SCN-

inhibi-tion assay, was further supported by

observa-tions showing that: (i) salivas which did not

possess MPO activity, nonetheless, exhibited

reduced peroxidase activity toward guaiacol in

the presenceof0.05 mMKSCN,

(ii)

saliva from

normalorxerostomic donorspossessed

iodide-oxidizing properties

which couldnotbe attribut-ed solely toLPO orMPO, and (iii) saliva from

one of the xerostomic patients which did not

possess LPOactivity, nonetheless, still

exhibit-edstrongiodide-oxidizing activity.

Thefactthat theenzymatic propertiesof TSP,

like LPO, differed substantially from those of

leukocyte MPO suggests that TSP is not of

leukocytic origin. On the other hand, the close

similarity in the enzymatic properties of LPO

and TSP, except for SCN- sensitivity, and the

persistenceof TSPactivityin saliva of a number of different donors suggests a salivary origin.

Although we consider TSP to be a separate

peroxidase, a relationship between TSP and LPO cannot be excluded on the basis of the

evidencepresented here. Since LPO is known to

exist inparotidorwhole saliva in more than one

molecular-weight form (18), it is possible that

TSP may represent a form of salivary LPO

which is sensitive toinhibition by SCN-.

We previously reported (6) that the iodide-oxidizing

activity

of saliva obtained from

pa-tients receiving

ionizing

radiation therapy for

head or neck tumors declinedrapidly

during

the

radiation treatment interval. However, in the

later postradiation phase of care, the residual

secretions from some, but not

all,

of these

individuals exhibited iodide-oxidizing activities

which were equivalent to, or exceeded, those

observed at pretreatment. Some

explanation

of this phenomenon was obtained from the two

xerostomic patients examined in this

study.

In

both cases, the high

peroxidase activity

was

clearly associated with increased levels of both TSP andMPO. The

high

levelsof MPO in these residual salivas could be the result of an in-creased leakage of crevicular

fluid,

which is known to be rich in leukocyte MPO (12; M. J.

Kowolik, M. Grant, J. A. Raeburn, Abstr. Int.

Assoc. Dent. Res., 59th Gen. Meet., 1981,

Abstr. no. 1203, p. 610). Since the residual

secretions presumably contain a higher relative

percentage of the minor glandular secretions,

the occurrence of high levels of TSP suggests

that this enzymatic activity might be of minor

glandular origin.

The major quantitative shifts which occur in specific microbial elements of the oral micro-biota inassociation with the onsetof radiation-induced xerostomia have been attributed, in

part, to a loss of salivary protective factors,

including LPO (3, 10). Although these

alter-ations usually lead to a greater incidence of postradiation caries in xerostomic patients,

re-cent studies (2, 4) have indicated that some

patients do not experience any postradiation caries activity. Salivas from these individuals

werefoundto possesshighersalivary

agglutina-tion titers to Streptococcus mutans and higher immunoglobulin A levelsthan salivas from

pa-tients whodeveloppostradiation caries activity. However, as shown here, the loss of salivary

LPO activitymay notnecessarily leadto aloss

ofthe protective influence attributable to

sali-varyperoxidases. The occurrence ofhighlevels

ofleukocyte MPOin postradiation salivas is of potential significance because this enzyme, in

the presence of SCN- and hydrogen peroxide,

mayexhibit eitherabacteriostaticor

bactericid-aleffect toward S. mutans (8). Thus, the nature

of the composition of the peroxidase activity

associated with postradiation saliva might also

influencethe intraoral microbial ecological

rela-tionshipswhich ultimately determine the sever-ity ofpostradiation caries activity. Studies are

nowin progress to further examine the

magni-tude and natureofthemodifications occurring in

salivary peroxidase activity during and after

radiation therapy and the effect of suchchanges

onthegrowth of the cariogenic streptococci.

VOL.18,1983

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ACKNOWLEDGMENT

This studywassupportedinpartby PublicHealth research grant 2-R01-DE-04278-07A from the National Institute of DentalResearch.

LITERATURECITED

1. Bakkenhist, A.R.J., R.Wever, T.Vulsma, H. Plat,and B.F. van Gelder. 1978. Isolation procedure and some properties ofmyeloperoxidase from human leucocytes. Biochim. Biophys. Acta 524:45-54.

2. Brown, L.R., S. Dreizen, T. E. Daly, J. B. Drane, S. Handler,L.J.Riggan,and D. A.Johnston. 1978. Interre-lations of oral microorganisms, immunoglobulins, and dental caries following radiotherapy. J. Dent. Res. 57:882-893.

3. Brown, L. R., S. Dreizen, S. Handler, and D. A. Johnston. 1975. Effect ofradiation-induced xerostomia on human oralmicroflora.J. Dent.Res. 54:740-750.

4. Brown, L.R., P. A. O'Neill, S. Dreizen, S. F. Handler, L.J.Riggan, and D. H. Perkins. 1981. Relationship be-tween saliva and serum agglutination titers and post-irradiation caries activity incancerpatients.J.Dent.Res. 60:10-18.

5. Chance, B.,and A.C.Maehly.1964. Peroxidaseassayby spectrophotometric measurements of the disappearance ofhydrogen donor or the appearance of their colored oxidation products. MethodsEnzymol.2:769-775. 6. Cowman, R.A., S. S. Baron, A. H. Glassman, M. E.

Davis, and A. M. Strosberg. 1983. Changes in protein composition of saliva from radiation-induced xerostomia patients andthe effectongrowth of oral streptococci. J. Dent. Res.62:336-340.

7. Hosoya, T., and M. Morrison. 1965. Theperoxidaseand other hemoproteins of thyroid microsomes. Biochem. Biophys.Res.Commun.20:27-32.

8. Kersten, H.W., W.R. Moorer, and R. Wever. 1981. Thiocyanate as a cofactor in myeloperoxidase activity

against Streptococcusmutans.J.Dent. Res.60:831-837. 9. Klebanoff, S.J. 1968. Myeloperoxidase-halide-hydrogen peroxide antibacterialsystem.J.Bacteriol.95:2131-2138. 10. Llory,H., A. Dammron, M. Gioanni, and R.J. Frank. 1972.Somepopulation changesin oral anaerobic microor-ganisms, Streptococcusmutansand yeastsfollowing irra-diation ofthesalivary glands. CariesRes.6:298-311. 11. Maguire, R.J.,andH. B.Dunford.1972. Kineticsofthe

oxidationofiodide ionby lactoperoxidase compoundII. Biochemistry 11:937-941.

12. Makinen,K.K.,andJ.Tenovuo. 1976.Chromatographic separationofhumansalivary peroxidases.ActaOdontol. Scand.34:141-150.

13. Makinen, K.K., J. Tenovuo, and A. Scheinin. 1976. Xylitol-induced increase oflactoperoxidase activity. J. Dent.Res.55:652-660.

14. Matheson,N.R.,P. S.Wong,andJ. Travis. 1981. Isola-tionandpropertiesofhumanneutrophilmyeloperoxidase. Biochemistry 20:325-330.

15. McRipley, R.J., and A.J. Sbarra. 1967. Role ofthe phagocyte in host-parasite interactions. XII. Hydrogen peroxide-myeloperoxidase bactericidal system in the phagocyte. J.Bacteriol. 94:1425-1430.

16. Pilz, H., J.S. O'Brien, and R. Heipertz. 1976. Human saliva peroxidase: microanalytical isoelectric fraction-ationand properties in normal persons and in caseswith neuronalceroid-lipofuscinosis. Clin.Biochem. 9:85-88. 17. Tenovuo, J. 1978. Lactoperoxidase-catalysed iodine

me-tabolism in human saliva. Arch. Oral Biol. 23:253-258. 18. Tenovuo, J. 1981. Different molecular forms ofhuman

salivary lactoperoxidase. Arch. Oral Biol.26:1051-1055. 19. Tenovuo, J., and M. L. E. Knuuittila. 1977.Antibacterial

effect ofsalivary peroxidases on a cariogenic strain of Streptococcusmutans. J. Dent. Res.56:1608-1613. 20. Tenovuo, J., and J. Valtakoski. 1976. The correlation

betweensalivary peroxidase activity, salivaryflow rate, andthe oxidation-reduction potential of human salivaand dental plaquesuspensions. Acta Odontol. Scand.

34:169-176.