Available online at www.sciencedirect.com

(2008) 584–590

Clinical Biochemistry 41

A novel assay system for myeloperoxidase activity in whole saliva

Wataru Sakamoto a,b,⁎, Yoshihiro Fujii a, Takashi Kanehira c, Kozo Asano d, Hiroshi Izumi e

a Institute of Well Being, Fuji Women's University, 061-3204 Ishikari, Hokkaido, Japanb Serotec Laboratory, 069-0822 Ebetsu, Hokkaido, Japan

c Preventive Dentistry, Graduate School of Dental Medicine Hokkaido University, 060-8589 Sapporo, Hokkaido, Japand Laboratory Applied Microbiology, Graduate School of Agriculture Hokkaido University, 060-8589 Sapporo, Hokkaido, Japan

e Department of Physiology, School of Dentistry, Health Sciences University of Hokkaido, 061-0293 Ishikari-Tobetsu, Hokkaido, Japan

Received 18 June 2007; received in revised form 17 December 2007; accepted 30 December 2007Available online 15 January 2008

Abstract

Objectives: The application of a novel assay system for the direct measurement of MPO (myeloperoxidase) activity in whole saliva.Design and methods: The assay system employs a novel sensitive substrate from 3,3′-diaminobenzidine (DAB) and guaiacol in the presence

of dapsone (4,4′-diaminodiphenylsulfone) to determine MPO activity in whole saliva using an original “sandwich” test-disk (DEAE-cellulosepaper and cellulose chromatography paper). The saliva (0.1 mL) was directly applied to the sandwich test-disk, and then 0.1 mL of the substratesolution containing 1 mM dapsone in 0.3 M Tris–HCl buffer (pH 7.5) was added. After incubation for 30 min at room temperature, absorbance onthe test-disk was measured at 460 nm with an optical analyzer.

Results: The assay system was shown to distinguish MPO from salivary peroxidase in whole mixed saliva and was sensitive, easy and cheap.The assays revealed that MPO activity in whole saliva from subjects with periodontal disease was significantly higher than in saliva from healthysubjects. There was also a significant positive correlation between MPO activity and the probing depth of subgingival pockets (r=0.736,pb0.001).

Conclusions: These results indicate that this novel assay system for measurement of MPO is a useful technique for predicting the progressionof periodontal disease.© 2008 The Canadian Society of Clinical Chemist. Published by Elsevier Inc. All rights reserved.

Keywords: MPO (myeloperoxidase); Salivary peroxidase; Saliva; DAB (3,3′-diaminobenzidine); Guaiacol; Dapsone (4,4′-diaminodiphenylsulfone); “Sandwich”test-disk; Periodontal disease

Introduction

Human whole saliva is a mixed fluid comprising secretionsfrom major and minor salivary glands, a serum-derived transu-dation from the gingival crevices as well as components fromoral microorganisms, leukocytes, and epithelial cells. In thiscomplex milieu, oral peroxidase is composed of two peroxidaseenzymes: salivary peroxidase and MPO [1]. MPO is a hemicenzyme having dual effects on peroxidase and chlorination. It isstored primarily in neutrophils and accounts for b5% of total

⁎ Corresponding author. Institute of Well Being, Fuji Women's University,061-3204 Ishikari, Hokkaido, Japan. Fax: +81 11 383 4322.

E-mail address: wsakamot@fujijoshi.ac.jp (W. Sakamoto).

0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemist.doi:10.1016/j.clinbiochem.2007.12.025

cell protein content in these cells, functioning not only as a hostdefense mechanism by efficiently mediating microbial killingsubstances but also contributing to the initiation and propagationof acute and chronic inflammatory reactions [2–4]. In fact, MPOin gingival crevicular fluid is reported to increase in infectiousperiodontal disease [5–7], initiated by bacteria that colonize thesupra- and subgingival environments [8]. Specifically, period-ontopathogenic bacteria stimulate local host responses thatenhance the production of prostaglandins and inflammatorycytokines, and the recruitment of inflammatory cells with therelease of lytic enzymes such as elastase and MPO, leading tosubsequent damages to periodontal tissue [9–12]. Therefore,MPO could participate in the initiation and progression of pe-riodontal disease because MPO-derived oxidants contribute totissue damage and the initiation and propagation of acute and

Published by Elsevier Inc. All rights reserved.

585W. Sakamoto et al. / Clinical Biochemistry 41 (2008) 584–590

chronic vascular inflammatory disease [13]. In general, period-ontal disease is characterized by chronic inflammatory lesionsand destruction of supportive periodontal tissues, although thesymptoms are typically mild during the progression of the dis-ease. Thus, subjects tend to ignore the condition in the earlyphase of periodontal disease until more severe symptoms appear,for example, increased tooth mobility or tooth loss. Multipletooth loss leads to impairment of essential functions contributingto quality of life, such as conversation and eating. Therefore,early detection of periodontal disease is an important factor inmaintaining quality of life. Several investigators have reportedthat substances such as IgA, IL-1β, elastase and MPO mayprovide a screening test for periodontal disease in whole salivaand gingival crevicular fluid [9,10,14,15]. However, severalbarriers have prevented the routine utilization of gingival cre-vicular fluid as a screening methodology [16,17]. For example,samples collected from a single site in the mouth may not reflectthe whole mouth status of the subjects. Sampling time is also aproblem as is the way the sample strip is inserted. In addition, themeasurement of total MPO content by enzyme-linked immu-noassays (ELISA) is not representative of the active fraction ofMPO and this technique is time consuming and expensive.On the other hand, colorimetric assays using several substratessuch as guaiacol and DAB (3,3′-diaminobenzidine) do not allowus to distinguish MPO activity from salivary peroxidase activity[18–20]. The ultimate goal of the present study was to measureMPO in whole saliva as a screening test for periodontal disease.Thus, we attempted to develop an easy and cheap method forassaying MPO activity in whole saliva and to examine the rela-tionship between the enzyme activity and periodontal disease.

Materials and methods

DAB (3,3′-diaminobenzidine), guaiacol, and dapsone (4,4′-diaminodiphenylsulfone) were purchased from Dojin ChemicalCo., Japan, Wako Pure Chemical Co., Japan and Sigma-AldrichInc., USA, respectively. All other chemicals were of the highestgrade available commercially.

Unstimulated whole mixed saliva was collected from peri-odontally healthy subjects (36 males, 20 females; 20–25 years)for 20 min after mouth-rinsing with water, as described pre-viously [21]. Pooled saliva was centrifuged at 10,000 rpm for10 min and the supernatants were used as salivary peroxidase.

MPO (specific activity: 180–220 U/mg protein) from humanleukocytes was purchased from Biodesign International Co.(USA) and was dissolved in pooled whole saliva and/or heat-inactivated saliva (100 °C, 60 min).

Assessment of periodontal status and saliva collection frompatients with periodontal disease

This group consisted of 27 patients (16 males, 11 females;18–80 years) examined at the dental hospital of Hokkaido Uni-versity. The patients' periodontal condition was measured byprobing pocket depth in mm using a periodontal probe (#5 type,YDM Co., Japan). Probing was performed at six sites per toothfor all teeth except the third molar, and the deepest value was

recorded for each. Saliva samples were collected for 10min fromthe subjects, whowere instructed not to eat, drink or perform oralhygiene activities such as tooth brushing and mouth-rinsingfor at least 90min prior to saliva collection. Theywere instructedto chew a piece of gum (CAT21 Chewing Pellet, Will DentCo, Japan) and spit stimulated whole saliva into graduated testtubes for 10 min. Collected saliva samples were centrifuged at10,000 rpm for 10 min and the supernatants were stored at−80 °C until used.

Microplate assay for salivary peroxidase activity

Salivary peroxidase activity was measured in a 96-well mic-roplate (Nunclon™ 167008; Nunc A/S, Denmark) using wholemixed saliva as salivary peroxidase. The reaction mixture con-tained 150 μL of 0.3MTris–HCl buffer (pH 7.5), 20 μL of salivaand 30 μL of hydrogen donor solution in the presence ofhydrogen peroxide solution (0.84 mM). The optical density wasdetermined at 450 nm using a microplate reader (Labsystems-Multiskan MS, Dainippon Pharm. Co., Japan).

Sandwich test-disk assay for peroxidase activity of MPO andsalivary peroxidase in whole saliva

The assay of enzyme activity was carried out using a sand-wich test-disk, a sensitive substrate and an optical device. Thesandwich test-disk wasmade of DEAE-cellulose paper, a weaklybasic anion exchanger (DE81; 23 mm diameter, 0.2 mm thick-ness; Whatman Int. Ltd., England) and cellulose chromatogra-phy paper (Toyo; 23 mm diameter, 0.7 mm thickness: ToyoRoshi Co., Japan). These papers were held in an aluminum ring.The sensitive substrate for peroxidase activity was composed of3.47 mM DAB, 176 mM guaiacol and hydrogen peroxide(0.84 mM and/or 4 mM) in 0.3 M Tris–HCl buffer (pH 7.5).Unless otherwise stated, the assay was performed as follows.The sample (0.1 mL) was directly applied to the sandwich test-disk and then 0.1 mL of the substrate solution was added. Afterincubation for 30 min at room temperature, the color density wasanalyzed using an optical device containing a light-emittingdiode (LED) at a wavelength of 460 nm and a photodetector(Nippon Denshoku Co., Japan), which was directed verticallyonto the sandwich test-disk. The light was reflected verticallyback through the photodetector. Thus, the enzyme activity wasexpressed as absorbance at 460 nm after the difference in ab-sorbance was subsequently calculated using a blank of heatedsaliva. Enzyme activity was expressed as the increase in A460per 30 min. MPO activity in whole mixed saliva was alsomeasured using the sensitive substrate described above contain-ing 1 mM dapsone, which was prepared by addition of 100 mMdapsone dissolved in 0.5 N HCl. MPO activity was expressed asabsorbance at 460 nm after 30 min of reaction, which was thedifference in absorbance using distilled water as a blank.

Statistical analysis

Student's t-test was used for the statistical analysis. Dataare expressed as mean±SD. Regression analysis was used to

Fig. 1. Comparison of substrates as hydrogen donors and the relationshipbetween reaction time and enzyme activity in human salivary peroxidase. Ex-perimental conditions are described in the text. The data represent the means±SD from three experiments:●, mixture of 3.47 mMDAB and 176 mM guaiacol;○, 176 mM guaiacol; □, 3.47 mM DAB. Fig. 3. Time course of enzyme activity of MPO from human neutrophils.

Experimental conditions are described in the text, except for the variousconcentrations of MPO, 0.84 mM hydrogen peroxide and incubation time. Thedata represent the means±SD from three experiments. ○, 8 ng protein MPO;●, 40 ng protein MPO; □, 200 ng protein MPO.

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determine the significance of correlation between two changes.pb0.05 was considered statistically significant.

Results

A novel system for assay of peroxidase activity

To create a highly sensitive substrate for peroxidase activity,we mixed guaiacol and DAB in anticipation of enhancement ofDAB oxidation by guaiacol metabolites in the presence of theperoxidase/hydrogen peroxide system. The assay system wastested in a 96-well microplate using whole mixed saliva assalivary peroxidase. Fig. 1 shows the relationship betweenreaction time and enzyme activity in human salivary peroxidase

Fig. 2. Comparison of reaction sites of MPO activity on three membranes.Experimental conditions are described in the text, except for using MPO (40 ngprotein) dissolved in inactivated saliva and 0.84 mM hydrogen peroxide. Thedata represent the means±SD from three experiments. DE81 (DEAE-cellulosepaper); P81 (cellulose phosphate paper); Toyo (cellulose chromatography paper).

using 3.47 mM DAB alone, 176 mM guaiacol alone, and amixture of DAB and guaiacol as a hydrogen donor. Our resultsshow that the mixture of DAB and guaiacol resulted in the mostintense increase in absorbance at 450 nm, reaching a plateau in30 min. On the other hand, the use of guaiacol or DAB aloneshowed less intense absorbance. Based on these results, we usedthe mixture of 3.47 mM DAB and 176 mM guaiacol in thepresence of hydrogen peroxide as a highly sensitive substratefor all other measurements.

Next, we attempted to develop a novel assay system forperoxidase activity using a sandwich test-disk, a sensitive sub-strate and an optical device. The purpose of the sandwich test-disk was to compare the specificity of three membranes: anionchromatography paper (DE81), cation chromatography paper(P81; cellulose phosphate; 0.23 mm thickness; Whatman Int.Ltd., England), and cellulose chromatography paper (Toyo) atthe site of the enzyme reaction. As shown in Fig. 2, the anionicpaper (DE81) used in the sandwich test-disk demonstrated themost intense color at the site of the enzyme reaction afterincubation for 30 min. The cation and cellulose chromatographypapers recorded 36±8% and 39±10% of the reaction of theanionic paper, respectively (n=3). Therefore, we used a sand-wich test-disk consisting of anionic chromatography and cellu-lose chromatography papers for all further measurements ofenzyme activity.

Time course of enzyme activity of MPO from human neutrophils

To investigate the time course of enzyme activity on thesandwich test-disk, MPO activity was measured using a novelsensitive substratemixture consisting of 3.47mMDAB, 176mMguaiacol and 0.84 mM hydrogen peroxide in 0.3 M Tris–HCl

Fig. 4. Effect of hydrogen peroxide on MPO activity. Experimental conditionsare described in Fig. 3, except for the use of 40 ng protein MPO, variousconcentrations of hydrogen peroxide, and the incubation time of 30 min. Thedata represent the means±SD from three experiments.

Fig. 5. Effects of dapsone on the peroxidase activity of MPO and salivaryperoxidase. Experimental conditions are described in the text, except for the useof the substrate solution composed of 3.47 mM DAB, 176 mM guaiacol, and4 mM hydrogen peroxide in 0.3 M Tris–HCl buffer (pH 7.5) containing dapsoneranging from 0 to 2.2 mM. The enzyme activity of each preparation is expressedas a percentage of the control without dapsone. The data represent the means±SD from three experiments. ●, salivary peroxidase (170 μg protein); ○, MPO(40 ng protein).

Fig. 6. Standard curve for MPO in whole saliva. MPO was dissolved in pooledwhole saliva from healthy subjects. Other experimental conditions are describedin the text, except for the use of substrate solution composed of 3.47 mM DAB,176 mM guaiacol, 4 mM hydrogen peroxide and 1 mM dapsone in 0.3 M Tris–HCl buffer (pH 7.5). The enzyme activity is expressed as the increase inabsorbance (A460) per 30 min. The data represent the means±SD from threeexperiments.

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buffer (pH 7.5). After 0.1 mL of MPO solution was directlyapplied to the sandwich test-disk, 0.1 mL of substrate solutionwas added and incubated for the appropriate time. The reactionwas allowed to proceed to completion, corresponding to themaximal absorbance at 460 nm. Using 8 ng, 40 ng, and 200 ng ofMPO, most of the colored reaction product was developedduring 20 min of incubation followed by a further small increasein absorbance at room temperature (Fig. 3). The results were notaffected by environmental temperature, showing that the alte-ration of enzyme activity was less than 5% between 20 and 35 °C(data not shown). These results indicate that incubation of30 min at room temperature is appropriate for the measurementof MPO activity on the sandwich test-disk. Thus, the assaythereafter was carried out with measurement at 30 min after thebeginning of the reaction.

Effect of hydrogen peroxide on MPO activity

The concentration of hydrogen peroxide is a critical factor inMPO assays. Therefore, we examined the optimum concentra-tion of hydrogen peroxide in the assay system. As shown inFig. 4, enzyme activity increased with increasing concentrationsof H2O2 (0.1 mM to 4.2 mM), but decreased at concentrationsover 8.4 mM. The apparent maximum activity was observed atapproximately 4.2 mM. We therefore chose to use a level of4.0 mM hydrogen peroxide for the standard assay system forMPO activity.

Effects of dapsone on the peroxidase activity of MPO andsalivary peroxidase

Human salivary peroxidase is synthesized and secreted bythe salivary glands, whereas MPO is derived from leukocytes,

which migrate into the oral cavity at the gingival crevices. Todistinguish MPO from salivary peroxidase in whole mixedsaliva, we used dapsone which has two primary aromatic aminemoieties. As expected, dapsone levels up to 2.2 mM had littleinhibitory effect on MPO activity, but inhibited salivary per-oxidase activity by 95% (Fig. 5). This suggests that dapsone is aselective inhibitor of salivary peroxidase, although it cannotcompletely inhibit it.

Fig. 8. Correlation between MPO activity and probing pocket depth. Theenzyme activity was measured as described in Fig. 7. Other experimentalconditions are described in the text (n=27; r=0.736; pb0.001).

588 W. Sakamoto et al. / Clinical Biochemistry 41 (2008) 584–590

Standard curve for MPO in whole saliva

To determine the activity of MPO in whole saliva on thesandwich test-disk, purified MPO from human neutrophils wasadded to pooled whole saliva from periodontally healthy sub-jects at concentrations ranging from 10 ng/mL to 900 ng/mL. Atypical standard curve was obtained by plotting the absorbancevalues at 460 nm as a function of standard MPO concentrations.When MPO concentrations were expressed in the semi-loga-rithmic form, a linear curve was obtained for MPO values from30 ng/mL to 450 ng/mL (Fig. 6). The linear regression obtainedfrom the standard curve after polynomial transformation showeda good correlation coefficient (r=0.971) with coefficient varia-tion (CV)=13.0%. These results showed that the assay systemwas reliable for the quantitative determination of salivary MPOin the presence of 1 mM dapsone.

Correlation between MPO activity and probing depth

Having shown that this novel assay system reflects MPOactivity and content in whole saliva, we next examined MPOactivity in saliva from subjects with and without periodontaldisease. Fig. 7 shows that there is a higher enzyme activity insaliva from subjects with periodontal disease than the healthysubjects. Interestingly, a significant positive correlation wasobserved between MPO activity and the probing depth of sub-gingival pockets (n=27, r=0.736, pb0.001), as shown inFig. 8. Specifically, the MPO activity of patients with probingpocket depths above 4 mm was significantly ( pb0.001) higher

Fig. 7. A typical representation of MPO activity in whole saliva from subjectswith and without periodontal disease. The enzyme activity was measured withsubstrate solution composed of 3.47 mM DAB, 176 mM guaiacol, 4 mMhydrogen peroxide and 1 mM dapsone in 0.3 M Tris–HCl buffer (pH 7.5). Otherexperimental conditions are described in the text. 1) MPO (40 ng protein) fromhuman leukocytes; 2) 100 μL of saliva from a subject with periodontal disease(probing pocket depth 4.6 mm); 3) 100 μL of pooled whole mixed saliva fromperiodontally healthy subjects; 4) blank (100 μL of distilled water).

(A460=0.5441±0.2375; n=6) than of those with pocket depthsof 2–3 mm (A460=0.1399±0.0605; n=13). In pooled wholesaliva from periodontally healthy subjects the mean A460 was0.0699±0.0252 (n=3).

Discussion

MPO functions not only in host defense by mediating effi-cient microbial killing, but also can contribute to progressivetissue damage in chronic inflammatory states such as athero-sclerosis [22] and periodontal disease [23,24]. Recently, it hasbeen reported that MPO levels increase at sites of inflammationand that the levels in gingival crevicular fluid are related to theseverity of periodontal disease [5–7]. Thus, MPO may be usedas a biomarker for periodontal disease. However, several bar-riers have prevented the routine utilization of gingival crevicularfluid and the measurement of MPO by ELISA as a screeningmethodology because this assay is inappropriate. Indeed theELISA test measures total (active and inactive) MPO and theenzyme activity of horseradish peroxidase conjugated to thesecondary antibody could interfere with the peroxidase activityof the MPO captured by the antibodies. Recently it was demon-strated that MPO captured by immobilized antibodies couldmaintain its activity [25]. In our preliminary experiments usinga commercially available ELISA kit, the addition of purifiedMPO to heated whole saliva showed accurate linearity rangingfrom 2 ng/mL to 60 ng/mL but this was not the case for unheatedsaliva. In fact, it should be noted that peroxidases are stronglyadsorbed from the aqueous solution onto glass, enamel and cellsurfaces [20]. On the other hand, various substrates are availablefor determination of MPO using colorimetric assays [18–20].However, a colorimetric method for measurement of oxidationof DAB has not yet been developed because very low levels ofperoxidase activity must be measured at the maximum sensi-tivity of the spectrophotometer but oxidized DAB is stableunder the usual assay conditions [19]. The guaiacol assay is oneof the most commonly used assays for peroxidase activity, but itis impossible to use the assay method at the bedside because the

589W. Sakamoto et al. / Clinical Biochemistry 41 (2008) 584–590

initial formation of the oxidized product is comparatively linearwith time only over the first 40 s, reaching a maximal levelat the rate of 43 s−1, followed by a slow and distinct decline[26]. Interestingly, tetraguaiacol produced from the oxidation ofguaiacol by MPO is ascribed to diguaiacol during the course ofthe peroxidation cycle, and diguaiacol subsequently undergoesfurther to serve as a hydrogen donor [26,27]. Thus, we at-tempted to create a novel sensitive substrate for peroxidase fromDAB and guaiacol. As expected, the combination of DAB andguaiacol increased the enzyme reaction 2–3-fold compared withDAB and guaiacol alone. Although the mechanism has not yetbeen clarified, redox cycles of guaiacol seemed to enhance theDAB oxidation in the presence of peroxidase and hydrogenperoxide. Previous reports from Deby-Dupont et al. and Kleba-noff have shown that the active form of MPO is oxidized byhydrogen peroxide to compound I characterized by a π-cationradical state. Compound I is subsequently reduced back intoferric MPO by two monoelectronic oxidations of an electrondonor via the formation of an intermediate non-radical state ofthe enzyme [3,4]. Therefore, diguaiacol produced from the redoxcycles of guaiacol and DAB will be used as electron donor in theactivity cycle of MPO. For these reasons, the mixture of DABand guaiacol appears to be a good combination to amplify MPOactivity on the sandwich test-disk. However, the details of themechanism should be further investigated. Next we attempted tomake the enzyme reaction proceed in the solid phase instead ofthe aqueous solution, to make the procedure easy and inex-pensive. In general, MPO is assumed to form a complex withanionic proteins such as albumin in serum, because it is a highlycationic protein with an isoelectric point higher than 10 [28,29].Thus, MPO purified from human leukocytes was dissolved inpooled whole saliva and/or inactivated saliva. As expected, theDE81 anionic paper of the sandwich test-disk showed the mostintense color of all the papers during the enzyme reaction,presumably because salivary peroxidase and MPO are adsorbedto it. We further attempted to develop a method to distinguishMPO from salivary peroxidase in whole mixed saliva usingdapsone, because the novel substrate is not specific for MPO.Several investigators have reported that dapsone inhibits per-oxidase activity in a limited set of conditions, i.e. at pH 5.4 withtetramethylbenzidine (TMB) as the substrate [30]. In fact, ac-cording to Thomas et al., the use of dapsone allowed identi-fication and quantification of MPO and salivary peroxidase inhuman mixed saliva [31]. We also demonstrated in the presentstudy that dapsone inhibited salivary peroxidase but notMPO byusing a novel substrate containing 1 mM dapsone in 0.3 M Tris–HCl buffer (pH 7.5). However, it is not clear by which mecha-nism dapsone inhibits salivary peroxidase but not MPO activity.This point should be further investigated. Our study using thesandwich test-disk and the novel highly sensitive substrate con-taining 1 mM dapsone demonstrated a linear curve with a semi-logarithmic relation between peroxidase activity and MPO con-tent in whole saliva, ranging from 30 ng/mL to 450 ng/mL.Regarding the assessment of periodontal disease severity byclinical examination, it is well known that the clinical attachmentlevel, pocket depth, bleeding upon probing, and radiographicbone loss are measured in the patients [15,32]. We finally ex-

amined the relationship between probing pocket depth andMPOactivity in whole saliva using the novel assay system, in order todevelop the potential rapid screening method for periodontaldisease. As expected, MPO activity in whole saliva from sub-jects with periodontal disease was higher than from healthysubjects, with a significant positive relationship between activitylevels and probing pocket depth. Consequently, our study sug-gests that MPO activity in whole saliva may reflect the severityof periodontal disease. These findings indicate that the novelassay system for MPO is a useful technique for predicting theprogression of periodontal disease.

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