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Clinical Biochemistry 41 (2008) 584 590

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
Institute of Well Being, Fuji Women's University, 061-3204 Ishikari, Hokkaido, Japan b Serotec Laboratory, 069-0822 Ebetsu, Hokkaido, Japan c Preventive Dentistry, Graduate School of Dental Medicine Hokkaido University, 060-8589 Sapporo, Hokkaido, Japan d Laboratory Applied Microbiology, Graduate School of Agriculture Hokkaido University, 060-8589 Sapporo, Hokkaido, Japan 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 2007 Available online 15 January 2008
a

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-cellulose paper and cellulose chromatography paper). The saliva (0.1 mL) was directly applied to the sandwich test-disk, and then 0.1 mL of the substrate solution containing 1 mM dapsone in 0.3 M TrisHCl buffer (pH 7.5) was added. After incubation for 30 min at room temperature, absorbance on the 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 healthy subjects. There was also a significant positive correlation between MPO activity and the probing depth of subgingival pockets (r = 0.736, p b 0.001). Conclusions: These results indicate that this novel assay system for measurement of MPO is a useful technique for predicting the progression of 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 secretions from major and minor salivary glands, a serum-derived transudation from the gingival crevices as well as components from oral microorganisms, leukocytes, and epithelial cells. In this complex milieu, oral peroxidase is composed of two peroxidase enzymes: salivary peroxidase and MPO [1]. MPO is a hemic enzyme having dual effects on peroxidase and chlorination. It is stored primarily in neutrophils and accounts for b 5% 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).

cell protein content in these cells, functioning not only as a host defense mechanism by efficiently mediating microbial killing substances but also contributing to the initiation and propagation of acute and chronic inflammatory reactions [24]. In fact, MPO in gingival crevicular fluid is reported to increase in infectious periodontal disease [57], initiated by bacteria that colonize the supra- and subgingival environments [8]. Specifically, periodontopathogenic bacteria stimulate local host responses that enhance the production of prostaglandins and inflammatory cytokines, and the recruitment of inflammatory cells with the release of lytic enzymes such as elastase and MPO, leading to subsequent damages to periodontal tissue [912]. Therefore, MPO could participate in the initiation and progression of periodontal disease because MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and

0009-9120/$ - see front matter 2008 The Canadian Society of Clinical Chemist. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.12.025

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chronic vascular inflammatory disease [13]. In general, periodontal disease is characterized by chronic inflammatory lesions and destruction of supportive periodontal tissues, although the symptoms are typically mild during the progression of the disease. Thus, subjects tend to ignore the condition in the early phase of periodontal disease until more severe symptoms appear, for example, increased tooth mobility or tooth loss. Multiple tooth loss leads to impairment of essential functions contributing to quality of life, such as conversation and eating. Therefore, early detection of periodontal disease is an important factor in maintaining quality of life. Several investigators have reported that substances such as IgA, IL-1, elastase and MPO may provide a screening test for periodontal disease in whole saliva and gingival crevicular fluid [9,10,14,15]. However, several barriers have prevented the routine utilization of gingival crevicular fluid as a screening methodology [16,17]. For example, samples collected from a single site in the mouth may not reflect the whole mouth status of the subjects. Sampling time is also a problem as is the way the sample strip is inserted. In addition, the measurement of total MPO content by enzyme-linked immunoassays (ELISA) is not representative of the active fraction of MPO and this technique is time consuming and expensive. On the other hand, colorimetric assays using several substrates such as guaiacol and DAB (3,3-diaminobenzidine) do not allow us to distinguish MPO activity from salivary peroxidase activity [1820]. The ultimate goal of the present study was to measure MPO in whole saliva as a screening test for periodontal disease. Thus, we attempted to develop an easy and cheap method for assaying MPO activity in whole saliva and to examine the relationship between the enzyme activity and periodontal disease. Materials and methods DAB (3,3-diaminobenzidine), guaiacol, and dapsone (4,4diaminodiphenylsulfone) were purchased from Dojin Chemical Co., Japan, Wako Pure Chemical Co., Japan and Sigma-Aldrich Inc., USA, respectively. All other chemicals were of the highest grade available commercially. Unstimulated whole mixed saliva was collected from periodontally healthy subjects (36 males, 20 females; 2025 years) for 20 min after mouth-rinsing with water, as described previously [21]. Pooled saliva was centrifuged at 10,000 rpm for 10 min and the supernatants were used as salivary peroxidase. MPO (specific activity: 180220 U/mg protein) from human leukocytes was purchased from Biodesign International Co. (USA) and was dissolved in pooled whole saliva and/or heatinactivated saliva (100 C, 60 min). Assessment of periodontal status and saliva collection from patients with periodontal disease This group consisted of 27 patients (16 males, 11 females; 1880 years) examined at the dental hospital of Hokkaido University. The patients' periodontal condition was measured by probing pocket depth in mm using a periodontal probe (#5 type, YDM Co., Japan). Probing was performed at six sites per tooth for all teeth except the third molar, and the deepest value was

recorded for each. Saliva samples were collected for 10 min from the subjects, who were instructed not to eat, drink or perform oral hygiene activities such as tooth brushing and mouth-rinsing for at least 90 min prior to saliva collection. They were instructed to chew a piece of gum (CAT21 Chewing Pellet, Will Dent Co, Japan) and spit stimulated whole saliva into graduated test tubes for 10 min. Collected saliva samples were centrifuged at 10,000 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 microplate (Nunclon 167008; Nunc A/S, Denmark) using whole mixed saliva as salivary peroxidase. The reaction mixture contained 150 L of 0.3 M TrisHCl buffer (pH 7.5), 20 L of saliva and 30 L of hydrogen donor solution in the presence of hydrogen peroxide solution (0.84 mM). The optical density was determined at 450 nm using a microplate reader (LabsystemsMultiskan MS, Dainippon Pharm. Co., Japan). Sandwich test-disk assay for peroxidase activity of MPO and salivary peroxidase in whole saliva The assay of enzyme activity was carried out using a sandwich test-disk, a sensitive substrate and an optical device. The sandwich test-disk was made of DEAE-cellulose paper, a weakly basic anion exchanger (DE81; 23 mm diameter, 0.2 mm thickness; Whatman Int. Ltd., England) and cellulose chromatography paper (Toyo; 23 mm diameter, 0.7 mm thickness: Toyo Roshi Co., Japan). These papers were held in an aluminum ring. The sensitive substrate for peroxidase activity was composed of 3.47 mM DAB, 176 mM guaiacol and hydrogen peroxide (0.84 mM and/or 4 mM) in 0.3 M TrisHCl buffer (pH 7.5). Unless otherwise stated, the assay was performed as follows. The sample (0.1 mL) was directly applied to the sandwich testdisk and then 0.1 mL of the substrate solution was added. After incubation for 30 min at room temperature, the color density was analyzed using an optical device containing a light-emitting diode (LED) at a wavelength of 460 nm and a photodetector (Nippon Denshoku Co., Japan), which was directed vertically onto the sandwich test-disk. The light was reflected vertically back through the photodetector. Thus, the enzyme activity was expressed as absorbance at 460 nm after the difference in absorbance was subsequently calculated using a blank of heated saliva. Enzyme activity was expressed as the increase in A460 per 30 min. MPO activity in whole mixed saliva was also measured using the sensitive substrate described above containing 1 mM dapsone, which was prepared by addition of 100 mM dapsone dissolved in 0.5 N HCl. MPO activity was expressed as absorbance at 460 nm after 30 min of reaction, which was the difference in absorbance using distilled water as a blank. Statistical analysis Student's t-test was used for the statistical analysis. Data are expressed as mean SD. Regression analysis was used to

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Fig. 1. Comparison of substrates as hydrogen donors and the relationship between reaction time and enzyme activity in human salivary peroxidase. Experimental conditions are described in the text. The data represent the means SD from three experiments: , mixture of 3.47 mM DAB and 176 mM guaiacol; , 176 mM guaiacol; , 3.47 mM DAB.

determine the significance of correlation between two changes. p b 0.05 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 of DAB oxidation by guaiacol metabolites in the presence of the peroxidase/hydrogen peroxide system. The assay system was tested in a 96-well microplate using whole mixed saliva as salivary peroxidase. Fig. 1 shows the relationship between reaction time and enzyme activity in human salivary peroxidase

Fig. 3. Time course of enzyme activity of MPO from human neutrophils. Experimental conditions are described in the text, except for the various concentrations of MPO, 0.84 mM hydrogen peroxide and incubation time. The data represent the means SD from three experiments. , 8 ng protein MPO; , 40 ng protein MPO; , 200 ng protein MPO.

using 3.47 mM DAB alone, 176 mM guaiacol alone, and a mixture of DAB and guaiacol as a hydrogen donor. Our results show that the mixture of DAB and guaiacol resulted in the most intense increase in absorbance at 450 nm, reaching a plateau in 30 min. On the other hand, the use of guaiacol or DAB alone showed less intense absorbance. Based on these results, we used the mixture of 3.47 mM DAB and 176 mM guaiacol in the presence of hydrogen peroxide as a highly sensitive substrate for all other measurements. Next, we attempted to develop a novel assay system for peroxidase activity using a sandwich test-disk, a sensitive substrate and an optical device. The purpose of the sandwich testdisk was to compare the specificity of three membranes: anion chromatography paper (DE81), cation chromatography paper (P81; cellulose phosphate; 0.23 mm thickness; Whatman Int. Ltd., England), and cellulose chromatography paper (Toyo) at the site of the enzyme reaction. As shown in Fig. 2, the anionic paper (DE81) used in the sandwich test-disk demonstrated the most intense color at the site of the enzyme reaction after incubation for 30 min. The cation and cellulose chromatography papers recorded 36 8% and 39 10% of the reaction of the anionic paper, respectively (n = 3). Therefore, we used a sandwich test-disk consisting of anionic chromatography and cellulose chromatography papers for all further measurements of enzyme activity. Time course of enzyme activity of MPO from human neutrophils

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

To investigate the time course of enzyme activity on the sandwich test-disk, MPO activity was measured using a novel sensitive substrate mixture consisting of 3.47 mM DAB, 176 mM guaiacol and 0.84 mM hydrogen peroxide in 0.3 M TrisHCl

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Fig. 4. Effect of hydrogen peroxide on MPO activity. Experimental conditions are described in Fig. 3, except for the use of 40 ng protein MPO, various concentrations of hydrogen peroxide, and the incubation time of 30 min. The data represent the means SD from three experiments.

Fig. 5. Effects of dapsone on the peroxidase activity of MPO and salivary peroxidase. Experimental conditions are described in the text, except for the use of the substrate solution composed of 3.47 mM DAB, 176 mM guaiacol, and 4 mM hydrogen peroxide in 0.3 M TrisHCl buffer (pH 7.5) containing dapsone ranging from 0 to 2.2 mM. The enzyme activity of each preparation is expressed as a percentage of the control without dapsone. The data represent the means SD from three experiments. , salivary peroxidase (170 g protein); , MPO (40 ng protein).

buffer (pH 7.5). After 0.1 mL of MPO solution was directly applied to the sandwich test-disk, 0.1 mL of substrate solution was added and incubated for the appropriate time. The reaction was allowed to proceed to completion, corresponding to the maximal absorbance at 460 nm. Using 8 ng, 40 ng, and 200 ng of MPO, most of the colored reaction product was developed during 20 min of incubation followed by a further small increase in absorbance at room temperature (Fig. 3). The results were not affected by environmental temperature, showing that the alteration of enzyme activity was less than 5% between 20 and 35 C (data not shown). These results indicate that incubation of 30 min at room temperature is appropriate for the measurement of MPO activity on the sandwich test-disk. Thus, the assay thereafter was carried out with measurement at 30 min after the beginning of the reaction. Effect of hydrogen peroxide on MPO activity The concentration of hydrogen peroxide is a critical factor in MPO assays. Therefore, we examined the optimum concentration of hydrogen peroxide in the assay system. As shown in Fig. 4, enzyme activity increased with increasing concentrations of H2O2 (0.1 mM to 4.2 mM), but decreased at concentrations over 8.4 mM. The apparent maximum activity was observed at approximately 4.2 mM. We therefore chose to use a level of 4.0 mM hydrogen peroxide for the standard assay system for MPO activity. Effects of dapsone on the peroxidase activity of MPO and salivary peroxidase Human salivary peroxidase is synthesized and secreted by the salivary glands, whereas MPO is derived from leukocytes,

which migrate into the oral cavity at the gingival crevices. To distinguish MPO from salivary peroxidase in whole mixed saliva, we used dapsone which has two primary aromatic amine moieties. As expected, dapsone levels up to 2.2 mM had little inhibitory effect on MPO activity, but inhibited salivary peroxidase activity by 95% (Fig. 5). This suggests that dapsone is a selective inhibitor of salivary peroxidase, although it cannot completely inhibit it.

Fig. 6. Standard curve for MPO in whole saliva. MPO was dissolved in pooled whole saliva from healthy subjects. Other experimental conditions are described in 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 in absorbance (A460) per 30 min. The data represent the means SD from three experiments.

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Standard curve for MPO in whole saliva To determine the activity of MPO in whole saliva on the sandwich test-disk, purified MPO from human neutrophils was added to pooled whole saliva from periodontally healthy subjects at concentrations ranging from 10 ng/mL to 900 ng/mL. A typical standard curve was obtained by plotting the absorbance values at 460 nm as a function of standard MPO concentrations. When MPO concentrations were expressed in the semi-logarithmic form, a linear curve was obtained for MPO values from 30 ng/mL to 450 ng/mL (Fig. 6). The linear regression obtained from the standard curve after polynomial transformation showed a good correlation coefficient (r = 0.971) with coefficient variation (CV) = 13.0%. These results showed that the assay system was reliable for the quantitative determination of salivary MPO in the presence of 1 mM dapsone. Correlation between MPO activity and probing depth Having shown that this novel assay system reflects MPO activity and content in whole saliva, we next examined MPO activity in saliva from subjects with and without periodontal disease. Fig. 7 shows that there is a higher enzyme activity in saliva from subjects with periodontal disease than the healthy subjects. Interestingly, a significant positive correlation was observed between MPO activity and the probing depth of subgingival pockets (n = 27, r = 0.736, p b 0.001), as shown in Fig. 8. Specifically, the MPO activity of patients with probing pocket depths above 4 mm was significantly ( p b 0.001) higher

Fig. 8. Correlation between MPO activity and probing pocket depth. The enzyme activity was measured as described in Fig. 7. Other experimental conditions are described in the text (n = 27; r = 0.736; p b 0.001).

(A460 = 0.5441 0.2375; n = 6) than of those with pocket depths of 23 mm (A460 = 0.1399 0.0605; n = 13). In pooled whole saliva from periodontally healthy subjects the mean A460 was 0.0699 0.0252 (n = 3). Discussion MPO functions not only in host defense by mediating efficient microbial killing, but also can contribute to progressive tissue damage in chronic inflammatory states such as atherosclerosis [22] and periodontal disease [23,24]. Recently, it has been reported that MPO levels increase at sites of inflammation and that the levels in gingival crevicular fluid are related to the severity of periodontal disease [57]. Thus, MPO may be used as a biomarker for periodontal disease. However, several barriers have prevented the routine utilization of gingival crevicular fluid and the measurement of MPO by ELISA as a screening methodology because this assay is inappropriate. Indeed the ELISA test measures total (active and inactive) MPO and the enzyme activity of horseradish peroxidase conjugated to the secondary antibody could interfere with the peroxidase activity of the MPO captured by the antibodies. Recently it was demonstrated that MPO captured by immobilized antibodies could maintain its activity [25]. In our preliminary experiments using a commercially available ELISA kit, the addition of purified MPO to heated whole saliva showed accurate linearity ranging from 2 ng/mL to 60 ng/mL but this was not the case for unheated saliva. In fact, it should be noted that peroxidases are strongly adsorbed from the aqueous solution onto glass, enamel and cell surfaces [20]. On the other hand, various substrates are available for determination of MPO using colorimetric assays [1820]. However, a colorimetric method for measurement of oxidation of DAB has not yet been developed because very low levels of peroxidase activity must be measured at the maximum sensitivity of the spectrophotometer but oxidized DAB is stable under the usual assay conditions [19]. The guaiacol assay is one of the most commonly used assays for peroxidase activity, but it is impossible to use the assay method at the bedside because the

Fig. 7. A typical representation of MPO activity in whole saliva from subjects with and without periodontal disease. The enzyme activity was measured with substrate solution composed of 3.47 mM DAB, 176 mM guaiacol, 4 mM hydrogen peroxide and 1 mM dapsone in 0.3 M TrisHCl buffer (pH 7.5). Other experimental conditions are described in the text. 1) MPO (40 ng protein) from human leukocytes; 2) 100 L of saliva from a subject with periodontal disease (probing pocket depth 4.6 mm); 3) 100 L of pooled whole mixed saliva from periodontally healthy subjects; 4) blank (100 L of distilled water).

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initial formation of the oxidized product is comparatively linear with time only over the first 40 s, reaching a maximal level at the rate of 43 s 1, followed by a slow and distinct decline [26]. Interestingly, tetraguaiacol produced from the oxidation of guaiacol by MPO is ascribed to diguaiacol during the course of the peroxidation cycle, and diguaiacol subsequently undergoes further to serve as a hydrogen donor [26,27]. Thus, we attempted to create a novel sensitive substrate for peroxidase from DAB and guaiacol. As expected, the combination of DAB and guaiacol increased the enzyme reaction 23-fold compared with DAB and guaiacol alone. Although the mechanism has not yet been clarified, redox cycles of guaiacol seemed to enhance the DAB oxidation in the presence of peroxidase and hydrogen peroxide. Previous reports from Deby-Dupont et al. and Klebanoff have shown that the active form of MPO is oxidized by hydrogen peroxide to compound I characterized by a -cation radical state. Compound I is subsequently reduced back into ferric MPO by two monoelectronic oxidations of an electron donor via the formation of an intermediate non-radical state of the enzyme [3,4]. Therefore, diguaiacol produced from the redox cycles of guaiacol and DAB will be used as electron donor in the activity cycle of MPO. For these reasons, the mixture of DAB and guaiacol appears to be a good combination to amplify MPO activity on the sandwich test-disk. However, the details of the mechanism should be further investigated. Next we attempted to make the enzyme reaction proceed in the solid phase instead of the aqueous solution, to make the procedure easy and inexpensive. In general, MPO is assumed to form a complex with anionic proteins such as albumin in serum, because it is a highly cationic protein with an isoelectric point higher than 10 [28,29]. Thus, MPO purified from human leukocytes was dissolved in pooled whole saliva and/or inactivated saliva. As expected, the DE81 anionic paper of the sandwich test-disk showed the most intense color of all the papers during the enzyme reaction, presumably because salivary peroxidase and MPO are adsorbed to it. We further attempted to develop a method to distinguish MPO from salivary peroxidase in whole mixed saliva using dapsone, because the novel substrate is not specific for MPO. Several investigators have reported that dapsone inhibits peroxidase activity in a limited set of conditions, i.e. at pH 5.4 with tetramethylbenzidine (TMB) as the substrate [30]. In fact, according to Thomas et al., the use of dapsone allowed identification and quantification of MPO and salivary peroxidase in human mixed saliva [31]. We also demonstrated in the present study that dapsone inhibited salivary peroxidase but not MPO by using a novel substrate containing 1 mM dapsone in 0.3 M Tris HCl buffer (pH 7.5). However, it is not clear by which mechanism dapsone inhibits salivary peroxidase but not MPO activity. This point should be further investigated. Our study using the sandwich test-disk and the novel highly sensitive substrate containing 1 mM dapsone demonstrated a linear curve with a semilogarithmic relation between peroxidase activity and MPO content in whole saliva, ranging from 30 ng/mL to 450 ng/mL. Regarding the assessment of periodontal disease severity by clinical examination, it is well known that the clinical attachment level, pocket depth, bleeding upon probing, and radiographic bone loss are measured in the patients [15,32]. We finally ex-

amined the relationship between probing pocket depth and MPO activity in whole saliva using the novel assay system, in order to develop the potential rapid screening method for periodontal disease. As expected, MPO activity in whole saliva from subjects with periodontal disease was higher than from healthy subjects, with a significant positive relationship between activity levels and probing pocket depth. Consequently, our study suggests that MPO activity in whole saliva may reflect the severity of periodontal disease. These findings indicate that the novel assay system for MPO is a useful technique for predicting the progression of periodontal disease. References
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