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RESEARCH REPORT |
Department of Oral Biology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel-Aviv University, Ramat-Aviv, Israel, 69978;
*corresponding author, melros{at}post.tau.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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0.002; Spearman) and tongue malodor (p 0.001; Spearman). ß–galactosidase activity and sulfide monitor measurements both factored significantly into multiple regression equations for odor judge scores, yielding multiple r-values ranging from 0.47 (p = 0.0007) to 0.60 (p < 0.0001). Analysis of the data presented indicates that ß–galactosidase activity in saliva is correlated with oral malodor.
KEY WORDS: ß-Galactosidase • oral malodor • diagnosis • glycoproteins • saliva
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Among the most common sources of protein for malodor production are salivary mucins and epithelial cell components (Kleinberg and Westbay, 1992; Yaegaki and Sanada, 1992), both of which contain numerous glycoproteins (Levine et al., 1987). Since the proteolysis of glycoproteins depends on initial removal of the carbohydrate side-chains (Gottschalk and Fazekas De St. Groth, 1960), we have considered the possibility that deglycosylation is an initial step in oral malodor production. ß-galactosidase is one of the important enzymes responsible for the removal of both O- and N-linked carbohydrate side-chains (De Jong and Van Der Hoeven, 1987; Van Der Hoeven and Camp, 1991; Homer et al., 1994). ß-galactosidase activity can be easily quantified with the use of chromogenic substrates (Gossrau, 1977).
In the present report, we have tested for the association between ß-galactosidase activity in saliva and oral malodor parameters (i.e., odor judge scores and volatile sulfide compounds [VSC]). The results suggest that: (i) ß-galactosidase activity is significantly associated with oral malodor as measured by odor judges; and (ii) both ß-galactosidase activity and VSC independently account for oral malodor levels.
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MATERIALS & METHODS |
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Experimental Protocol
Participants were asked to refrain from eating or drinking for 2 hrs prior to measurements. Measurements (described in detail below) included malodor-related parameters (odor judges' scores and volatile sulfide measurements), and ß-galactosidase activity. All measurements were conducted during late afternoon hours.
Measurements
ß-galactosidase activity
ß-galactosidase activity was assayed with use of the chromogenic substrate 5–bromo-4-chloro-3-indolyl-ß-D–galactopyranoside (X-Gal; Gossrau, 1977). Two stock solutions were prepared. The first was prepared by the dissolution of 100 mg of X-Gal (Sigma, St. Louis, MO, USA) in 5 mL of N,N–dimethylformamide (Sigma, St. Louis, MO, USA). The second was prepared by the dissolution of 100 mg of isopropyl-ß-D–thiogalactopyranoside (IPTG, Sigma, St. Louis, MO, USA) in 2 mL of distilled water.
Six-mm-diameter paper discs were prepared with Whatman chromatography paper 3MM (Whatman Ltd, Maidstone, England). Equal volumes of the two stock solutions described above were mixed vigorously, and then 20 µL of the mixture were allowed to absorb onto each paper disc. The discs were dried overnight at 37°C.
Whole unstimulated saliva was collected from each subject. Saliva samples (20 µL) were immediately applied to each disc, and the results were recorded by a judge blinded to all other results after 10 minutes' incubation at room temperature as follows: 0 – no color, 1 – faint blue color, 2 – moderate- to dark-blue color.
Sulfide monitor
Determination of intra-oral headspace volatile sulfur compounds (VSC) was carried out by means of a portable sulfide monitor (model 1170, Interscan) as previously described (Rosenberg et al., 1991a,b). Subjects were asked to refrain from talking for 5 min prior to measurements. The monitor was zeroed on ambient air, and the measurements were performed by the insertion of a disposable /-inch plastic straw approximately 4 cm into the partially opened oral cavity. Subjects were asked to breathe through their noses during measurements. Results were recorded as peak ppb hydrogen sulfide equivalents.
Organoleptic measurements
Whole-mouth malodor and tongue dorsum malodor were scored by two odor judges, who were blinded to one another's scores as well as to the other data. Judge 1 (MR) and Judge 2 are both experienced odor judges, having participated in 18 and five clinical studies, respectively. For judge scoring of whole-mouth malodor, subjects were instructed to exhale briefly through the mouth, at a distance of approximately 10 cm from the nose of the judge. Tongue malodor was scored with the use of a plastic spoon to scrape and scoop material from the far posterior region of the tongue dorsum. The malodor emanating from the spoon was estimated by both judges, sequentially. Odor levels were assessed on a continuous scale of 0 (no appreciable odor) to 5 (extremely foul odor), with descriptions of intermediate scores as follows: 1, barely noticeable odor; 2, slight, but clearly noticeable odor; 3, moderate odor; and 4, strong odor. In this manner, judges had the freedom to assess odors as falling between two given intermediate scores (e.g., 2.75).
Reproducibility of the Paper Disc Assay
To test reproducibility of the paper disc assay, we carried out the following experiment. Samples of fresh whole saliva from ten volunteers were applied in triplicate to impregnated paper discs as described above. This was repeated on two separate days. Scores were determined in a random fashion by two judges blinded to the identity of the discs and each other's scores.
Statistical Analysis
We used Spearman correlation coefficients to determine the levels of association between and among the various parameters. Analysis of variance (ANOVA) was used for comparison of the color assay results in terms of the other parameters. The between-examiner reliability (internal consistency) of the malodor judges was assessed through the calculation of intraclass correlation coefficients (Winer, 1971).
We carried out stepwise multiple regression analysis to test the contributions of the color assay and the sulfide monitor in accounting for the odor judges' scores. As in previous studies, sulfide monitor readings were transformed to natural logarithms so that near-normal distribution could be obtained. The reproducibility of the paper disc assay was analyzed by ANOVA, with comparison of the variance among (i) the triplicate measurements, (ii) the results obtained on the two separate days, and (iii) the scores of the individual subjects.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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).
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The strength of association among the various parameters was assessed by Spearman correlations (Table 2). ß–galactosidase levels, as measured by means of the paper disc assay, were significantly associated with both judges' scores for whole-mouth and tongue odor (p 0.002 and p 0.001, respectively). In general, correlations between odor judges' scores and the paper disc assay were higher than the corresponding correlations between odor judges' scores and the sulfide monitor measurements (r values ranging from 0.38 to 0.48 as opposed to 0.18 to 0.46) (Table 2). A relatively strong association (r = 0.67) was observed when the whole-mouth odor judges' scores of the two judges were compared (Table 2). To test for the reliability (between-examiner reliability) in odor-judge scoring of whole-mouth malodor, we carried out a modified ANOVA test (Winer, 1971), yielding a similar intraclass correlation of r = 0.665. As shown in previous studies, odor judges' scores and sulfide monitor levels were significantly associated with one another, although the correlations were slightly lower than in some previous reports (Rosenberg et al., 1991a,b; Goldberg et al., 1994; Kozlovsky et al., 1994).
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). Both sulfide monitor readings and color assay scores were factored into the regression equation for both judges' scores for both whole-mouth and tongue odors, yielding multiple r values ranging from 0.47 to 0.60.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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0.0007).
In a separate study of 38 subjects (data not shown), statistically significant correlations were similarly found in comparisons of odor judge scores of oral malodor and ß-galactosidase activity in saliva, based on spectrophotometric measurement of enzyme activity in liquid phase. In that study, a significant correlation was also observed in comparisons of ß-galactosidase activity and volatile sulfides, whereas the correlation between these two measurements in the present study was not significant (p = 0.17, Table 2
). This discrepancy warrants further study. In the present investigation, the paper disc assay and the sulfide monitor scores appeared to constitute independent parameters, both factoring significantly in accounting for odor judge scores. One possible explanation is that the paper disc assay is primarily associated with the Gram-positive microbiota that produce ß-galactosidase but not volatile sulfides. In contrast, the sulfide monitor may be more closely associated with the Gram-negative microbiota that elaborate significant quantities of volatile sulfides, but low levels of ß-galactosidase (Persson et al., 1990; Kleinberg and Codipilly, 1995).
Although assessment of oral malodor by human judges is currently the most common method for the measurement of bad breath, it suffers from several drawbacks, including the lack of suitable standards with which to calibrate, test, train, and compare odor judges (Rosenberg and McCulloch, 1992). In the present study, reliability of odor judgment was confirmed by between-examiner reliability analysis, yielding an intraclass correlation of r = 0.665. Nevertheless, the development of simple, objective measurement tools is necessary, for both research and clinical assessment. At present, few such tests are available. Examples include the portable sulfide monitor (Rosenberg et al., 1991a,b) and the BANA test (De Boever et al., 1994; Kozlovsky et al., 1994).
To our knowledge, this is the first study showing a correlation between ß-galactosidase activity and odor judge scores of oral malodor. ß-galactosidase is one of the important enzymes responsible for the initial degradation of oral mucins, facilitating subsequent proteolysis by the removal of O- and N-linked carbohydrate side-chains (De Jong and Van Der Hoeven, 1987; Van Der Hoeven and Camp, 1991; Homer et al., 1994). Putrefaction of saliva increases when ß-galactosidase is added, and is inhibited in the presence of inhibitors of the enzyme (Sterer and Rosenberg, manuscript in preparation). The paper disc assay used here has several potential advantages: (i) Easily visible results are available in a matter of minutes; (ii) the test is non-invasive, simple to perform, and does not require valuable chairside time; (iii) no auxiliary equipment is necessary; (iv) test results, when combined with VSC measurements, provide higher correlations with odor judge scores; and (v) the assay was found to be reproducible over time, and among multiple samples. Further investigations are necessary to determine the ability of the present assay to correlate with oral-malodor-associated parameters, in different subject populations and following various interventions.
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ACKNOWLEDGMENTS |
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Received March 20, 2001; Last revision December 27, 2001; Accepted January 16, 2002
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REFERENCES |
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De Boever EH, De Uzeda M, Loesche WJ (1994). Relationship between volatile sulfur compounds, BANA-hydrolyzing bacteria and gingival health in patients with and without complaints of oral malodor. J Clin Dent 4:114–119.[Medline]
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Van Der Hoeven JS, Camp PGM (1991). Synergistic degradation of mucin by Streptococcus oralis and Streptococcus sanguis in mixed chemostat cultures. J Dent Res 70:1041–1044.
Winer BJ (1971). Statistical principles in experimental design. 2nd ed. New York, NY: McGraw-Hill, Inc., p. 290.
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