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β-Galactosidase Activity in Saliva is Associated with Oral MalodorDepartment of Oral Biology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel-Aviv University, Ramat-Aviv, Israel, 69978; Correspondence: *corresponding author, melros{at}post.tau.ac.il
Deglycosylation of oral mucins may be a critical initial step leading to their subsequent proteolysis and putrefaction. The present study was undertaken to determine whether activity in saliva of a major glycosidic enzyme (β–galactosidase) is associated with oral malodor in a group of 64 subjects. Enzyme activity was detected by the use of a chromogenic substrate (X-Gal) impregnated on paper discs. Malodor-related measurements included two odor judges' assessments of whole-mouth and tongue malodor, and volatile sulfide levels measured by a portable sulfide monitor (Interscan Corp.). β–galactosidase assay scores were significantly associated with both odor judges' scores for whole-mouth (p  0.002; Spearman) and tongue malodor (p 0.001; Spearman). β–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 β–galactosidase activity in saliva is correlated with oral malodor.
Key Words: β-Galactosidase • oral malodor • diagnosis • glycoproteins • saliva
Oral malodor, a common condition, usually derives from microbial activity in the mouth itself. It is considered to be due to protein degradation, followed by subsequent breakdown of certain amino acids (e.g., methionine, cysteine, tryptophan, and lysine), yielding malodorous volatile products (e.g., methylmercaptan, hydrogen sulfide, indole, skatole, and cadaverine) (Berg and Fosdick, 1946; McNamara et al., 1972; Goldberg et al., 1994). Previous lines of evidence have suggested that Gram-negative species produce the malodor. Gram-negative, rather than Gram-positive, species tend to produce foul odors in vitro following growth on various amino acids and proteins (Persson et al., 1990; Kleinberg and Codipilly, 1995). When saliva is incubated and allowed to putrefy, Gram-negative species predominate (McNamara et al., 1972). 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. β-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). β-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 β-galactosidase activity in saliva and oral malodor parameters (i.e., odor judge scores and volatile sulfide compounds [VSC]). The results suggest that: (i) β-galactosidase activity is significantly associated with oral malodor as measured by odor judges; and (ii) both β-galactosidase activity and VSC independently account for oral malodor levels.
Subjects We tested the potential relationship between β-galactosidase and oral malodor by carrying out initial measurements among a convenience sample of 64 subjects (mean age, 23 ± 2.0 yrs, 35 females) who were participating in a comparison of anti-malodor products. Subjects were recruited by newspaper and university advertisements. Those who were smokers or took antibiotics within one month prior to the study were excluded. The human subjects protocol was reviewed and approved by the Helsinki committee for human subject research, Tel Aviv University, and informed consent was obtained from all participants.
Experimental Protocol
Measurements 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
Organoleptic measurements
Reproducibility of the Paper Disc Assay
Statistical Analysis 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.
The study sample was comprised of 64 subjects, with 4 missing data values for paper disc scores (N = 60); mean levels (± standard deviation) of the oral-malodor-related measurements are summarized (see Table 1  ).
Increasing intensity of oral malodor was accompanied by concomitant increases in the levels of the β–galactosidase color assay (Fig.). Subjects with paper disc scores of 2 (moderate-to dark-blue color) had significantly higher scores in all four odor-judge evaluations (mouth and tongue odors scored by each of the two judges) as compared with those subjects with paper disc scores of 0 (no color), with p values (ANOVA) ranging from 0.0004 to 0.019. Subjects with paper disc scores of 1 (faint blue color) had significantly higher levels of tongue odor scores from judge 1 (p = 0.0004) and whole-mouth odor scores from judge 2 (p = 0.0011) as compared with those subjects with paper disc scores of 0 (no color).
The strength of association among the various parameters was assessed by Spearman correlations (Table 2
To determine whether odor judges' scores could be explained in terms of both VSC and β-galactosidase activity, we carried out stepwise multiple regression analyses (Table 3 ). 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.
Results carried out in a separate experiment (data not shown) showed that the paper disc assay was reproducible over two separate days of measurement, as well as with respect to multiple simultaneous measurements. Variance was low when the results of the separate days were compared, as well as when triplicate scores were compared (0.067 and 0.050, respectively), in contrast to the much higher variance observed when subjects' scores were compared (2.585).
In the present study, we have tested the premise that β-galactosidase activity in saliva is related to oral malodor. Spearman regression analysis showed that enzyme activity was significantly associated with both odor judges' scores of whole-mouth and tongue dorsum malodor. Furthermore, significantly higher malodor levels were found in subjects with higher paper disc scores. Multiple regression analysis showed that both β-galactosidase activity and sulfide levels factored independently in accounting for the various odor judge scores, yielding multiple r-values ranging from 0.47 to 0.60 (p 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 β-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 β-galactosidase activity and volatile sulfides, whereas the correlation between these two measurements in the present study was not significant (p = 0.17, Table 2 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 β-galactosidase activity and odor judge scores of oral malodor. β-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 β-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.
We thank Ilana Gelernter for statistical evaluations, and Howard M. Proskin, University of Rochester, for valuable discussions. We are grateful to Yardena Mazor for excellent technical assistance. The study was supported (including consultation fees to authors) by Ramot Ltd. (Tel Aviv University Authority for Applied Research and Development) and InnoScent Ltd. Received for publication March 20, 2001. Revision received December 27, 2001. Accepted for publication January 16, 2002.
Journal of Dental Research, Vol. 81, No. 3,
182-185 (2002)
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