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The relationship between 48-h dental plaque accumulation in young human adults and the concentrations of hypothiocyanite, ‘free’ and ‘total’ lysozyme, lactoferrin and secretory immunoglobulin a in saliva
Archs oral Bid Vol.37,No. 1,pp.23-28,1992 Printed in Great Britain. All rights reserved
0003~9969/92 S5.00+0.00 Copyright 0 1992 Pcrgamon Rcss plc
THE RELATIONSHIP BETWEEN 48-h DENTAL PLAQUE ACCUMULATION IN YOUNG HUMAN ADULTS AND THE CONCENTRATIONS OF HYPOTHIOCYANITE, ‘FREE’ AND ‘TOTAL’ LYSOZYME, LACTOFERRIN AND SECR.ETORY IMMUNOGLOBULIN A IN SALIVA R. A. JALIL,* F. P. ASHLEY? and R. F. WILSON United Medical and Dental Schools of Guy’s and St Thomas’s Hospitals, London SE1 9RT, U.K. (Accepted 18 July 1991)
Summary-Samples of resting and stimulated whole saliva and stimulated parotid saliva were collected from 40 young adults. One week later, after 48 h on a standardized diet without oral hygiene, all available plaque was collected for dry weighing. An inverse relationship was found between the ‘free’ lysozyme concentration in stimulated parotid saliva and plaque dry weight (r = -0.46, p < 0.01). There were no other statistically significant correlation coefficients between concentrations of individual salivary constituents and plaque dry weight. However, cluster analysis of constituents in resting whole saliva revealed three groups of subjects with different salivary profiles, and in particular with different concentrations of both IgA and hypothiocyanite. Subsequent analysis revealed differences in plaque dry weight between the groups, demonstrating the potential biological significance of cluster membership based on salivary factors. Key words: saliva., hypothiocyanite,
lysozyme, lactoferrin, secretory IgA, plaque.
INTRODUCIION
these components in the maintenance of oral health. Rudney and Smith (1985) and Rudney (1989) defined groups of subjects with different salivary profiles through cluster analysis, which allows subjects to be grouped objectively by their overall similarity. Rudney (1989) suggested that respiratory tract infection was associated with raised salivary concentrations of lysozyme, lactoferrin and salivary peroxidase. He recommended further work, including studies of whole saliva, to determine the biological significance of such cluster groups. Investigation of the relationship between salivary variables and plaque accumulation is complicated by the wide variation seen between individuals in the amount of plaque present in the mouth, owing to factors such as oral hygiene habits, diet and the condition of the teeth and supporting tissues. Standardization of these factors might make it easier to demonstrate any relationship between salivary variables and plaque accumulation. Our aims now were (1) to investigate whether the concentration of any single salivary variable was related to observed differences in 48-h plaque accumulation in young adults; and (2) to identify groups of subjects sharing similar salivary profiles and determine whether there were differences between these groups with regard to plaque formation. Saliva was analysed for hypothiocyanite, ‘free’ and ‘total lysozyme, lactoferrin and secretory IgA concentrations. Flow rate was also investigated because it affects the composition of saliva and influences the total amount of these defence factors delivered into the mouth.
Saliva is known to affect the adherence, metabolism and multiplication cvf bacteria (Mandel, 1979; Tenovuo et al., 1987). However, the relationship between the accumulation of dental plaque and the concentration of salivary defence factors such as hypothiocyanite, lysozyme, lactoferrin and secretory IgA has not been investigated extensively. Simonsson et al. (1987) compared two groups of i_ndividuals with ‘slow’
tinins. No significant difference in the concentration of these antimicrobial factors was found when these extreme groups were compared. However, Raeste and Tuompo (1976) have reported an inverse relationship between lysozyme in whole saliva and plaque scores in children. Rudney and Smith (11985)have drawn attention to the synergistic and antagonistic interactions among salivary antimicrobial1 proteins in vitro. They considered it was more appropriate to look at the overall salivary profile rather than single salivary constituents in order to reflect the interactions of
*Current address: Faculty of Dentistry, University of Malaya, Malaysia. tTo whom all correspondence should be addressed.
Abbrmiation: ELBA, enzyme-linked immunosorbent assay.
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R. A. JALIL et al. MATERIALS
AND METHOD!3
The subjects were 40 dental students, 14 women and 26 men. Subjects were only included in the study if they had good general health and less than 10 per cent of selected gingival sites bled on probing. The sites examined were the mesiobuccal and distobuccal aspects of the upper teeth and the mesiolingual and distolingual aspects of the lower teeth. Saliva was collected from each subject at 12 : 00 h, immediately before lunch. They were asked to refrain from drinking, eating or smoking for at least 1 h before the collection. Three samples were collectedresting and stimulated whole saliva and stimulated parotid saliva. For resting whole saliva, subjects were instructed to refrain from all tongue and jaw movements except when transferring saliva every 30 s into an ice-chilled graduated tube. The time taken to collect 2.4 ml was recorded. Stimulated whole saliva was collected by chewing paraffin wax (Orion Diagnostica, Espoo, Finland) at a constant rate of one chew/s, with saliva being transferred into a tube every 15 s. The side on which the wax was chewed was changed every 15 s. Saliva collected during the first 2 min was discarded and the time for the collection of the next 2.4 ml was recorded. After this, parotid secretion was collected from one gland in each subject via a cup (Carlsson and Crittenden, 1910) placed over the orifice of the Stensen’s duct and connected by polyethylene tubing (TSR, Slaughter Ltd, Upminster, Essex, U.K.) to a graduated tube standing in ice. Stimulation was provided by chewing paraffin wax as before but only on the side of the mouth where the collecting device was placed. The time taken to collect approx. 2.4 ml was noted. Portions of 200 ~1 of saliva were used for estimation of hypothiocyanite, which was analysed immediately. In addition, 800 ~1 of saliva were centrifuged at 20,OOOg for 20 min at 4°C and the supernatant stored at - 17°C for later analysis of ‘free’ lysozyme. Portions of 200~1 of saliva were frozen at - 17°C for estimation of ‘total’ lysozyme, lactoferrin and secretory IgA at a later date. Hypothiocyanite was assayed by the method of Pruitt et al. (1982). The analysis is based on the oxidation of pre-reduced 5-thio-2-nitrobenzoic acid by hypothiocyanite. Lysozyme estimation was by the lysoplate method of Osserman and Lawlor (1966). The method is based on the ability of the enzyme to lyse Micrococcus fysodeikticus. In brief, samples are introduced to wells in agarose diffusion plates containing the organism, and zones of lysis are related to concentration of the enzyme in the samples. Human urine lysozyme (Kallestad Laboratories, Brill, Bucks., U.K.) was used as a standard. Estimation of ‘free’ lysozyme was made directly on the salivary supernatant without further treatment (Papadopoulos, 1983). For estimation of ‘total’ lysozyme, saliva was diluted with 0.025 M HCl. Lactoferrin concentration in saliva was analysed by ELISA. Human colostral lactoferrin (Sigma Chemical Co., Poole, Dorset, U.K.) was used as a standard (Dipaola and Mandel, 1980). Secretory IgA was also quantitated by ELISA, using a colostral IgA standard comprising predominantly 11 S dimers (Sigma Chemical Co., Poole, Dorset, U.K.). The
precise volume of each sample was measured after thawing, and the flow rate determined. After a baseline plaque removal, the subjects were instructed to abstain from all oral hygiene during the next 48 h. They were also asked not to eat fibrous foods such as apples, to have three main meals a day, and to include a sugar intake at each meal. In order to standardize between-meal sugar intake, boiled sweets were provided to be eaten mid-morning and mid-afternoon, and a bar of chocolate was provided as a late-evening snack. No other food or drink was permitted, except sugar-free beverages. At the end of the 48 h, all available plaque was collected from each subject as two samples: one from the lower anterior teeth and the other from the remaining tooth surfaces except the third molars. Plaque was removed with Younger Good 7/8 curettes (HuFreidy, Chicago, IL) and transferred to labelled polypropylene vials with caps (TAAB Laboratories, Aldermaston, Berks., U.K.). They were immediately frozen at - 17°C before estimation of dry weight, which was done after drying the samples in a desiccator for 48 h. The total dry weight for each subject was calculated from the two samples. Plaque was collected as two samples because of the requirements of a separate investigation into the calcium and phosphorus concentrations of plaque and saliva (Ashley et al., 1991). concentrations not Hypothiocyanite were estimated in stimulated whole saliva as it is essential to analyse this constituent immediately after collection and a limited number of samples could be processed at one time. Data analysis
Mean values, SDS, frequency distributions and Pearson’s correlation coefficients were determined. In partial correlation coefficients were addition, calculated to take into account variation in the data attributable to flow rate. The concentrations of all constituents except the total lysozyme in stimulated whole saliva required transformation before the calculation of correlation coefficients. Square-root transformation was used for hypothiocyanite and total lysozyme in resting whole saliva, and transformation to the natural logarithm for all other variables. Multiple regression analysis was done with plaque dry weight as the dependent variable and salivary components as independent variables. Principal-component analysis was used to identify major patterns of intercorrelation (Pimentel, 1979). The choice of variables for this analysis followed that of Rudney and Smith (1985) as far as possible, with the substitution of hypothiocyanite for peroxidase, which we did not assay. Cluster analysis was then done, subjects being grouped according to the similarity coefficient based on the principal-component analysis. Thus subjects in each cluster group have salivary profiles based on a combination of variables that are more similar to each other than the profiles of subjects in other groups. Subjects were divided into groups in a sequential, step-wise procedure. The first three cluster groups to emerge with nine or more further. Differences subjects were investigated between cluster groups were investigated by analysis of variance, followed by analysis of contrasts.
Plaque accumulation and salivary defence factors
25
Table 1. Mean concentrations of hypothiocyanite, free and total lysozyme, lactoferrin and secretory IgA in resting and stimulated whole saliva and stimulated parotid saliva ?Z=4O Hypothiocyanite
(JIM)
‘Free’ lysozyme bg/ml) ‘Total’ lysozyme @g/ml) Lactoferrin @g/ml) Secretory IgA @g/ml)
Resting whole saliva
Stimulated whole saliva
Stimulated parotid saliva
19.03 (12.24) 6.71 (2.96) 49.85 (23.47) 7.30 (5.17) 90.30 (41.56)
N.A.
23.01 (26.66)
3.26 (1.72) 37.59 (15.72) 5.72 (3.43) 43.47 (25.59)
(Z) 6.46 (4.25) 2.17 (2.98) 28.47 (16.36)
SD in parentheses. N.A. = not assayed. Table 2. Pearson’s correlation coefficients between variables in resting whole saliva, stimulated whole saliva and stimulated parotid saliva and plaque dry weight
Hylpothiocyanite ‘Free’ lysozyme ‘Total’ lysozyme Lactoferrin Secretory IgA **p
Resting whole saliva
Stimulated whole saliva
Stimulated parotid saliva
-0.20 -0.14 -0.23 0.20 0.21
N.A. -0.23 -0.06 0.04 0.15
0.02 -0.46** -0.21 0.02 0.22
R:l?SULTS The mean dry weight of plaque per subject was 6.26mg (SD = 3.45). Mean flow rates of resting whole saliva, stimulateld whole saliva and stimulated parotid saliva were 0.43, 1.47 and 0.47 ml/min, respectively. Table 1 presents the mean concentrations of hypothiocyanite, ‘free’ and ‘total’ lysozyme, lactoferrin and secretory IgA in resting and stimulated whole saliva and in stimulated parotid saliva. Table 2 presents Pearson’s correlation coefficients between the individual salivary variables and the dry weight of plaque collected at the end of the 48 h. ‘Free’ lysozyme in stimulated parotid saliva was the only variable that had a statistically significant relationship with dry ,weight (r = -0.46, p < 0.01). None of the other correlations reached levels of statistical significance. The relationship between ‘free’ lysozyme in stimulated parotid saliva and plaque dry weight remained statistically significant when partial correlation coefficients were calculated to take into consideration the effect of flow rate. Multiple regression analysis provided no evidence for further statistically significant relationships between plaque and salivary components. Pearson’s correlation coefficients between the salivary variables formed a pattern of intercorrelation that was difficult to interpret. For this reason, an alternative description of the relationships was obtained by principal-component analysis and the results of the analysis are presented in Table 3. In principal-component analysis, linear combinations of the observed variables are formed in an attempt to produce a reduced number of new mathematical components that represent the underlying character
of the multivariate data more simply and form the basis of a salivary profile for use in cluster analysis. The first principal component (PCI) is the combination of the original variables that accounts for the greatest proportion of variance in the multivariate data. The second principal component (PC2) accounts for the next largest proportion of the variance and is not correlated with the first component. The first few principal components generally account for most of the variance in the data set. Varimax rotation was used to simplify the principal-component matrices, by minimizing the number of variables that have high loadings on a single component and enhancing their interpretability. Although 40 cases were processed, only 39 were used in this analysis because one had a missing variable. The variable loadings indicate the correlation between the original variables and each principal component. The eigenvalues give an
Table 3. Principal-component analysis for salivary variables in 39 young adults Loading after varimax* rotation Variables Flow rate Hypothiocyanite Lysozyme Lactoferrin Secretory IgA Eigenvalues Total variance (%)
PC1
PC2
Communalities
-0.24 -0.44 0.80 0.80 0.80 2.19 43.80
0.84 -0.60 -0.19 -0.03 0.24 1.16 23.20
0.77 0.56 0.68 0.65 0.69
*A procedure for maximizing interpretability patterns-see text.
of loading
R. A. JAL.IL et al.
26
indication of the proportion of the total variance accounted for by the principal component, which must have an eigenvalue of 1.0 or greater to be considered statistically significant. Communalities indicate the proportion of the variance of each variable that is accounted for by the principal components. Here, two principal components accounted for a statistically significant proportion of the variance. Together they accounted for about 67% of the total variance, and for between 56 and 77% of the variance in each of the variables. The pattern of loadings provided a description of relationships among salivary variables. The high loadings of lysozyme, lactoferrin and secretory IgA on PC1 suggested that variation in lysozyme, lactoferrin and secretory IgA was largely independent of variation in salivary hypothiocyanite and flow rate. Both hypothiocyanite and flow rate loaded heavily on PC2. Cluster analysis was then used to group subjects according to their salivary profile in relation to the results of principal-components analysis. The aim was to achieve a few relatively homogeneous groups of subjects with similar salivary profiles. Subjects in each cluster group were similar to each other but distinct from subjects in the other cluster groups. Three groups of subjects emerged, having 13 subjects in group 1, 9 in group 2 and 11 in group 3. An estimate of 100% classification-function efficiency for the data indicated that the groups were totally independent, with no overlap. Six other subjects were either not classified into groups because they had a unique pattern of intercorrelation or they formed groups with extremely small numbers and had to be excluded. Table 4 shows the characteristics of the subjects in the three main cluster groups. Although clustergroup membership was based on the principal-components analysis, analysis of variance indicated that the groups also differed with regard to some of the original variables that had been used in the principalcomponent analysis. There were statistically significant differences in mean secretory IgA (p < 0.001) and hypothiocyanite (p < 0.05) concentrations. Analysis of contrasts revealed that mean hypothioTable 4. Mean concentrations (SD) for three different groups of subjects clustered by flow rate, hypothiocyanite, ‘total’ lysozyme, lactoferrin and secretory IgA levels in restinn whole saliva together with plaque dry weight &our,
Flow rate (ml/min) Hypothiocyanite OtM) ‘Free’ lvsozvme
wh
1
Group 2
13 0.49
n
-
‘Total’ lvsozvme
(0.31) 13.88 (7.78) 6.67
*
(3.33) 42.48 (18.24) 6.09 (4.01) 85.06 (13.80) 7.78 (3.14)
Olgbl) Secretory IgA @g/ml) Plaque dry weight (mg) *p < 0.05; ***p < 0.001.
*** *
Group 3
9 0.43
11 0.35
(0.12) 24.73
(0.14) 19.5 (9.87) 6.47
(3.62) 48.58
(1.66) 46.57
(12.48) (12.80) 4.49 8.13 (4.67) (3.34) 47.61 *** 132.27 (12.34) (10.24) 4.63 5.75 (3.42) (1.84)
cyanite concentrations in Groups 1 and 2 were significantly different (p < 0.05), whilst all three groups were significantly different with regard to mean secretory IgA (p < 0.001). Analysis of variance also showed statistically significant differences in mean plaque dry weight between the groups: this variable had not been used in the principal-components analysis that formed the basis for cluster-group formation. The difference between Groups 1 and 2 was statistically significant, and that between Groups 1 and 3 approached statistical significance (p = 0.06). Stimulated whole saliva was not cluster analysed as hypothiocyanite was not assayed. For stimulated parotid saliva the number of subjects who emerged in the three major groups (17, 6, 5) was not considered appropriate for further analysis. DISCUSSION
It has been generally assumed that hypothiocyanite, lysozyme, lactoferrin and secretory IgA play a part in the maintenance of oral health. Their precise role in controlling the oral flora with regard to plaque accumulation is more difficult to assess. Secretory IgA may interfere with adherence (Olson, Bleiweiss and Small, 1972) and inhibit glucosyltransferase activity (Evans and Genco, 1973) in some oral bacteria. Other components may be bactericidal or reduce metabolic activity (Mandel and Ellison, 1985) and thus affect multiplication in establishing plaque. We collected plaque under experimental conditions from young adults and care was taken to control factors that are known to modify plaque formation, in particular oral hygiene and the frequency of sugar consumed during the study. In spite of this, we found no significant associations between individual salivary factors and plaque accumulation, with the exception of ‘free’ lysozyme in stimulated parotid saliva, which was inversely related to plaque dry weight. There do not appear to be any previous reports of a similar relationship for stimulated parotid saliva. However, our finding is consistent with studies on the protective role of lysozyme in vitro. Lysozyme affects the integrity of the cell wall and the viability of some bacteria (Pollock ef al., 1976) and may also aggregate organisms (Bleiweiss et al., 1971; Pollock et al., 1976) preparing them for removal from the mouth. Although the correlations between ‘free’ lysozyme and plaque in both resting and stimulated whole saliva were negative, they did not reach levels of statistical significance. Raeste and Tuompo (1976) reported an inverse relationship between lysozyme in whole saliva and plaque scores in children, but it is not clear which fraction of lysozyme they analysed. The problem with investigating individual components of saliva is that subjects with identical levels of a given antimicrobial protein may be quite different with regard to other antimicrobial proteins, and these others may affect the component of interest in a variety of ways. Interaction between salivary proteins may be an important reason for previous difficulties in relating variation in levels of individual salivary defence factors to oral health and disease. The evidence from in vitro work suggests that interactive
Plaque accumulation and salivary defence factors tive effects may vary with concentrations of the proteins involved (Ruldney and Smith, 1985), and salivary concentrations of hypothiocyanite, lysozyme, lactoferrin and secretory IgA show considerable variation between subjects. It is likely, therefore, that patterns of interaction will also vary between individuals. F:udney, Kajander and Smith (1985) and Grahn et a!. (1988) have emphasized the need to appreciate that the saliva components probably never operate in isolation in viva. Rudney and Smith (1985) have pioneered the use of cluster analysis as a method of looking at the overall salivary profill:. Selection of variables for cluster analysis was made on the basis of their work. Three groups of subjects were identified (n = 13, 9, 11) with similar profiles in resting whole saliva. Analysis of variance revealed differences in plaque dry weight in the three groups. The group that had the lowest mean dry .weight had the highest mean concentration of hypothiocyanite and the lowest mean concentration of secretory IgA. Thus, although there was no evidence of a relationship between hypothiocyanite in saliva and plaque amount when hypothiocyanite was considered in isolation, cluster analysis offered some supporting evidence for a possible protective role, as suggested by the work of Tenovuo and Anttonen (1980) in vitro. The reactive ion oxidizes susceptible sulphydryl groups in bacterial proteins and interferes with the metabolism and respiration of many difFerent types of micro-organism (Tenovuo and Pruitt, 1984). It is tempting to ex:plain the association of a low plaque dry weight with a low secretory IgA concentration in terms of a lower antigenic challenge associated with smaller numbers of bacteria (Grahn et al., 1988). However, the situation is probably complicated as the group that had the highest mean values for secretory IgA had intermediate values for plaque dry weight. Indeed, the fact that these salivary components only showed a degree of association with plaque dry weight through cluster analysis suggests that some interaction with other components may be modifying their effect. Such interaction may be synergistic or antagonistic. The potential for collaborative antimicrobial activity as well as inhibitory effects has been suggested by studies in vitro. The generation of products in the peroxidase system may be enhanced by secretory IgA (Tenovuo et al., 1982), lactoferrin (Moldoveanu et al., 1983) and lysozyme (Arnold et al., 1984). Hypothiocyanite has been reported to potentiate the effects of lysozyme (Pollock et ul., 1979). Non-specific sIgA can bind to lactoferrin (Watanabe er al., 1984) and this may enhance iron sequestration (Spik et al., 1978). In contrast, lactoferrin activity may be blsocked by specific IgA (Cole et ul., 1976) and inhibited by peroxidase (Lassiter et al., 1987). These will require further investigation to establish whether such interaction occurs in uivo. Multivariate approaches such as cluster analysis will be important in these enquiries. We conclude that concentrations of ‘free’ lysozyme in stimulated parotid ;saliva were inversely related to the dry weight of 48-h plaque. In addition, cluster analyses showed that combinations of some of the variables might be of importance in relation to plaque formation. Secretory IgA and hypothiocyanite in
AOB 3711-B.
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resting whole saliva appeared to be the most important determinants of cluster membership. The question arises as to whether the influence of these salivary factors would still be apparent under more normal conditions without withdrawal of oral hygiene or the imposition of dietary restrictions. However, our findings show the potential biological significance of investigating the overall salivary profile by cluster analysis.
REFERENCES
Arnold R. R., Russell J. E., Devine S. M., Adamson M. and Pruitt K. M. (1984) Antimicrobial activity of the secretory innate defense factors lactoferrin, lactoperoxidase and lysozyme. In: Curiology Today (Ed. Guggenheim B.), pp. 74-88. Karger, Basel. Ashley F. P., Coward P. Y., Jalil R. A. and Wilson R. F. (1991) Relationship between calcium and inorganic phosphorus concentrations of both resting and stimulated saliva and dental plaque in children and young adults. Archs oral Biol. 36, 431434.
Bleiweiss A. S., Craig R. A., Coleman S. E. and Van de Rijn I. (1971) The streptococcal cell wall: structure, antigenic composition, and reactivity with lysozyme. J. dent. Res. 50, 1118-1129. Carlsson A. V. and Crittenden A. L. (1910) The relation of ptyalin concentration to the diet and to the rate of secretion of the saliva. Am. J. Physiol. 26, 169-177. Cole M. F., Arnold R. R., Mestecky J., Prince S., Kulhavy R. and McGhee J. R. (1976) Studies with human lactoferrin and Streptococcus mutans. In: Microbial Aspects of Dental Caries II (Eds Stiles H. M., Loesche W. J. and O’Brien T. C.), pp. 359-373. Information Retrieval, Washington, DC. Dipaola C. and Mandel I. D. (1980) Lactoferrin concentration in human parotid saliva as measured by an enzyme-linked immunosorbent assay (ELISA). J. dent. Res. 59, 1463-1465. Evans R. T. and Genco R. J. (1973) Inhibition of glucosyltransferase activity by antisera to known serotypes of Streptococcus mutans. Infect. Immun. 7, 237-241.
Grahn E., Tenovuo J., Lehtonen O.-P., Eerola E. and Vilja P. (1988) Antimicrobial systems of human whole saliva in relation to dental caries, cariogenic bacteria and gingival inflammation in young adults. Acta odon?. stand. 46, 67-74. Lassiter M. O., Newsome A. L., Sams L. D. and Arnold R. R. (1987) Characteristion of lactoferrin interaction with Streptococcus mutans. J. dent. Res. 66, 48&485.
Mandel I. D. (1979) In defence of the oral cavity. In: Saliua and Dental Caries (Eds Kleinberg I., Ellison S. A. and Mandel I.), pp. 473491. Information Retrieval, Washington, DC. Mandel I. D. and Ellison S. A. (1985) The biological significance of the nonimmunoglobulin defense factors. In: The Lactoperoxidase system (Eds Pruitt K. M. and Tenovuo J. OI), pp. I-14.-Dekkei, New York. Moldoveanu Z., Tenovuo J., Pruitt K. M., Mansson-Rahentulla B. and Mestecky J. (1983) Antibacterial properties of milk: IgA-peroxidase-lactoferrin interactions. Ann. N.Y. Acad. Sci. 409, 848-850.
Olson G. A., Bleiweiss A. S. and Small P. A. (1972) Adherence inhibition of Streptococcus mutans: an assay reflecting the possible role of antibody in dental caries prophylaxis. Infect. Immun. 5, 419427. Osserman E. F. and Lawlor D. P. (1966) Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J. exp. Med. 124, 921-952. Papadopoulos N. M. (1983) Free and total lysozyme determined in parotid saliva. Clin. Chem. 29, 1551.
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Pimentel R. A. (1979) Morphometrics: the Multivariate Analysis of Biological Data. Kendall/Hunt, Dubuque, IA. Pollock J. J., Iacano V. J., Bicker H. G., MacKay B. J., Katona L. I.. Taichman L. B. and Thomas E. (1976) The binding, aggregation and lytic properties of lysozyme. In: Microbial Asoects of Dental Caries II (Eds Stiles H. M.. Loesche W. J. and O’Brien T. d.), pp. 325-352: Information Retrieval, Washington, DC. Pollock J. J., Goodman Bicker G., Katona K. I., Cho M. I. and Iacono V. J. (1979) Lysozyme bacteriolysis. In: Saliva and Denfal Caries (Eds Kelinberg I., Ellison S. A. and Mandel I. D.), pp. 42w7. Information Retrieval, Washington, DC. Pruitt K. M., Tenovuo J., Fleming W. and Adamson M. (1982) Limiting factors for the generation of hypothiocyanite ion, an antimicrobial agent in human saliva. Caries Res. 16, 315-323.
Raeste A. M. and Tuompo H. (1976) Lysozyme activity and flow rate of mixed saliva in children, adolescents and adults. Stand. J. dent. Res. 84, 418422. Rudney J. D. (1989) Relationships between human parotid saliva lysozyme, lactoferrin, salivary peroxidase and secretory immunoglobulin A in a large sample population. Archs oral Biol. 34, 499-506. Rudney J. D. and Smith Q. T. (1985) Relationships between levels of lysozyme, lactoferrin, salivary peroxidase and secretory immunoglobulin A in stimulated parotid saliva. Infect. Immun. 49, 46W75.
Rudney J. D., Kajander K. C. and Smith Q. T. (1985)
Correlations between human salivary levels of lysozyme, lactoferrin, salivary peroxidase and secretory immunoglobulin A with different stimulatory states and over time. Archs oral Biol. 30, 765-771. Simonsson T., Ronstrom A., Rundegren J. and Birkhed D. (1987) Rate of plaque formation--some clinical and biochemical characteristics of ‘heavy’ and ‘light’ plaque fonners. Scand. J. dent. Res. 95, 97-103. Spik G., Cheron A., Montreuil J. and Birkhed D. (1978) Bacteriostasis of a milk-sensitive strain of E. coli by immunoglobulins and iron-binding proteins in association. immunology 35, 663671. Tenovuo J. and Anttonen T. (1980) Peroxidase-catalysed hypothiocyanite production in human salivary sediment in relation to oral health. Caries Res. 14, 269-275. Tenovuo J. and Pruitt J. M. (1984) Relationship of the human saliva ueroxidase system to oral health. J. oral Path. 13, 573-584.
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Tenovuo J., Moldoveanu Z., Mestecky J., Pruitt K. M. and Mansson-Rahentulla B. (1982) Interaction of specific and innate factors of immunity: IgA enhances the &timicrobial effect of the lactoperoxidase system against Strepfococcus mutans. J. Immunol. 12s. 72673 1. Tenovuo J., Grahn E., Lehtonen O.-P., Hyyppa T., Karhuvaara L. and Vilja P. (1987) Antimicrobial factors in saliva: ontogeny and relation to oral health. J. dent. Res. 66, 47-79.
Watanabe T., Nagura H., Watanabe K. and Brown W. (1984) The binding of human milk lactoferrin to immunoglobulin A. FEBS L.&t. 168, 202207.
0003~9969/92 S5.00+0.00 Copyright 0 1992 Pcrgamon Rcss plc
THE RELATIONSHIP BETWEEN 48-h DENTAL PLAQUE ACCUMULATION IN YOUNG HUMAN ADULTS AND THE CONCENTRATIONS OF HYPOTHIOCYANITE, ‘FREE’ AND ‘TOTAL’ LYSOZYME, LACTOFERRIN AND SECR.ETORY IMMUNOGLOBULIN A IN SALIVA R. A. JALIL,* F. P. ASHLEY? and R. F. WILSON United Medical and Dental Schools of Guy’s and St Thomas’s Hospitals, London SE1 9RT, U.K. (Accepted 18 July 1991)
Summary-Samples of resting and stimulated whole saliva and stimulated parotid saliva were collected from 40 young adults. One week later, after 48 h on a standardized diet without oral hygiene, all available plaque was collected for dry weighing. An inverse relationship was found between the ‘free’ lysozyme concentration in stimulated parotid saliva and plaque dry weight (r = -0.46, p < 0.01). There were no other statistically significant correlation coefficients between concentrations of individual salivary constituents and plaque dry weight. However, cluster analysis of constituents in resting whole saliva revealed three groups of subjects with different salivary profiles, and in particular with different concentrations of both IgA and hypothiocyanite. Subsequent analysis revealed differences in plaque dry weight between the groups, demonstrating the potential biological significance of cluster membership based on salivary factors. Key words: saliva., hypothiocyanite,
lysozyme, lactoferrin, secretory IgA, plaque.
INTRODUCIION
these components in the maintenance of oral health. Rudney and Smith (1985) and Rudney (1989) defined groups of subjects with different salivary profiles through cluster analysis, which allows subjects to be grouped objectively by their overall similarity. Rudney (1989) suggested that respiratory tract infection was associated with raised salivary concentrations of lysozyme, lactoferrin and salivary peroxidase. He recommended further work, including studies of whole saliva, to determine the biological significance of such cluster groups. Investigation of the relationship between salivary variables and plaque accumulation is complicated by the wide variation seen between individuals in the amount of plaque present in the mouth, owing to factors such as oral hygiene habits, diet and the condition of the teeth and supporting tissues. Standardization of these factors might make it easier to demonstrate any relationship between salivary variables and plaque accumulation. Our aims now were (1) to investigate whether the concentration of any single salivary variable was related to observed differences in 48-h plaque accumulation in young adults; and (2) to identify groups of subjects sharing similar salivary profiles and determine whether there were differences between these groups with regard to plaque formation. Saliva was analysed for hypothiocyanite, ‘free’ and ‘total lysozyme, lactoferrin and secretory IgA concentrations. Flow rate was also investigated because it affects the composition of saliva and influences the total amount of these defence factors delivered into the mouth.
Saliva is known to affect the adherence, metabolism and multiplication cvf bacteria (Mandel, 1979; Tenovuo et al., 1987). However, the relationship between the accumulation of dental plaque and the concentration of salivary defence factors such as hypothiocyanite, lysozyme, lactoferrin and secretory IgA has not been investigated extensively. Simonsson et al. (1987) compared two groups of i_ndividuals with ‘slow’
tinins. No significant difference in the concentration of these antimicrobial factors was found when these extreme groups were compared. However, Raeste and Tuompo (1976) have reported an inverse relationship between lysozyme in whole saliva and plaque scores in children. Rudney and Smith (11985)have drawn attention to the synergistic and antagonistic interactions among salivary antimicrobial1 proteins in vitro. They considered it was more appropriate to look at the overall salivary profile rather than single salivary constituents in order to reflect the interactions of
*Current address: Faculty of Dentistry, University of Malaya, Malaysia. tTo whom all correspondence should be addressed.
Abbrmiation: ELBA, enzyme-linked immunosorbent assay.
23
24
R. A. JALIL et al. MATERIALS
AND METHOD!3
The subjects were 40 dental students, 14 women and 26 men. Subjects were only included in the study if they had good general health and less than 10 per cent of selected gingival sites bled on probing. The sites examined were the mesiobuccal and distobuccal aspects of the upper teeth and the mesiolingual and distolingual aspects of the lower teeth. Saliva was collected from each subject at 12 : 00 h, immediately before lunch. They were asked to refrain from drinking, eating or smoking for at least 1 h before the collection. Three samples were collectedresting and stimulated whole saliva and stimulated parotid saliva. For resting whole saliva, subjects were instructed to refrain from all tongue and jaw movements except when transferring saliva every 30 s into an ice-chilled graduated tube. The time taken to collect 2.4 ml was recorded. Stimulated whole saliva was collected by chewing paraffin wax (Orion Diagnostica, Espoo, Finland) at a constant rate of one chew/s, with saliva being transferred into a tube every 15 s. The side on which the wax was chewed was changed every 15 s. Saliva collected during the first 2 min was discarded and the time for the collection of the next 2.4 ml was recorded. After this, parotid secretion was collected from one gland in each subject via a cup (Carlsson and Crittenden, 1910) placed over the orifice of the Stensen’s duct and connected by polyethylene tubing (TSR, Slaughter Ltd, Upminster, Essex, U.K.) to a graduated tube standing in ice. Stimulation was provided by chewing paraffin wax as before but only on the side of the mouth where the collecting device was placed. The time taken to collect approx. 2.4 ml was noted. Portions of 200 ~1 of saliva were used for estimation of hypothiocyanite, which was analysed immediately. In addition, 800 ~1 of saliva were centrifuged at 20,OOOg for 20 min at 4°C and the supernatant stored at - 17°C for later analysis of ‘free’ lysozyme. Portions of 200~1 of saliva were frozen at - 17°C for estimation of ‘total’ lysozyme, lactoferrin and secretory IgA at a later date. Hypothiocyanite was assayed by the method of Pruitt et al. (1982). The analysis is based on the oxidation of pre-reduced 5-thio-2-nitrobenzoic acid by hypothiocyanite. Lysozyme estimation was by the lysoplate method of Osserman and Lawlor (1966). The method is based on the ability of the enzyme to lyse Micrococcus fysodeikticus. In brief, samples are introduced to wells in agarose diffusion plates containing the organism, and zones of lysis are related to concentration of the enzyme in the samples. Human urine lysozyme (Kallestad Laboratories, Brill, Bucks., U.K.) was used as a standard. Estimation of ‘free’ lysozyme was made directly on the salivary supernatant without further treatment (Papadopoulos, 1983). For estimation of ‘total’ lysozyme, saliva was diluted with 0.025 M HCl. Lactoferrin concentration in saliva was analysed by ELISA. Human colostral lactoferrin (Sigma Chemical Co., Poole, Dorset, U.K.) was used as a standard (Dipaola and Mandel, 1980). Secretory IgA was also quantitated by ELISA, using a colostral IgA standard comprising predominantly 11 S dimers (Sigma Chemical Co., Poole, Dorset, U.K.). The
precise volume of each sample was measured after thawing, and the flow rate determined. After a baseline plaque removal, the subjects were instructed to abstain from all oral hygiene during the next 48 h. They were also asked not to eat fibrous foods such as apples, to have three main meals a day, and to include a sugar intake at each meal. In order to standardize between-meal sugar intake, boiled sweets were provided to be eaten mid-morning and mid-afternoon, and a bar of chocolate was provided as a late-evening snack. No other food or drink was permitted, except sugar-free beverages. At the end of the 48 h, all available plaque was collected from each subject as two samples: one from the lower anterior teeth and the other from the remaining tooth surfaces except the third molars. Plaque was removed with Younger Good 7/8 curettes (HuFreidy, Chicago, IL) and transferred to labelled polypropylene vials with caps (TAAB Laboratories, Aldermaston, Berks., U.K.). They were immediately frozen at - 17°C before estimation of dry weight, which was done after drying the samples in a desiccator for 48 h. The total dry weight for each subject was calculated from the two samples. Plaque was collected as two samples because of the requirements of a separate investigation into the calcium and phosphorus concentrations of plaque and saliva (Ashley et al., 1991). concentrations not Hypothiocyanite were estimated in stimulated whole saliva as it is essential to analyse this constituent immediately after collection and a limited number of samples could be processed at one time. Data analysis
Mean values, SDS, frequency distributions and Pearson’s correlation coefficients were determined. In partial correlation coefficients were addition, calculated to take into account variation in the data attributable to flow rate. The concentrations of all constituents except the total lysozyme in stimulated whole saliva required transformation before the calculation of correlation coefficients. Square-root transformation was used for hypothiocyanite and total lysozyme in resting whole saliva, and transformation to the natural logarithm for all other variables. Multiple regression analysis was done with plaque dry weight as the dependent variable and salivary components as independent variables. Principal-component analysis was used to identify major patterns of intercorrelation (Pimentel, 1979). The choice of variables for this analysis followed that of Rudney and Smith (1985) as far as possible, with the substitution of hypothiocyanite for peroxidase, which we did not assay. Cluster analysis was then done, subjects being grouped according to the similarity coefficient based on the principal-component analysis. Thus subjects in each cluster group have salivary profiles based on a combination of variables that are more similar to each other than the profiles of subjects in other groups. Subjects were divided into groups in a sequential, step-wise procedure. The first three cluster groups to emerge with nine or more further. Differences subjects were investigated between cluster groups were investigated by analysis of variance, followed by analysis of contrasts.
Plaque accumulation and salivary defence factors
25
Table 1. Mean concentrations of hypothiocyanite, free and total lysozyme, lactoferrin and secretory IgA in resting and stimulated whole saliva and stimulated parotid saliva ?Z=4O Hypothiocyanite
(JIM)
‘Free’ lysozyme bg/ml) ‘Total’ lysozyme @g/ml) Lactoferrin @g/ml) Secretory IgA @g/ml)
Resting whole saliva
Stimulated whole saliva
Stimulated parotid saliva
19.03 (12.24) 6.71 (2.96) 49.85 (23.47) 7.30 (5.17) 90.30 (41.56)
N.A.
23.01 (26.66)
3.26 (1.72) 37.59 (15.72) 5.72 (3.43) 43.47 (25.59)
(Z) 6.46 (4.25) 2.17 (2.98) 28.47 (16.36)
SD in parentheses. N.A. = not assayed. Table 2. Pearson’s correlation coefficients between variables in resting whole saliva, stimulated whole saliva and stimulated parotid saliva and plaque dry weight
Hylpothiocyanite ‘Free’ lysozyme ‘Total’ lysozyme Lactoferrin Secretory IgA **p
Resting whole saliva
Stimulated whole saliva
Stimulated parotid saliva
-0.20 -0.14 -0.23 0.20 0.21
N.A. -0.23 -0.06 0.04 0.15
0.02 -0.46** -0.21 0.02 0.22
R:l?SULTS The mean dry weight of plaque per subject was 6.26mg (SD = 3.45). Mean flow rates of resting whole saliva, stimulateld whole saliva and stimulated parotid saliva were 0.43, 1.47 and 0.47 ml/min, respectively. Table 1 presents the mean concentrations of hypothiocyanite, ‘free’ and ‘total’ lysozyme, lactoferrin and secretory IgA in resting and stimulated whole saliva and in stimulated parotid saliva. Table 2 presents Pearson’s correlation coefficients between the individual salivary variables and the dry weight of plaque collected at the end of the 48 h. ‘Free’ lysozyme in stimulated parotid saliva was the only variable that had a statistically significant relationship with dry ,weight (r = -0.46, p < 0.01). None of the other correlations reached levels of statistical significance. The relationship between ‘free’ lysozyme in stimulated parotid saliva and plaque dry weight remained statistically significant when partial correlation coefficients were calculated to take into consideration the effect of flow rate. Multiple regression analysis provided no evidence for further statistically significant relationships between plaque and salivary components. Pearson’s correlation coefficients between the salivary variables formed a pattern of intercorrelation that was difficult to interpret. For this reason, an alternative description of the relationships was obtained by principal-component analysis and the results of the analysis are presented in Table 3. In principal-component analysis, linear combinations of the observed variables are formed in an attempt to produce a reduced number of new mathematical components that represent the underlying character
of the multivariate data more simply and form the basis of a salivary profile for use in cluster analysis. The first principal component (PCI) is the combination of the original variables that accounts for the greatest proportion of variance in the multivariate data. The second principal component (PC2) accounts for the next largest proportion of the variance and is not correlated with the first component. The first few principal components generally account for most of the variance in the data set. Varimax rotation was used to simplify the principal-component matrices, by minimizing the number of variables that have high loadings on a single component and enhancing their interpretability. Although 40 cases were processed, only 39 were used in this analysis because one had a missing variable. The variable loadings indicate the correlation between the original variables and each principal component. The eigenvalues give an
Table 3. Principal-component analysis for salivary variables in 39 young adults Loading after varimax* rotation Variables Flow rate Hypothiocyanite Lysozyme Lactoferrin Secretory IgA Eigenvalues Total variance (%)
PC1
PC2
Communalities
-0.24 -0.44 0.80 0.80 0.80 2.19 43.80
0.84 -0.60 -0.19 -0.03 0.24 1.16 23.20
0.77 0.56 0.68 0.65 0.69
*A procedure for maximizing interpretability patterns-see text.
of loading
R. A. JAL.IL et al.
26
indication of the proportion of the total variance accounted for by the principal component, which must have an eigenvalue of 1.0 or greater to be considered statistically significant. Communalities indicate the proportion of the variance of each variable that is accounted for by the principal components. Here, two principal components accounted for a statistically significant proportion of the variance. Together they accounted for about 67% of the total variance, and for between 56 and 77% of the variance in each of the variables. The pattern of loadings provided a description of relationships among salivary variables. The high loadings of lysozyme, lactoferrin and secretory IgA on PC1 suggested that variation in lysozyme, lactoferrin and secretory IgA was largely independent of variation in salivary hypothiocyanite and flow rate. Both hypothiocyanite and flow rate loaded heavily on PC2. Cluster analysis was then used to group subjects according to their salivary profile in relation to the results of principal-components analysis. The aim was to achieve a few relatively homogeneous groups of subjects with similar salivary profiles. Subjects in each cluster group were similar to each other but distinct from subjects in the other cluster groups. Three groups of subjects emerged, having 13 subjects in group 1, 9 in group 2 and 11 in group 3. An estimate of 100% classification-function efficiency for the data indicated that the groups were totally independent, with no overlap. Six other subjects were either not classified into groups because they had a unique pattern of intercorrelation or they formed groups with extremely small numbers and had to be excluded. Table 4 shows the characteristics of the subjects in the three main cluster groups. Although clustergroup membership was based on the principal-components analysis, analysis of variance indicated that the groups also differed with regard to some of the original variables that had been used in the principalcomponent analysis. There were statistically significant differences in mean secretory IgA (p < 0.001) and hypothiocyanite (p < 0.05) concentrations. Analysis of contrasts revealed that mean hypothioTable 4. Mean concentrations (SD) for three different groups of subjects clustered by flow rate, hypothiocyanite, ‘total’ lysozyme, lactoferrin and secretory IgA levels in restinn whole saliva together with plaque dry weight &our,
Flow rate (ml/min) Hypothiocyanite OtM) ‘Free’ lvsozvme
wh
1
Group 2
13 0.49
n
-
‘Total’ lvsozvme
(0.31) 13.88 (7.78) 6.67
*
(3.33) 42.48 (18.24) 6.09 (4.01) 85.06 (13.80) 7.78 (3.14)
Olgbl) Secretory IgA @g/ml) Plaque dry weight (mg) *p < 0.05; ***p < 0.001.
*** *
Group 3
9 0.43
11 0.35
(0.12) 24.73
(0.14) 19.5 (9.87) 6.47
(3.62) 48.58
(1.66) 46.57
(12.48) (12.80) 4.49 8.13 (4.67) (3.34) 47.61 *** 132.27 (12.34) (10.24) 4.63 5.75 (3.42) (1.84)
cyanite concentrations in Groups 1 and 2 were significantly different (p < 0.05), whilst all three groups were significantly different with regard to mean secretory IgA (p < 0.001). Analysis of variance also showed statistically significant differences in mean plaque dry weight between the groups: this variable had not been used in the principal-components analysis that formed the basis for cluster-group formation. The difference between Groups 1 and 2 was statistically significant, and that between Groups 1 and 3 approached statistical significance (p = 0.06). Stimulated whole saliva was not cluster analysed as hypothiocyanite was not assayed. For stimulated parotid saliva the number of subjects who emerged in the three major groups (17, 6, 5) was not considered appropriate for further analysis. DISCUSSION
It has been generally assumed that hypothiocyanite, lysozyme, lactoferrin and secretory IgA play a part in the maintenance of oral health. Their precise role in controlling the oral flora with regard to plaque accumulation is more difficult to assess. Secretory IgA may interfere with adherence (Olson, Bleiweiss and Small, 1972) and inhibit glucosyltransferase activity (Evans and Genco, 1973) in some oral bacteria. Other components may be bactericidal or reduce metabolic activity (Mandel and Ellison, 1985) and thus affect multiplication in establishing plaque. We collected plaque under experimental conditions from young adults and care was taken to control factors that are known to modify plaque formation, in particular oral hygiene and the frequency of sugar consumed during the study. In spite of this, we found no significant associations between individual salivary factors and plaque accumulation, with the exception of ‘free’ lysozyme in stimulated parotid saliva, which was inversely related to plaque dry weight. There do not appear to be any previous reports of a similar relationship for stimulated parotid saliva. However, our finding is consistent with studies on the protective role of lysozyme in vitro. Lysozyme affects the integrity of the cell wall and the viability of some bacteria (Pollock ef al., 1976) and may also aggregate organisms (Bleiweiss et al., 1971; Pollock et al., 1976) preparing them for removal from the mouth. Although the correlations between ‘free’ lysozyme and plaque in both resting and stimulated whole saliva were negative, they did not reach levels of statistical significance. Raeste and Tuompo (1976) reported an inverse relationship between lysozyme in whole saliva and plaque scores in children, but it is not clear which fraction of lysozyme they analysed. The problem with investigating individual components of saliva is that subjects with identical levels of a given antimicrobial protein may be quite different with regard to other antimicrobial proteins, and these others may affect the component of interest in a variety of ways. Interaction between salivary proteins may be an important reason for previous difficulties in relating variation in levels of individual salivary defence factors to oral health and disease. The evidence from in vitro work suggests that interactive
Plaque accumulation and salivary defence factors tive effects may vary with concentrations of the proteins involved (Ruldney and Smith, 1985), and salivary concentrations of hypothiocyanite, lysozyme, lactoferrin and secretory IgA show considerable variation between subjects. It is likely, therefore, that patterns of interaction will also vary between individuals. F:udney, Kajander and Smith (1985) and Grahn et a!. (1988) have emphasized the need to appreciate that the saliva components probably never operate in isolation in viva. Rudney and Smith (1985) have pioneered the use of cluster analysis as a method of looking at the overall salivary profill:. Selection of variables for cluster analysis was made on the basis of their work. Three groups of subjects were identified (n = 13, 9, 11) with similar profiles in resting whole saliva. Analysis of variance revealed differences in plaque dry weight in the three groups. The group that had the lowest mean dry .weight had the highest mean concentration of hypothiocyanite and the lowest mean concentration of secretory IgA. Thus, although there was no evidence of a relationship between hypothiocyanite in saliva and plaque amount when hypothiocyanite was considered in isolation, cluster analysis offered some supporting evidence for a possible protective role, as suggested by the work of Tenovuo and Anttonen (1980) in vitro. The reactive ion oxidizes susceptible sulphydryl groups in bacterial proteins and interferes with the metabolism and respiration of many difFerent types of micro-organism (Tenovuo and Pruitt, 1984). It is tempting to ex:plain the association of a low plaque dry weight with a low secretory IgA concentration in terms of a lower antigenic challenge associated with smaller numbers of bacteria (Grahn et al., 1988). However, the situation is probably complicated as the group that had the highest mean values for secretory IgA had intermediate values for plaque dry weight. Indeed, the fact that these salivary components only showed a degree of association with plaque dry weight through cluster analysis suggests that some interaction with other components may be modifying their effect. Such interaction may be synergistic or antagonistic. The potential for collaborative antimicrobial activity as well as inhibitory effects has been suggested by studies in vitro. The generation of products in the peroxidase system may be enhanced by secretory IgA (Tenovuo et al., 1982), lactoferrin (Moldoveanu et al., 1983) and lysozyme (Arnold et al., 1984). Hypothiocyanite has been reported to potentiate the effects of lysozyme (Pollock et ul., 1979). Non-specific sIgA can bind to lactoferrin (Watanabe er al., 1984) and this may enhance iron sequestration (Spik et al., 1978). In contrast, lactoferrin activity may be blsocked by specific IgA (Cole et ul., 1976) and inhibited by peroxidase (Lassiter et al., 1987). These will require further investigation to establish whether such interaction occurs in uivo. Multivariate approaches such as cluster analysis will be important in these enquiries. We conclude that concentrations of ‘free’ lysozyme in stimulated parotid ;saliva were inversely related to the dry weight of 48-h plaque. In addition, cluster analyses showed that combinations of some of the variables might be of importance in relation to plaque formation. Secretory IgA and hypothiocyanite in
AOB 3711-B.
27
resting whole saliva appeared to be the most important determinants of cluster membership. The question arises as to whether the influence of these salivary factors would still be apparent under more normal conditions without withdrawal of oral hygiene or the imposition of dietary restrictions. However, our findings show the potential biological significance of investigating the overall salivary profile by cluster analysis.
REFERENCES
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Bleiweiss A. S., Craig R. A., Coleman S. E. and Van de Rijn I. (1971) The streptococcal cell wall: structure, antigenic composition, and reactivity with lysozyme. J. dent. Res. 50, 1118-1129. Carlsson A. V. and Crittenden A. L. (1910) The relation of ptyalin concentration to the diet and to the rate of secretion of the saliva. Am. J. Physiol. 26, 169-177. Cole M. F., Arnold R. R., Mestecky J., Prince S., Kulhavy R. and McGhee J. R. (1976) Studies with human lactoferrin and Streptococcus mutans. In: Microbial Aspects of Dental Caries II (Eds Stiles H. M., Loesche W. J. and O’Brien T. C.), pp. 359-373. Information Retrieval, Washington, DC. Dipaola C. and Mandel I. D. (1980) Lactoferrin concentration in human parotid saliva as measured by an enzyme-linked immunosorbent assay (ELISA). J. dent. Res. 59, 1463-1465. Evans R. T. and Genco R. J. (1973) Inhibition of glucosyltransferase activity by antisera to known serotypes of Streptococcus mutans. Infect. Immun. 7, 237-241.
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_
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Watanabe T., Nagura H., Watanabe K. and Brown W. (1984) The binding of human milk lactoferrin to immunoglobulin A. FEBS L.&t. 168, 202207.