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Objective. To provide a comprehensive overview of published evidence on the impact of erythritol, a noncaloric polyol bulk sweetener, on oral health.Methods. A literature review was conducted regarding the potential effects of erythritol on dental plaque (biofilm), dental caries, and periodontal therapy. The efficacy of erythritol on oral health was compared with xylitol and sorbitol.Results. Erythritol effectively decreased weight of dental plaque and adherence of common streptococcal oral bacteria to tooth surfaces, inhibited growth and activity of associated bacteria likeS. mutans, decreased expression of bacterial genes involved in sucrose metabolism, reduced the overall number of dental caries, and served as a suitable matrix for subgingival air-polishing to replace traditional root scaling.Conclusions. Important differences were reported in the effect of individual polyols on oral health. The current review provides evidence demonstrating better efficacy of erythritol compared to sorbitol and xylitol to maintain and improve oral health.

The World Health Organization (WHO) classifies oral diseases in two different ways: officially dental caries (K02) and gingivitis as well periodontal diseases (K.05) are diseases in the digestive system. In epidemiological surveys the so-called DMF-index values (D= decayed or M= missing or F= filled tooth/surface) are used in determining past and present caries experience and the Community Periodontal Index (CPI) of WHO is used for the region around the teeth. Prevalence may be defined as the proportion of a population that has a disease at a specific point in time. In epidemiology, a subjectspecific approach is the only accepted practice which means that a patient has one or multiple diseases, the rest of the population being healthy. A certain “cut-off” value normally differentiates the healthy subjects from diseased ones. In oral epidemiology the index values are used to determine the “seriousness” of the oral diseases. Calibrated dentists/examiners may be educated in recording dental caries and attachment loss exactly at a high level of precision but unless these scientific recordings are “diagnoses”, the observations represent disease detections and assessments only without providing any prevalence or incidence values of oral diseases. The reason for that is hidden in the fact that the tools for oral health determinations are different from those for the diagnosis of oral diseases by WHO.

Objective: The aim of this study was to test the efficacy of long-term, daily intake of erythritol and xylitol candy, compared with sorbitol candy, on the development of enamel and dentin caries lesions. Methods: The study was a double-blind randomized controlled prospective clinical trial. Altogether 485 primary school children, first- and second-graders at baseline, from southeastern Estonia participated in this 3-year intervention. Each child consumed four erythritol, xylitol or sorbitol (control) candies three times per school day. The daily intake of polyol was about 7.5 g. The International Caries Detection and Assessment System (ICDAS) was used in the clinical examinations by four calibrated examiners at baseline and at 12, 24 and 36 months. Results: The annual examination analyses and the follow-up analyses confirmed that the number of dentin caries teeth and surfaces at 24 months follow-up and surfaces at 36 months follow-up was significantly lower in the mixed dentition in the erythritol group than in the xylitol or control group. Time of enamel/dentin caries lesions to develop and of dentin caries lesions to progress was significantly longer in the erythritol group compared to the sorbitol and xylitol groups. Also the increase in caries score was lower in the erythritol group than in the other groups. Conclusions: In the follow-up examinations, a lower number of dentin caries teeth and surfaces was found in the erythritol group than in the xylitol or control groups. Time to the development of caries lesions was longest in the erythritol group. Trial registration: ClinicalTrials.gov Identifier NCT01062633.

The objective of the present paper is to report results from oral biologic studies carried out in connection with a caries study.Samples of whole-mouth saliva and dental plaque were collected from initially 7- to 8-year-old subjects who participated in a 3-year school-based programme investigating the effect of the consumption of polyol-containing candies on caries rates. The subjects were randomized in three cohorts, consumed erythritol, xylitol, or sorbitol candies. The daily polyol consumption from the candies was approximately 7.5 g.A significant reduction in dental plaque weight from baseline (p0.05) occurred in the erythritol group during almost all intervention years while no changes were found in xylitol and sorbitol groups. Usage of polyol candies had no significant or consistent effect on the levels of plaque protein, glucose, glycerol, or calcium, determined yearly in connection with caries examinations. After three years, the plaque of erythritol-receiving subjects contained significantly (p0.05) lower levels of acetic acid and propionic acid than that of subjects receiving xylitol or sorbitol. Lactic acid levels partly followed the same pattern. The consumption of erythritol was generally associated with significantly (p0.05) lower counts of salivary and plaque mutans streptococci compared with the other groups. There was no change in salivary Lactobacillus levels.Three-year consumption of erythritol-containing candies by initially 7- to 8-year old children was associated with reduced plaque growth, lower levels of plaque acetic acid and propionic acid, and reduced oral counts of mutans streptococci compared with the consumption of xylitol or sorbitol candies.

Objectives: This demonstration programme tested topical use of xylitol as a possible oral health promoting regimen in infants at a Finnish Public Health Centre in 2002–2011. Methods: Parents (usually mothers) began once- or twice-daily administration of a 45% solution of xylitol (2.96 m) onto all available deciduous teeth of their children at the age of approximately 6–8 months. The treatment (xylitol swabbing), which continued till the age of approximately 36 months (total duration 26–28 months), was carried out using cotton swabs or a children’s toothbrush; the approximate daily xylitol usage was 13.5 mg per each deciduous tooth. Results: At the age of 7 years, caries data on the deciduous dentition of 80 children were compared with those obtained from similar, untreated children (n = 90). Xylitol swabbing resulted in a significant (P < 0.001) reduction in the incidence of enamel and dentine caries compared with the comparison subjects (relative risk 2.1 and 4.0, respectively; 95% confidence intervals 1.42–3.09 and 2.01–7.98, respectively). Similar findings were obtained when the children were 5 or 6 years old. The treatment reduced the need of tooth filling relative risk and 95% confidence intervals at 7 years: 11.86 and 6.36–22.10, respectively; P < 0.001). Compared with untreated subjects, the oral counts of mutans streptococci were reduced significantly (P < 0.001). Conclusions: Considerable improvement in dental health was accomplished in infants participating in a topical at-home xylitol administration experiment, which was offered to families in the area by the Public Health Centre as a supplement to standard oral health care. Caregiver assessment of the programme was mostly rated as high or satisfactory.

Sugar substitution with low-calorie carbohydrates constitutes a well-grounded approach to controlling energy intake to prevent obesity or certain diseases, and reducing the incidence of bacteria-associated diseases such as dental caries. This review discusses latest applications of alditol-type sugar substitutes with emphasis placed on erythritol (a tetritol) and xylitol (a pentitol). Especially xylitol has been shown to exert interesting biochemical effects (such as ammonia formation) in the dental biofilm. Understanding the oral biologic processes involved presumes referring to certain physicochemical aspects of alditols (such as complex formation with Ca(II), protein stabilization, hydroxyl radical scavenging, and others), which will be briefly discussed. This review also comments on the gastrointestinal effects associated with enteral administration of alditols, discusses the synergy between chlorhexidine and alditols, and the use of alditols in periodontal treatments (i.e. non-sweet applications of alditols), and focuses on European Union regulations on sugar substitutes. The article also attempts to rectify defective information regarding these sweeteners. A part of this information has passed unnoticed, since the data involved were published in supplements to regular volumes in the 1970s. The present review emphasizes the oral biologic significance of the xylitol-associated shift observed in the biology of the oral biofilm (dental plaque): from carbohydrate dominance to one where nitrogen metabolism plays an important role.

Manuscript. Published version available at http://dx.doi.org/10.1159/000450762 Objective: Assess the effect of daily consumption of erythritol, xylitol, and sorbitol candies on caries development in mixed dentition during a three-year intervention and three years after the intervention. Methods: 485 Estonian first and second grade primary school children participated. Children were randomly allocated to an erythritol, xylitol, or sorbitol (control) group. Polyol-containing candies were administered on school days with a daily polyol consumption of 3x2.5 g. Yearly, caries development was assessed by calibrated dentists using the ICDAS criteria. Six years after initiation of the study and three years after cessation of daily polyol consumption, 420 participants were re-examined to identify potential long-term effects of polyol consumption. Survival curves were generated at the end of the intervention period and three years post intervention. The model included age of the subjects, schools, tooth surface ages and years of surface exposure to intervention. ICDAS scoring system based events included enamel/dentine caries development, dentine caries development, increase in caries score, and dentist intervention. Results: At the end of the intervention, time to enamel/dentine caries development, dentine caries development, increase in caries score, and dentist intervention was significantly longer in the erythritol group as compared to the sorbitol group. Except for increase in caries score, all effects persisted three years after cessation of daily polyol consumption. Conclusions: A caries preventive effect of three-year erythritol consumption as compared to sorbitol was established in children with mixed dentition. The effect persisted up to three years after the end of the intervention. Trial registration: ClinicalTrials.gov Identifier NCT01062633.

Purified rat liver aminopeptidase B preparations were found to contain two molecular forms of the enzyme which can be separated by preparative electrophoresis on poly-acrylamide columns. The one with higher electrophoretic mobility represents the actual liver enzyme and the one with lower electrophoretic mobility was seen to be derived most likely from hemolyzed red blood cells constantly present in liver homo-genates. This was proved by purifying aminopeptidase B directly from isolated rat erythrocytes with the same procedure earlier used for the liver enzyme. The enzymic properties of the three enzymes (two derived from livers and one derived from erythrocytes) were seen to be largely the same. The similarity was especially considerable in the kinetics of the enzyme-catalyzed reactions, where substrate inhibition was seen to be the most characteristic feature.

Aim: To investigate the use of xylitol-containing chewing-gums in public elementary schools as a means to affect the growth of salivary and plaque mutans streptococci and salivary lactobacilli. Design: Over a period of 24 months, 750 8- to 9-year-old children chewed xylitol (X group; n = 255) or xylitol-sorbitol (XS group; n = 264) gum on school days (454 days over 2 years), or chewed no gum at all (C group; n = 231). Consumption of xylitol in the X and the XS groups was 6.6 and 5.4g per day, respectively. Use of gum took place at school in four daily episodes of which three were supervised by teachers. Following the 24-month chewing-gum period, the subjects were re-examined after 15 months (total follow-up period: 39 months) at which time an extraneous comparison group (ExC; n = 117) was also examined. The numbers of subjects examined after 39 months were: X, 239; XS, 248; C 217. Methods: Salivary and plaque levels of mutans streptococci and the salivary levels of aerobically cultured aciduric bacteria (mostly representing lactobacilli) were determined using the Orion Diagnostica Dentocult SM and LB test kits, respectively. Results: There were statistically significant differences after 24 and 39 months between the two xylitol chewing-gum groups and the C group with regard to salivary and plaque mutans streptococci and salivary lactobacilli: the use of xylitol-containing chewing-gums significantly reduced these bacterial scores. The reductions were statistically more significant in the X group than in the XS group. The bacterial scores of groups C and ExC were similar. Conclusions: Long-term use of xylitol-containing chewing-gum can reduce the growth of mutans streptococci in saliva and dental plaque, and lactobacilli-type bacteria in saliva, even if xylitol is used only on school days. The results also suggest that xylitol gum use can have a long-term, delayed growth-retarding effect on these micro-organisms, since reduced bacterial growth was still observed 15 months following the termination of xylitol use. The results indicate a close biochemical relationship between xylitol and mutans streptococci, and suggest that a similar relationship may exist regarding aerobically cultured aciduric bacteria present in saliva.

The World Health Organization (WHO) determines dental caries in two different ways (1). The official determination has been specified by the International Statistical Classification of Diseases and Other Health Related Problems (ICD) (2) and the other one is an epidemiological determination presented by Oral Health Surveys (3). Another problem arises from the fact that dental caries affects both permanent and primary teeth and occurs simultaneously during the period of mixed dentition before and during elementary school years. Indeed dental caries is the same disease in both primary and permanent teeth. Nonetheless, the scientific attitude that caries is primarily an infectious disease and that the host has only a minor role prevails in analyzing the carious process. The terms involved in dental caries include caries activity when referring to the presence and virulence of cariogenic infection and caries susceptibility when speaking about the host resistance covering primary and permanent teeth as well as saliva. Caries occurs in subjects who have an imbalance between favorable caries-causing activity and unfavorable susceptibility resulting in a diseased subject. This means that caries normally affect younger patients and in the elderly they occur in the form of root caries on the tooth’s cementum. A similar imbalance also exists in osteoporotic-penic subjects where the diagnostic outcome and disease consists of bone fractures. Thus, carious lesions in the dentition of carious subjects can be treated as either a diagnostic outcome or a symptom.

1 The extracellular zinc-aminopeptidase of Aeromonas proteolyticarevealed, upon nitration with tetranitro-methane, one highly reactive tyrosyl residue. Nitration was accompanied by a loss of approximately 80% of the original activity. When the residual activity was taken into account, the true inactivation constant of nitration was 0.0265 min−1 at pH8.0. The rate of inactivation was less at lower pH values and competitive inhibitors of the aminopeptidase provided partial protection against nitration. Loss of activity (80%) paralleled nitration of one tyrosine. 2 Treatment of the enzyme with diazosulphanilic acid at pH 9.0 resulted in an 80% reduction in activity and in the azotization of one tyrosyl residue. Both tetranitromethane and diazosulphanilic acid apparently reacted with the same essential and reactive tyrosyl residue; mononitroaminopeptidase was not azotized or further inactivated by diazosulphanilic acid. In contrast, monoazo-aminopeptidase upon prolonged treatment with tetranitromethane, was completely inactivated and three additional tyrosines were nitrated. 3 Diethylpyrocarbonate incubated with the enzyme at pH 6.0 modified two histidyl residues with no loss of enzyme activity. A zinc-depleted enzyme, resulting from dialysis against 1, 10-o-phenanthroline was treated with diethylpyrocarbonate resulting in modification of one additional histidyl residue and one tyrosyl residue as compared with the native enzyme. Treatment of a zinc-depleted enzyme with diazosulphanilic acid resulted in one additional modified tyrosyl residue, suggesting the involvement of these residues in zinc-ligand formation. 4 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, at pH 5.3, caused up to 80%, inactivation of the aminopeptidase. When the residual activity was considered, the true inactivation constant, k, was found to be 0.096 min-1. Inactivation proceeded much more readily at lower pH values and was sharply reduced at pH 7.0. Hydroxylamine treatment of the partially inactivated preparation did not reverse the inactivation. Substrates and competitive inhibitors partially protected the aminopeptidase from inactivation by the carbodiimide. The results strongly indicate involvement of a carboxylic acid residue in the mechanism of Aeromonasaminopeptidase, a result substantiated by experiments using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Treatment of the enzyme with different concentrations of this reagent showed that one carboxyl group in the active site reacts with one molecule of the reagent. 5 These results show that the active site of A. proteolytica aminopeptidase contains at least one tyrosyl and one carboxylic acid residue as does the active site of another zinc metalloprotease, carboxypeptidase A.

The effect of chewable saliva-stimulants on Streptococcus mutans levels in dental plaque and paraffin-stimulated whole saliva among participants who were mentally disabled was investigated. Over 64-days, 98 participants chewed one of four saliva-stimulating tablets five times/day. The tablets contained one of the following: xylitol (X) or sorbitol (S), or 1:1 mixtures of xylitol and erythritol (XE) or sorbitol and erythritol (SE). Consumption of xylitol and sorbitol in Groups X and S was 5.4 grams/day/ subject, and of each polyol in Groups XE and SE, consumption was 2.7 g/day/subject. Interproximal dental plaque and stimulated whole saliva were sampled at baseline, at Day 36, and Day 64. There was a statistically significant reduction of S. mutans in plaque and saliva counts in Groups X and XE. The percentage of S. mutans in total streptococci increased significantly in dental plaque in Group S but decreased in the other groups. The results suggest that xylitol-containing saliva stimulants may be more effective than sorbitol-containing products in controlling some caries-associated parameters in people who are mentally disabled. Also a relationship may exist between the pentitol-type xylitol and S. mutans, and erythritol may exert a specific biochemical effect on this organism, although further studies are needed.

The effect of 2–month usage of saliva–stimulating pastils containing either erythritol or xylitol was studied in a cohort of 30 subjects assigned to the respective polyol groups (n = 15). The daily consumption level of both polyols was 5.2 g, used in 5 daily chewing episodes. The mean weight of total plaque mass (collectable during a standard period of 3 min from all available tooth surfaces) was reduced significantly in the xylitol–group, while no such effect was observed in the erythritol–group. This reduction in plaque mass was accompanied by a significant reduction in the turbidity readings (A660) of aqueous plaque suspensions; no such effect was observed in the erythritol–group. However, plaque protein levels did not differ between baseline and endpoint in either polyol group. The plaque and salivary levels of Streptococcus mutans and plaque levels of total streptococci were reduced significantly in the xylitol–group, while no such effect was detected in the erythritol–group. However, either polyol regimen had no effect on plaque levels of S. sobrinus. The results suggest that systematic use of xylitol–containing saliva stimulants may be more effective in controlling some oral–hygiene–related and caries–associated parameters than similar use of erythritol–containing products. The results also speak for a special relationship between xylitol and S. mutans. However, owing to the great potential of erythritol as a caries–reducing agent – based on the tetritol nature of erythritol – the present laboratory results should be considered preliminary and subject to verifying clinical studies.

Theresults ofthe Turku studies were soonrecognized, firstin the then-SovietUnion, wherexylitol hadbeenroutinely usedin the diets ofdiabetic subjects. Atwo-year study conducted inthe state ofKazakhstan demonstrated a >70%reduction in theoverall caries incidence in the xylitol group compared with thesucrose group. The next trials were those conducted under theauspices ofthe WorldHealth Organization. TwoWHOstudieswere successfully completed: one in French Polynesia (1981-84), andanotherin Hungary, intheearly 1980s. Theformertrialinvolved a 32-month

Habitual xylitol gum-chewing may have a long-term preventive effect by reducing the caries risk for several years after the habitual chewing has ended. The goal of this report was (1) to determine if sorbitol and sorbitol/xylitol mixtures provide a long-term benefit, and (2) to determine which teeth benefit most from two-year habitual gum-chewing-those erupting before, during, or after habitual gum-chewing. Children, on average 6 years old, chewed gums sweetened with xylitol, sorbitol, or xylitol/sorbitol mixtures. There was a "no-gum" control group. Five years after the two-year program of habitual gum-chewing ended, 288 children were re-examined. Compared with the no-gum group, sorbitol gums had no significant long-term effect (relative risk [RR], 0.65; 95% confidence interval [c.i.], 0.39 to 1.07; p < 0.18). Xylitol gum and, to a lesser extent, xylitol/sorbitol gum had a long-term preventive effect. During the 5 years after habitual gum-chewing ended, xylitol gums reduced the caries risk 59% (RR, 0.41; 95% c.i., 0.23 to 0.75; p < 0.0034). Xylitol-sorbitol gums reduced the caries risk 44% (RR, 0.56; 95% c.i., 0.36 to 0.89; p < 0.02). The long-term caries risk reduction associated with xylitol strongly depended on when teeth erupted (p < 0.02). Teeth that erupted after 1 year of gum-chewing or after the two-year habitual gum use ended had long-term caries risk reductions of 93% (p < 0.0054) and 88% (p < 0.0004), respectively. Teeth that erupted before the gum-chewing started had no significant long-term prevention (p < 0.30). We concluded that for long-term caries-preventive effects to be maximized, habitual xylitol gum-chewing should be started at least one year before permanent teeth erupt.

Several studies indicate that xylitol (X) consumption is associated with certain biochemical changes in dental plaque and whole saliva. In making X-containing saliva stimulants more cost-effective and palatable, manufacturers may use maltitol syrup (MS, which normally contains some sorbitol and higher polyols) or polydextrose (PD, a polysaccharide molecule with a mass22 kDa) as bulking agents. Combinations of X with MS and PD have not been tested regarding their salivary effects. One hundred and eighty-eight young subjects (mean age, 22 years) of both sexes were divided into three groups of equal size for a 4-month study. The subjects in one group used X-MS dragees (in 7 daily episodes; 8 g X per day), while the subjects in another group used X-PD dragées in as many daily episodes (8 g X per day). Subjects in the third (comparison) group did not receive saliva stimulants. Paraffin-stimulated whole saliva samples were collected at baseline, after 2 months, and at endpoint. The usage of X-MS was associated with a significant (P0.05) reduction in the salivary sucrase activity. After 4 months, the activity of enzymes hydrolyzing N(alpha)-benzoyl-DL-arginyl-p-nitroaniline was significantly reduced in all groups, while the levels of free sialic acid were reduced in group X-PD only (P0.05). These salivary changes most likely reflected microbial shifts in the oral cavity and suggest that information from saliva studies may be of avail when deciding which bulking agents should be used in xylitol-based saliva stimulants.

An exploratory study investigated the root caries incidence in Department of Veterans Affairs patients with exposed root surfaces. For a period of six to 30 months, the subjects were systematically assigned to groups which used chewable dragees or chewing gums that contained either xylitol or sorbitol as bulk sweeteners. The mean treatment time was 1.8 years (standard deviation = 0.8). The consumption levels of both polyols was up to 8.5 g daily, used typically in five episodes during a 16-hour period. The subjects were examined every six months in connection with their standard scheduled visits at the Veterans Affairs Medical Center. The risk for a root-surface lesion in the xylitol group was only 19% of that for a surface in the sorbitol group (relative risk, 0.19; 95% confidence interval, 0.06-0.62; p < or = 0.0065). Simultaneous study in periodontal patients showed that both polyols significantly reduced the gingival index scores, and slightly (but not significantly) reduced the plaque index scores. Collectively, both studies suggest that frequent daily consumption of chewable, saliva-stimulating products containing essentially nonfermentable or slowly fermentable dietary carbohydrate sweeteners (xylitol and sorbitol) may have an oral-health-improving effect in Department of Veterans Affairs Medical Center patients. It is necessary to evaluate if these procedures would be efficacious in larger and expanded patient cohorts.

HPLC on a reversed phase column, amino acid sequencing and mass spectrometry were used to determine the structure of two human gingival crevicular exudate oligopeptides (Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-Val-Thr-Ala-Leu and Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) which were shown to have been derived from the beta-chain of hemoglobin. These sequences may simply represent two degradation products of the beta-chain. However, their preservation in an exudate characterized by active peptidolysis may also prompt the question about their possible more specific role.

The ability of washed whole cells of Treponema denticola ATCC 35405 to hydrolyze (inactivate) substance P, bradykinin, and angiotensin I was studied. Substance P was attacked primarily at the Phe-8-Gly-9 bond by a chymotrypsin-like proteinase (CTLP), at Pro-4-Gln-5 by an endo-acting prolyl oligopeptidase (POPase), and at Gln-5-Gln-6 by an endopeptidase (FALGPA-peptidase). Bradykinin was cleaved at Phe-5-Ser-6 by the FALGPA-peptidase and at Pro-7-Phe-8 by the POPase. Angiotensin I was rapidly converted to angiotensin II by the CTLP, and both angiotensin I and angiotensin II were further hydrolyzed at Pro-7-Phe-8 by the POPase. All these enzymes were assumed to be cell associated and were easily extracted with a mild (0.05 to 0.1%) Triton X-100 treatment. Because it was conceivable that the hydrolysis of substance P at the Phe-8-Gly-9 bond was catalyzed by a CTLP described earlier (V.-J. Uitto, D. Grenier, E. C. S. Chan, and B. C. McBride, Infect. Immun. 56:2717-2722, 1988), the enzyme was purified to homogeneity by means of conventional fast protein liquid chromatography procedures. For kinetic studies, Phe-8(4-nitro)-substance P (NSP) (absorption maximum at 309.2 nm, epsilon = 545 M-1 cm-1) was synthesized to replace substance P as a substrate in kinetic studies. In reversed-phase chromatography, both NSP and substance P gave identical results with both whole cells and the purified enzyme. The CTLP has a mass of 95 kDa, and its activity is suggested to be based on an active seryl residue, on an active imidazole group, and on an active carboxyl group but not on metal cations. The enzyme hydrolyzes N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (SAAPFNA, a typical chymotrypsin substrate) at a high rate and several proteins, such as calf thymus histone, human plasma fibrinogen, milk caseins, and gelatin. Among the substrates tested, substance P showed the highest affinity (Km = 0.22 mM) for the purified enzyme. Depending on conditions, clinically applicable chlorhexidine levels (3.2 mmol/liter, or 0.2%) strongly activated (up to fourfold) the hydrolysis of SAAPFNA by whole cells and the purified CTLP. The hydrolysis of NSP by whole cells and purified CTLP was slightly inhibited by chlorhexidine. The results demonstrated the versatility and the effectiveness of the outer membrane of T. denticola in occasioning a rapid breakdown and inactivation of human bioactive peptides and other peptidolytic catalyses.(ABSTRACT TRUNCATED AT 400 WORDS)

An endo-acting oligopeptidase (OPase) was purified to homogeneity from the cells of Treponema denticola ATCC 35405--a human oral spirochete--by a procedure that comprised a mild Triton X-100 extraction (which disintegrates the outer membrane but leaves the cells morphologically intact) and four successive fast protein liquid chromatographic steps of the extract. The activity of this oligopeptidase (formerly named "trypsin-like" enzyme and "BANA-peptidase") together with the proteinase activities of T. denticola and Porphyromonas gingivalis is utilized in a diagnostic test for human periodontal infections, but the enzyme's chemical nature has not been studied. The enzyme is a cell-associated 78-kDa protein with an isoelectric point of 6.1, and its estimated minimum peptide length was 688 amino acid residues. The OPase does not hydrolyze proteins, but hydrolyzes -X-Arg-p-nitroaniline peptides between arginine and the chromogen, the optimum pH of hydrolysis covering a broad pH range (7 to 9). The OPase is not a metalloenzyme, although 1.0 mmol/liter Ca(II) increases the rate of the hydrolysis of all substrates. Ca(II) did not affect the values of the Michaelis constant. The OPase activity is not dependent on reactive SH-groups, but is suggested to depend on the catalytic triad COOH. . .His. . .Ser. The N-terminal sequence for the first 29 amino acid residues is MKQSDFEKPPIAEIKETRFEKFGKTRIDN. The purified enzyme is very sensitive to chlorhexidine acetate (mixed inhibition; Ki = 0.85 microM) and somewhat less sensitive to bacitracin (Ki(app) = 27.5 microM). The present OPase is considered to belong to the serine peptidases, functionally resembling trypsin except that the OPase does not hydrolyze proteins. The OPase may be regarded as an oligopeptidase, the substrate specificity profile of which resembles to a certain extent that of some members of the coagulation cascade.

Dental caries explorers may become contaminated during routine caries examinations with pathogenic organisms and thereby potentially transmit infections from one tooth to another within a patient. The purpose of this study was to test the hypothesis that the contamination status of explorers influenced the caries risk of second molars. Two explorer contamination statuses were defined: (1) contamination status 1 – explorers which had probed a carious molar just prior to examining the second molar versus explorers which had probed a sound molar prior to examining the second molar, and (2) contamination status 2 – sterile explorers versus explorers which had probed several teeth. Caries examinations were performed by 4 dentists on a cohort of 4th grade students in Belize City. The examination dates and sample sizes (n) were: September-October 1989 (n = 1,277), January 1991 (n = 1,111), and January 1992 (n = 961), and January-February 1993 (n = 861). Within this cohort, there were 221 subjects who (1) had at least one pit and fissure carious onset on a caries-free second molar, (2) had no evidence of dental treatments, and (3) were examined by the same examiner during the entire study. After adjusting for confounding variables, the examination of a second molar with a dental caries explorer in either contamination status 1 or 2 had no substantial effect on the caries risk (rate ratio 0.95, 95% confidence interval: 0.77–1.18, and rate ratio 1.18,95% confidence interval: 0.89–1.56, respectively). If a true rate ratio of 1.7 or greater was associated with the contamination status 1 and 2, these analyses had more than 99 and 80% probability of detecting it, respectively. Conclusions: Examining a sound second molar with a contaminated dental explorer either does not affect the caries risk, or results in such a small increase in caries risk that it can only be reliably identified in studies where the exposure of sound teeth to contaminated dental explorers is randomized.

This study aims to assess the caries experience among first- and second-grade children in the elementary schools of southeastern Estonia.A representative sample of 485 children was studied. The mean age of children in the first grade was 7.8 years (SD = 0.35) and in the second grade 8.8 years (0.38). The clinical examinations using ICDAS criteria were completed by four calibrated examiners. The inter- and intra-examiner consistency of the examiners was high (surface and tooth-based kappa0.9).The mean caries experience of dentinal caries lesions was 0.8 (D 4-6 MFT) and 1.6 (D 4-6 MFS) among the first graders and 1.1 (D 4-6 MFT) and 1.6 (D 4-6 MFS) among the second graders. The mean caries enamel lesions among the first graders was 1.6 (D1-3T) and 2.2 (D1-3S) and among the second graders 2.1 and 3.0, respectively. The most affected surfaces were the occlusal surfaces of the lower first molars. The prevalence of sealants was very low-only 2.4% of the first molars were sealed among the first graders and 3.9% among the second graders. There were no statistically significant differences in caries experience or in the provision of restorative treatment between the schools.Caries experience is high in southeastern Estonia. Preventive programs are urgently needed.

The use of polyol (sugar alcohol) sweeteners in oral biologic research has predominantly stemmed from their anticipated non-cariogenicity in human nutrition and their inclusion in certain oral hygiene products (such as dentifrices) as humectants. The wide-spread use of polyols in food manufacturing, and in pharmacologic and cosmetic products, presumes that the oral effects of polyols must be well researched. The metabolism and safety of most of the common dietary polyols is indeed well known. Occasional reports on polyols applied in biomaterials research have also been published. Most alditol-based information published in the field of dentistry has generated from caries-associated research. Most studies have unfortunately dealt with xylitol and D-glucitol (sorbitol) only, leaving researchers unaware of the potential of other alditols. From the dental and nutritional point of view, however, naturally only alditols with sufficient sweetness have raised interest, since they have been originally used as sweeteners, i.e. to replace sugar in confectioneries and other food items. Several medical applications, however, rely on the osmotic effects of alditols, such as Dglucitol and D-mannitol. What makes polyols chemically such interesting research objects in biomedicine? The chemical and physiologic rationales behind the interest in polyols generate from their ubiquitous presence in nature, their promising applications in various fields of medicine and technology, and their well-known molecular structure and metabolism. The latter aspects have been reviewed in numerous articles and can be summarized as follows: i. The common dietary polyols, such as erythritol, xylitol, D-glucitol, D-mannitol, and related alditols (the term used in the subsequent text to include the above and similar molecules) are normal constituents of virtually all living tissues in the plant and animal kingdoms, although significant differences exist between species, and, regarding plants, also between seasons. Since simple straight-chain polyols can be regarded as reduced forms of corresponding aldoses and ketoses, it is possible that the corresponding alditol molecules preceded aldoses and ketoses in the chemical evolution in the early oxygenpoor environment of the Earth. Synthetic dietary disaccharide polyols include maltitol and lactitol. Hydrogenated glucose or maltose syrups may contain higher polyols consisting of more than two monosaccharide units. Industrial-scale polyol syrups often derive their sweetness from D-glucitol and maltitol, with traces of reducing sugars also contributing to sweetness. ii. The above alditol molecules are characterized by the absence of a reducing carbonyl group, by the chemical reducing power they can exert in biologic environments (owing to the “extra” hydrogen atoms present in the alditol molecules), by their complexation

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.

The analysis of caries incidence in clinical trials has several challenging features: (1) The distribution of the number of caries onsets per patient is skewed, with the majority of patients having few or no cavities; (2) the number of surfaces at risk varies (i) over time and (ii) between patients, due to eruption and exfoliation patterns, dental diseases, and treatments ; (3) surfaces within a patient differ in their caries susceptibility, and (4) caries onsets within a patient are correlated due to shared host factors. Recent statistical developments in the area of correlated data analyses permit incorporation of some of these characteristics into the analyses. With Poisson regression models, the expected number of caries onsets can be related to the number of surfaces at risk, the time they have been at risk, and surface- and subject-specific explanatory variables. The parameter estimated in these models is an epidemiological measure of disease occurrence: the disease incidence rate (caries rate) or the rate of change from healthy (sound) to diseased (carious). Differences and ratios of these rates provide standard epidemiological measures of excess risk. To illustrate, Poisson regression models were used for exploratory analyses of the Ylivieska xylitol study.

Caries has traditionally been assessed with WHO criteria including only obvious caries lesions. ICDAS has been developed to detect also the enamel caries lesions. This study aims to study caries and the associations of the number of caries lesions between the permanent and primary molars with ICDAS in the mixed dentition of the first and second grade primary school children. The clinical examinations of 485 children were conducted by four examiners with high reproducibility (inter- and intraexaminer kappas >0.9). The mean number of caries lesions—especially dentine caries—seemed to be higher in the second primary molars than in the first permanent molars. There were significant correlations between the number of lesions on occlusal and lingual surfaces between the primary and permanent molars. Enamel caries lesions, restorations, and caries experience did not increase according to age. Therefore, caries might be increasing in this population. As a conclusion, ICDAS recording seems to give appropriate information from the occurrence of caries lesions and its correlations between the primary and permanent teeth and surfaces.

The cells of a human oral spirochete, Treponema denticola ATCC 35405, and of seven clinical isolates of this organism obtained from the subgingival dental plaque of periodontitis patients were studied for their ability to grow in an aerobic and an anaerobic environment, and for their profile of peptidohydrolase and oxidoreductase enzymes. The growth yield of aerobically grown cultures was either comparable to or higher than that of anaerobically grown ones regardless of whether prereduced broth, freshly prepared broth or oxidized broth was used. However, elimination of certain supplements from the growth media resulted in poor growth regardless of the nature of the gaseous environment. The microscopic morphology and motility of the cells were not affected by differences in the gaseous atmosphere. Quantitative studies on several peptidohydrolase activities suggest that anaerobically grown cells displayed higher specific activity especially toward N alpha-L-prolyl-2-naphthylamine, indicating that increased synthesis of proline iminopeptidase enzymes (or enzyme) of the cells was associated with anaerobic growth conditions. The formation of enzymes hydrolysing N alpha-benzoyl-DL-arginyl-2-naphthylamine (and the corresponding p-nitroaniline) was not affected to the same extent. Growth experiments suggest that T. denticola ATCC 35405 is a facultatively anaerobic spirochete instead of an obligate anaerobe as reported in previous literature. The quantitative enzyme studies suggest that the gaseous growth atmosphere of the cells can exert a selective effect on the activity levels of certain peptidolytic enzymes of this organism. Such effects were not observed when the whole cells were studied by means of qualitative or semi-quantitative enzyme tests. The activities of catalase, peroxidase and superoxide dismutase of the cells were low and variable. Because of this, it was not possible to relate these oxidoreductase activities to the composition of the gaseous atmosphere.

About 65% of the original 258 children who participated in 1982-1984 in a caries prevention program involving the use of xylitol chewing gum were retrieved in 1989 for a follow-up study. Ninety-five subjects from the original xylitol (X) group and 70 subjects from the original control (no-gum, C) group were available. In 1984, when the children completed the program at the age of 13-14 years, the caries scores were significantly lower in children who had used xylitol gums daily, compared with the C group. In 1989, 5 years after the discontinuation of the gum program, the difference between the X and C groups had continued to increase in favor of the X group. These effects were explained by assuming that the X gum program had facilitated the establishment of a low-virulent bacterial flora on the surfaces of the teeth, and especially on those teeth that erupted during the trial proper. This type of results are possibly helpful when evaluating cost-benefit ratios of caries prevention.

Significant improvement in the dental caries status of children in the United States has been seen in recent decades. The percentage of children who are free from dental caries, however, rapidly decreases with age. By the time young people enter colleges or universities, only a few are caries free. Colleges and universities are places where the young are simultaneously, and perhaps for the last time, reachable as large cohorts before their lives undergo further "individualization." This makes college age a time that is crucial for channeling information about proper oral health practices by organizing mass-directed strategies for prevention of dental caries. It is now clear that dental caries can effectively be prevented. This calls for placing strong emphasis on dietary behavior and on the use of sugar substitutes as part of the strategy. Scientific evidence also suggests that the addition of small daily quantities of xylitol, a natural carbohydrate sweetener, to the diet of children and young adults causes significant reduction in the incidence of dental caries. On the basis of detailed long-term clinical trials carried out during the past 20 years, it appears that a reduction ranging from 30% to more than 85% in dental caries can be achieved simply by using a few xylitol chewing gums daily over a period of 1 to 3 years. In individual cases, virtually total, long-term protection against caries has been observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction: Dental caries is a diet-associated disease which continues to be a serious health problem in most industrialized and developing countries. Strategies to maximize caries prevention should automatically consider the use of sugar substitutes. It is important that public health authorities are made cognizant of the availability of new polyol-type sugar substitutes. Review Summary: Clinical studies have shown that xylitol, a natural, physiologic sugar alcohol of the pentitol type, can be used as a safe and effective caries-limiting sweetener. Habitual use of xylitol-containing food and oral hygiene adjuvants has been shown to reduce the growth of dental plaque, to interfere with the growth of caries-associated bacteria, to decrease the incidence of dental caries, and to be associated with remineralization of caries lesions. Numerous public regulatory bodies have endorsed the use of xylitol as a caries-limiting agent. Other sugar alcohols that have been successfully used as sugar substitutes include D-glucitol (sorbitol), which, however, owing to its hexitol nature, normally has no strong effect on the mass and adhesiveness of bacterial plaque and on the growth of mutans streptococci. A tetritol-type alditol, erythritol, has shown potential as a non-cariogenic sugar substitute. Combinations of xylitol and erythritol may reduce the incidence of caries more effectively than either alditol alone. Conclusions: Partial sugar substitution with polyols is an important dietary tool in the prevention of dental caries that should be used to enhance existing fluoride-based caries prevention programmes. The most effective method of conveying this information to the public is through a proper health claim for these alditols in food labelling. The present review summarizes clinical and biochemical aspects of the above three dietary polyols and emphasizes the role of sugar substitution as a potential health-promoting strategy.

An endopeptidase was purified to homogeneity from the cell extracts of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised dialysis, anion exchange fast protein liquid chromatography (FPLC), hydroxylapatite FPLC, immobilized metal affinity FPLC, FPLC chromatofocusing, and two consecutive gel permeation FPLC steps. The enzyme is a 62-kDa protein with an isoelectric point of 6.5-7.0. Experiments with enzyme inhibitors suggest that this enzyme is a metallopeptidase and that its activity is not dependent on sulfhydryl or serine residues. The enzyme is active on furylacryloyl-Leu-Gly-Pro-Ala (FALGPA; pH optimum near 6.25), bradykinin (Bk), and several Bk-related peptides. In FALGPA, the cleavage site is the Leu-Gly bond. An imino acid is absolutely necessary in position P'2. The shortest hydrolyzed peptide was FALGPA, the hydrolysis of which is strongly and competitively inhibited by Bk (Ki = 5.0 microM). The pyrophosphate ion and phosphoramidon also inhibited the hydrolysis of FALGPA. The enzyme does not hydrolyze all typical synthetic collagenase substrates, Azocoll, Azocasein, or Type I and Type IV collagens, or any other proteins tested. In Bk-related peptides, the hydrolyzed bond was Phe5-Ser6. Since a Bk antagonist and a Bk-potentiating pentapeptide also were good substrates, it is possible that the enzyme hydrolyzes Bks and related peptides only because of the coincidental, specific amino acid sequence of those substrates. A proposal is made that since a substantial portion of the amino acid sequence of FALGPA is present in collagen (and additionally acknowledging that the furylacryloyl residue structurally resembles that of proline), the natural substrates of this enzyme may be small, soluble collagen fragments produced by other enzymes from periodontal connective tissue, and that such peptides are important for the nutrition and pathogenicity of T. denticola.

Remineralization of minor enamel defects is a normal physiological process that is well known to clinicians and researchers in dentistry and oral biology. This process can be facilitated by various dietary and oral hygiene procedures and may also concern dentin caries lesions. Dental caries is reversible if detected and treated sufficiently early. Habitual use of xylitol, a sugar alcohol of the pentitol type, can be associated with significant reduction in caries incidence and with tooth remineralization. Other dietary polyols that can remarkably lower the incidence of caries include erythritol which is a tetritol-type alditol. Based on known molecular parameters of simple dietary alditols, it is conceivable to predict that their efficacy in caries prevention will follow the homologous series, that is, that the number of OH-groups present in the alditol molecule will determine the efficacy as follows: erythritol >/= xylitol > sorbitol. The possible difference between erythritol and xylitol must be confirmed in future clinical trials.

Clinical trials and laboratory studies involving the administration of oral health treatments and foods have benefited from the observance of the so-called crossover study design. Field experience and a growing number of laboratory experiments have shown, however, that ‘blind’ reliance on crossover designs may in some instances lead to unexpected results and erroneous conclusions. Some dietary substances, antibiotic agents, and even fluoride applications may have long-term effects that call into question the appropriateness of the washout periods between treatments. Studies have also been conducted on compounds that have turned out to display synergistic effects. When long-term and synergistic effects are simultaneously present in trials involving a crossover design, difficulties may arise in the interpretation of results. This communication uses as an example the long-term clinical and microbiological effects of xylitol and suggests that inclusion of the crossover practice in clinical trials should be separately and carefully contemplated in each instance.

Dental caries is caused by a diet‐associated pathologic process affecting the teeth. This is an infectious, transmissible disease, the infecting agents being specific micro‐organisms, with a streptococcal species, i.e. Streptococcus mutans (or other members of the so‐called mutans streptococci), being especially cariogenic under certain dietary conditions. The transmissibility of the disease is reflected, for example, in the fact that young children usually receive their S. mutans infection from their mothers. According to present scientific knowledge, dental caries should be fully preventable. Maximisation of preventive efforts is of fundamental importance; relying on only one prevention procedure, like a fluoride‐based method, is insufficient. Relying solely on reparative philosophy is wrong. As to dietary approaches, mere prohibition of sugar as the only educational measure is hypocritical. Maximisation of prevention automatically includes the use of sugar substitutes. Of these, xylitol, a natural, physiologic carbohydrate sweetener, has been rigorously tested in long‐term human clinical trials, and has been found to be safe and effective. An effective way to use xylitol is as xylitol‐containing chewing gum, lozenges or chewable tablets. This prevention procedure can easily be promoted in environments where children or young adults are reachable, i.e. at schools, universities, garrisons, public health centres and related institutions. Results from clinical studies suggest that less than 10 grams of xylitol per day, consumed in about five chewing episodes, significantly increases protection against dental caries. This approach, a single‐component modification of diet, can be used to complete existing prevention methods, both at individual and community level.

A soluble endopeptidase was purified from rat bone using ammonium sulphate precipitation followed by Fast Protein Liquid Chromatography on gel, anion exchange, and chromatofocusing columns. The enzyme was silver stain-pure (SDS-PAGE) and its apparent molecular weight was 60,000. In general, the enzyme favored the hydrolysis of the X-Y bond in substrates with the sequence of -Pro-X-Y-Pro-, where either X or Y (or both) were hydrophobic residues. The presence of imino acid residues near the scissile bond favored hydrolysis. The enzyme was strongly inactivated by metal chelating agents and p-hydroxymercuribenzoic acid, indicating that SH-groups may be necessary for full activity of the enzyme and that the enzyme may be a metallopeptidase.

The effect of 4 to 6 years of habitual use of xylitol gum (greater than or equal to 2 times per day) on the viable counts of mutans streptococci in both plaque and saliva was evaluated in a cross-sectional study of participants of the Ylivieska xylitol study (Finland, 1982-1984). Paraffin-stimulated saliva was collected first, whereafter the remaining plaque was collected from all available tooth surfaces and pooled. The habitual xylitol consumers (n = 15) had less plaque than the controls (n = 21). The total viable counts of mutans streptococci in the plaque collected from the habitual xylitol users were about 10% of those determined in controls (p less than 0.01). The salivary mutans streptococci levels were relatively high in both habitual xylitol consumers and controls, as determined using a plate count and with an adhesion-based assay; no significant differences were detected between these groups. Thus, habitual xylitol consumption appeared not to be associated with a reduction in the total numbers of mutans streptococci in saliva. The plaque and the mutans streptococci were, however, more loosely bound to the tooth surfaces of the habitual xylitol users. The results of the plate count and the adhesion-based assay for salivary mutans streptococci were in good agreement for both habitual xylitol consumers and the controls. This suggests that it was the accumulation of plaque rather than the adhesiveness of mutans streptococci that was affected by the habitual consumption of xylitol.

The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.

The relationship between the caries status and the microbiological status [mutans streptococci (MS)] in the proximal areas of lower right and upper left molars was investigated in subjects of the Ylivieska xylitol study in Finland (1982-1985) in a cross-sectional follow-up in 1988. Those children who had used xylitol chewing gum regularly since 1982 showed significantly lower caries indices in 1988, including the proximal caries scores. The presence of MS (expressed in log10 colony-forming units) in the proximal areas studied was significantly lower in habitual xylitol consumers. Carious interproximal areas of all subjects had significantly higher total levels of MS than clinically and radiographically sound interproximal areas. The present study supports the idea of a close association between MS and dental caries, and shows that the total level of MS in the human dentition can be influenced by xylitol. It is likely that the long-term (between 1982 and 1988) habitual xylitol consumption had decreased the prevalence of MS in the interproximal areas of the present subjects and, subsequently, decreased the proximal and overall caries indices.

Summary The benzoylarginine peptidase of Treponema denticola (strain ASLM; a human oral spirochaete) was progressively and irreversibly inactivated by 1-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, a carboxyl-group reagent. At acidic pH values, reaction of one mole of the modifier per active site of the enzyme resulted in total inactivation of the enzyme. Assuming that this modifier is a specific carboxy) reagent, the data suggest that the inactivation of the T. denticola benzoylarginine peptidase was caused by the modification of one carboxyl group located close to the active site of the enzyme. Results obtained with Woodward's reagent K (N-ethyl-5-phe nylisoxazolium 3' -sulphonate) supported these findings. Carbethoxylation with diethylpyrocarbonate effectively inactivated the enzyme, and addition of hydroxylamine at pH 7.0 restored the activity almost totally, suggesting that the pyrocarbonate had reacted with tyrosyl or histidyl residues.