Saliva Sediment Research Articles

HYGIENIC ASPECTS OF ENDO- AND EXOGENIC METHODS OF PREVENTION OF CARIES AND THEIR EFFICIENCY IN THE REMINERALIZATION OF TEETH ENAMEL

According to the dental survey, the prevalence of caries in 33-45 years aged adults amounts to 98%. Studies of the effectiveness of prevention of tooth decay have shown the effectiveness of this direction. But the exogenous methods do not guarantee the restoration of the balance between the processes of demineralization and remineralization of the enamel. Demineralizing factors will prevail if the concentration of essential minerals in the mixed saliva is reduced. Therefore, it is necessary to develop endogenous techniques that will promote the process of remineralization of enamel and normalization of the ion-molecular balance. The aim of the study was to compare endogenous or exogenous methods in order to saturate mixed saliva with mineral complexes in preventing the development of primary demineralization of hard tooth tissue. Material and methods. The chemical composition of the mixed saliva sediment in 27 patients was analyzed by IR spectroscopy. To record the measurement of IR spectra, a Vertex-70 spectrometer (Bruker, Germany) and a PLATINUMATR full internal reflectance attachment with a diamond prism were used, as well as a clinical determination of the enamel remineralization rate (COSRE test) was performed. Results and Discussion. The relationship between the use of endogenous and exogenous methods of saturation of mixed saliva with mineral complexes and changes occurring in its molecular composition has been revealed, in favor of the effectiveness of endogenous methods.

Rapid Detection of the mt3243A > G Mutation Using Urine Sediment in Elderly Chinese Type 2 Diabetic Patients

Objective In this study, we aimed to identify mt3243A > G mutation carriers in a group of Chinese elderly type 2 diabetic patients by a rapid and noninvasive diagnostic system. Methods DNA was extracted from blood, saliva, and urine sediment samples. The mutation screening and quantitation of heteroplasmy were performed by high-resolution melting (HRM) curve and pyrosequencing, respectively. Patients with mt3243A > G mutation underwent a detailed audiometric, ophthalmologic, neurological, and cardiac examination. Results Two patients (2/1041) carrying the mt3243A > G mutation were detected among all type 2 diabetic patients. In patient 1, the heteroplasmy was 0.8%, 2.8%, and 14.7% in peripheral blood leukocytes, saliva, and urine sediment, respectively. In patient 2, the heteroplasmy was 5.3%, 8.4%, and 37.7% in peripheral blood leukocytes, saliva, and urine sediment, respectively. Both of the two patients showed hearing impairment. Abnormal ophthalmologic conditions and hyperintensity on T2-weighted magnetic resonance images were showed in patient 1. Conclusion The occurrence of mt3243 A > G mutation was 0.2% in Chinese elderly type 2 diabetic patients. Moreover, detection of mt3243 A > G mutation in urine sediment with high-resolution melting (HRM) curve and pyrosequencing is feasible in molecular genetic diagnosis.

Acidogenicity and acidurance of dental plaque and saliva sediment from adults in relation to caries activity and chlorhexidine exposure

BackgroundThe ecological plaque hypothesis for the etiopathogenesis of caries implies a microbial shift towards a more aciduric dental plaque microbiota, due to a frequent carbohydrate intake. Acid tolerance has been suggested as an important property of the caries-associated bacteria and several in vitro studies with mixed cultures indicated that a low pH rather than the carbohydrate availability is responsible for microbiota shifts associated with the development of dental caries.ObjectiveTo examine 1) the acidogenic potential (amount lactate produced per mg plaque and minute, at pH 7.0 or pH 5.5) and the aciduric potential (acidogenic potential at pH 5.5/acidogenic potential at pH 7.0) of dental plaque and salivary sediment taken from caries-active or caries-free adults, and 2) the effect of a short-term chlorhexidine treatment on these potentials.DesignDental plaque and saliva sediment samples were taken from caries-free and caries-active subjects and suspended in Ringer's solution containing 1% sucrose and buffered with 0.5 M 3-[N-morpholino]propanesulfonic acid (MOPS), pH 7.0, or 3-[N-morpholino]ethanesulfonic acid (MES), pH 5.5. After incubation at 37°C for 10–20 min, the concentration of lactic acid in the suspension was determined by an enzymatic assay. The acid production of dental plaque was also determined after a period of mouth rinsing with 0.2% chlorhexidine.ResultsBoth dental plaque and salivary sediment from caries-free subjects exhibited significantly lower acidogenic potentials at both pHs compared to caries-active volunteers. The opposite was observed with the aciduric potential. Chlorhexidine treatment significantly reduced all three potentials but had no effect on the relative proportion of bacteria grown on acidic agar.ConclusionsCaries-active adults have an oral microbiota characterised by an increased catabolic velocity for sugar. The increase is more pronounced at neutral than acidic pH. Exposure to chlorhexidine, through mouthwash, temporarily decreases the acidogenicity of the microbiota.

Leukocyte Myeloperoxidase and Salivary Lactoperoxidase: Identification and Quantitation in Human Mixed Saliva

Human salivary lactoperoxidase (HS-LP) is synthesized and secreted by the salivary glands, whereas myeloperoxidase (MPO) is found in PMN leukocytes, which migrate into the oral cavity at gingival crevices. HS-LP levels vary with changes in salivary gland function, but increased numbers of MPO-containing leukocytes indicate infection or inflammation of oral tissues. To determine the contribution of each enzyme to the peroxidase activity of mixed-saliva samples, activity was assayed at pH 5.4 with tetramethylbenzidine as the substrate, with and without the inhibitor dapsone (4,4'-diaminodiphenylsulfone). Dapsone blocked the activity of HS-LP but not MPO. The enzymes were also separated and partially purified from the soluble portion of saliva samples and from detergent extracts of the saliva sediment. Chromatographic properties of the proteins were similar to those of LP from bovine milk (BM-LP) and MPO from human leukocytes. The identity and amounts of the enzymes were confirmed by the absorption spectra and by immunoblotting with antibodies to BM-LP and human MPO. Eosinophil peroxidase (EPO), a distinct enzyme found in eosinophilic leukocytes, was not detected by chromatography or with antibodies to human EPO. On average, 75% of the activity in samples from normal donors was due to MPO and 25% to HS-LP. When corrected for the lower specific activity of HS-LP in this assay, the average amount of MPO (3.6 micrograms/mL) was twice the amount of HS-LP (1.9 micrograms/mL). The amount of MPO corresponded to 1 x 10(6) PMN leukocytes/mL of saliva. The enzymes were distributed differently: Eighty-nine percent of the HS-LP was in the soluble portion of saliva, and 78% of the MPO was in the sediment, which contained 51% of the total activity. In contrast to results obtained with PMN leukocytes from blood, detergent was not required for MPO activity to be measured in saliva, indicating that the enzyme was accessible to peroxidase substrates. The results indicate that MPO is responsible for a large portion of peroxidase-catalyzed reactions in mixed saliva. The unique function of HS-LP may be carried out within the salivary glands, prior to secretion into the oral cavity.

C31G, a new agent for oral use with potent antimicrobial and antiadherence properties.

C31G, an equimolar mixture of alkyl dimethyl glycine and alkyl dimethyl amine oxide, was evaluated for antimicrobial and antiadherence properties. The efficacy of C31G, its two components, and several commercial mouth rinses was determined in assays measuring inhibition of glycolysis, inhibition of bacterial adherence, and MICs. Inhibition of glycolysis was determined by using a saliva sediment model, with glycolytic activity expressed as the change in pH relative to that of a control. Adherence studies were undertaken with Streptococcus sobrinus 6715 to measure inhibition of adherence to nichrome wires. MICs were determined against selected microorganisms by standard methods. C31G demonstrated broad-spectrum antimicrobial properties, with activity against both gram-positive and gram-negative organisms and Candida albicans, a yeast. C31G inhibited both glycolysis by salivary bacteria and adherence of Streptococcus strains to wire mesh. C31G was more effective in the assays conducted than any commercial formulation tested and was as effective as chlorhexidine. A synergistic effect was demonstrated between the individual components of C31G, and no loss of activity was noted when it was formulated into a mouth rinse vehicle.

ヒト混合唾液中の酵素活性 歯口清掃中止による変化

The purpose of this study was to elucidate 19 enzymatic activities in whole mixed saliva and their changes in men without oral hygiene.Ten healthy, unmedicated male students with excellent oral hygiene and clinically normal gingiva, between the ages of 22 and 26 years, were the experimental subjects. At the start of the experimental period, whole mixed saliva and parotid saliva were collected and the clinical parameters were recorded for each of them. Then, the experimental no-cleansing period of 7 days was started, and whole mixed saliva was collected and the clinical parameters were recorded every day. Enzymatic activities were determined using API ZYM systems (MONTALIEU VERCIEU, France).Alkaline phosphatase, butyrate esterase, caprylate esterase-lipase, myristate lipase, leucine aminopeptidase, valine aminopeptidase, and trypsin activities in whole mixed saliva supernatant were significantly higher at the end of the no-cleansing period than at the start of experiment. Myristate lipase, valine aminopeptidase, and trypsin were detected in whole mixed saliva sediment, but not in parotid saliva. This suggests that these enzyme activities in whole mixed saliva are useful in estimating the oral hygiene status.

Peroxidase antimicrobial system of human saliva: requirements for accumulation of hypothiocyanite.

Human saliva was fractionated to determine the components required for production and accumulation of the antimicrobial oxidizing agent, hypothiocyanite ion (OSCN-). The required components were: 1) peroxidase activity and thiocyanate ion (SCN-), 2) the saliva sediment, which produced hydrogen peroxide (H2O2) in the presence of oxygen and a divalent cation, and 3) heat-stable factors of the saliva supernatant. The supernatant factors were separated into high- and low-mol wt fractions. The high-mol wt fraction contained both peptide and carbohydrate, and its activity was partially inhibited by proteolytic treatment. The low-mol wt fraction contained carbohydrate and could be replaced by a number of mono- and di-saccharides. Glucosamine and N-acetyl glucosamine were the most effective, whereas neutral sugars such as sucrose were less effective. Sucrose competed with glucosamine, so that lower levels of OSCN- were obtained with increasing amounts of sucrose. The sugars stimulated production of H2O2 by the saliva sediment. Production of H2O2 was greater in the presence of glucosamine than of neutral sugars. Also, the ratio of OSCN- accumulation to H2O2 production was greater in the presence of glucosamine. The results suggest that peroxidase-mediated antimicrobial activity is modulated by the carbohydrate composition of whole saliva and by certain protein and glycoprotein components.

Characterization of trypsin-like enzymes from human saliva isolated by use of affinity chromatography.

Paraffin-stimulated whole mixed saliva was collected from 6 individuals and pooled. Three trypsin-like enzymes from saliva supernatant and two from the saliva sediment were isolated by use of affinity chromatography on a soybean trypsin inhibitor Sepharose 4 B column. The isoelectric points for these enzymes were pI 8.0, 6.8 and 6.4 from the supernatant and 6.4 and 6.2 from the sediment. The enzymes were characterized with respect to pH-optimum, pH-stability, temperature stability, substrate specificity and influence of metal ions and inhibitors. The Michaelis constants were determined for these enzymes on the substrates BAEE and TAME.

Relations between early dental calculus production and calcium and phosphate parameters of salivary fractions.

A pilot study was carried out to obtain an idea of the strength of the relations between early mandibular calculus production in humans and amounts and concentrations of bound calcium and phosphate in three salivary fractions. The salivary fractions were: (1) the spontaneous precipitate from incubated whole saliva, (2) the spontaneous precipitate from incubated centrifugal (20,000 X g) saliva supernatant and (3) the fresh centrifugal saliva sediment. Strong and highly significant correlations were found for the amount and concentration of calcium in the fresh sediment and whole saliva precipitate fractions. Although the correlations for the calcium parameters of the saliva supernatant precipitate were not significant, there were significant differences in these parameters between low and high calculus formers as determined by Student's t test. Relations between calculus production and amount and concentration of phosphate were weaker and not significant for all fractions. The data suggest that the amount or concentration of calcium in fresh saliva sediment is an important determinant of early mandibular calculus production.

The distribution of proteolytic and alkaline phosphatase activities in human saliva treated with sodium deoxycholate.

Sodium deoxycholate was used to release peptidase and alkaline phosphatase activities from saliva sediment. The degree of the release was determined at a variety of detergent concentrations and incubation periods. By use of 0.4% of this detergent 80% of the alkaline phosphatase found in the sediment was released. A twofold increase in peptidase in the soluble fraction (10,000 g supernatant) and a 10% increase in protein resulted in the same concentration of sodium deoxycholate. The distribution of the enzymes in saliva supernatant and the enzymes released from sediment were determined on the substrates gelatin, poly-L-lysine HBr, haemoglobin, a-N-benzoyl-L-arginine ethyl ester-HCl, p-tosyl-L-arginine methyl ester-HCl, L-lysine-p-nitroanilide HBr and p-nitrophenyl phosphate. The study indicates that the peptidase(s) soluble in human saliva without detergent are derived from saliva sediment.

Some properties of proteolytic enzymes in human saliva.

Paraffin stimulated whole mixed saliva from seven subjects was collected. The peptidases found in the supernatant and released from the saliva sediment were characterized by the following properties: pH-optimum on gelatin and haemoglobin as substrates, pH-stability and the influence of inhibitors and activators. Saliva was precipitated with acetone, treated with sodium deoxycholate and fractionated on Bio-gel P-100 and P-150. The data obtained indicate the presence of several peptidases in human saliva hydrolyzing the substrates α-N-benzoyl-L-arginine ethyl ester-HCl, p-tosyl-L-arginine methyl ester-HCl, L-Iysine-p-nitroanilide dihydrobromide and gelatin.

Development of an in-vitro plaque model from human salivary sediment suspensions

Agar-coated glass slides which were alternately immersed in and drained of salivary sediment (prepared from fresh pooled human saliva) became coated with a cellular deposit. When the coated slide and its deposit was incubated aerobically or anaerobically in sucrose or glucose, glycolytic reactions occurred within 3–4 hr. Microscopic examinations of the deposit before and after incubation revealed populations of microorganisms similar to those reported in 1–2 day old human dental plaque. Optimum conditions for deposit collection, including salivary sediment concentrations, collecting-time intervals and carbohydrate concentrations in incubation media were determined. Based on these results, slides coated with brain-heart infusion agar, rotated in a suspension of human saliva sediment concentrated 4 times, collected a deposit similar to in situ dental plaque. Under these optimum conditions, chlorhexidine, a clinically effective inhibitor of plaque formation, added to the sediment prevented microbial deposits and glycolytic reactions.

Odour production by human salivary fractions and plaque

The odour-producing activity of whole saliva, saliva sediment and supernate, and plaque was assessed on the basis of odour intensity, volatile oxidizable sulphur production, and hydrogen transfer activity. Studies on saliva fractions indicate that the cellular elements in the sediment are a primary source of participating enzymes. Saliva sediment exhibited only trace activity in the absence of supernate. This supernatefree sediment was still capable of producing intensely objectionable odour provided that the rate-limiting step is by-passed, as was demonstrated by the addition of cysteine. Heat-inactivated supernate completely restored the hydrogen transfer and most of the odour-producing activity of the viable sediment. In contrast, the activity of supernate in the absence of viable sediment or in the presence of heat-inactivated sediment was barely detectable within the time limits studied. The addition of cysteine failed to restore the odour production activity in these latter systems. Plaque exhibited a strong potential for odour production in the presence of appropriate substrates. While plaque inoculated into saliva supernate yielded only feeble activity, the addition of cysteine, cystine, and methionine markedly intensified odour and volatile sulphur compound production by these supplemented systems. Potent odour-producing activity was also demonstrated with plaque-inoculated synthetic test media. The amperometric and mass spectrometric analysis of volatile compositions revealed the presence of large concentrations of sulphur-containing compounds (hydrogen sulphide, methyl mercaptan, dimethyldisulphide, and sulphur dioxide) in the head-space of putrefied media systems. The evidence derived from these studies suggests that plaque and saliva elaborate similar malodorous products of putrefaction.

The effect of saliva on the metabolism of the oral flora

The ability of washed saliva sediment to metabolise glucose is much less than that of whole saliva. Supernatant saliva and the parotid gland secretion both contain substances which enhance glycolysis. Separation of saliva on Sephadex columns showed that the active substances are not confined in a single fraction. Fractionation of saliva with ethanol has demonstrated that material soluble in 80 per cent ethanol possesses most of the activity. The behaviour of the saliva can be simulated by mixtures of amino acids which have been reported present in saliva, but only at concentrations greater than those occurring in the normal secretion. It is suggested that the enhancement of glycolysis by saliva is due to the stimulation of anabolic processes by the supply of amino acids and peptides which are necessary for the synthesis of cellular components. These processes require the energy produced by glycolysis, and when anabolism is interrupted energy requirements are less and the metabolism of glucose will be diminished.

THE DIAGNOSIS OF METACHROMATIC LEUCODYSTROPHY DURING LIFE.

The authors report the study of saliva sediment in 4 cases of juvenile metachromatic leucodystrophy belonging to the same family (and else in a sister of one of these cases presenting the characteristic neurological picture but with no metachromasia demonstrable by the Austin test in urine or by biopsies), in 6 normal relatives of the patients with Scholz disease, in 9 cases of various diseases of the nervous system, and in 10 normal subjects. The presence of metachromatic bodies staining in a pinkish red colour with acid blue toluidine dye was demonstrated in the saliva sediment of the 4 cases of metachromatic leucodystrophy. In 2 of these patients biopsies of the parotid gland, stained with cresyl violet dye, showed the presence of intracellular brownish metachromatic bodies. In these 2 cases the study of cerebrospinal fluid sediment also disclosed the presence of metachromatic bodies. Furthermore, a chromatographic qualitative test for metachromatic lipids yielded positive results in saliva, cerebrospinal fluid, and urine sediments. The conclusion was drawn that the search for metachromatic bodies in cerebrospinal fluid and mainly in saliva sediment may be of help in disclosing or ratifying the diagnosis of metachromatic leucodystrophy during life.