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Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.

Autoradiography

Determination of ABO blood groups from saliva and saliva stains by an indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies.

The detection of A, B and H blood group substances (ABH-BGS) in saliva and in saliva stains has been investigated quantitatively by an indirect ELISA using a horseradish peroxidase conjugate in combination with the use of monoclonal antibodies. Through this method, the reaction specificity to BGS in the saliva was very high and its detection sensitivity was found to be approximately 1,000 times greater than has been achieved in a hemagglutination-inhibition test. The monoclonal anti-A and anti-B reagents reacting with both secretor and non-secretor saliva in a hemagglutination-inhibition test and in this ELISA method were selected from among commercial monoclonal antibodies. However, no monoclonal anti-H reagent was found to react with non-secretor saliva. The BGS level was determined by the use of calibration curves of A, B and H standard BGS from human gastric mucosa and was expressed in units, based on the inhibition titer of the standard BGS. In 230 saliva samples, ABH-BGS were detectable, except for H BGS in non-secretor saliva. The BGS levels in saliva stains experimentally prepared were found to be approximately proportional to the levels in the original saliva. As for actual and aged stains, it was possible to detect BGS in most cigarette butts and in aged stains, however, such detection proved impossible in saliva samples from postage stamp.

ABO Blood-Group System

Adrenal status assessed by direct radioimmunoassay of cortisol in whole saliva or parotid saliva.

We describe a direct radioimmunoassay for cortisol in 10-microliter volumes of parotid saliva or whole saliva. Binding proteins are absent from these fluids, as demonstrated by the excellent correlation between results for samples assayed directly and by a comparison procedure involving extraction with 1,2-dichloroethane. The direct assay is specific, precise, and had a lower limit of sensitivity of 4 pg per assay tube. Comparison of cortisol concentrations in plasma, parotid saliva, and whole saliva in persons undergoing investigations for assessing adrenal function, including stimulation with cosyntropin (Synacthen) and suppression with dexamethasone, indicated that changes in plasma cortisol concentration were accurately and immediately reflected in saliva from either the parotidgland or whole saliva. A marked circadian rhythm has also been demonstrated for cortisol in parotid-gland saliva and whole saliva. We had to modify the 1,2-dichloroethane extraction procedure for accurate determination of cortisol in parotid saliva and whole saliva of patients undergoing treatment with metyrapone.

Adrenal Glands

Saliva secretion rate and saliva composition as a model to determine the effect of antidepressant drugs on cholinergic and noradrenergic transmission.

Three principally different antidepressant drugs--amitriptyline (with effects on both noradrenaline and serotonin reuptake), maprotiline (a selective inhibitor of reuptake of noradrenaline) and zimelidine (a selective inhibitor of reuptake of serotonin) were tested after a single dose and, as concerns maprotiline and zimelidine, after long-term use in healthy volunteers as regards the effects on saliva secretion rate and saliva composition. Amitriptyline gave a strong decrease in both unstimulated and stimulated saliva secretion rate, indicating a strong anticholinergic effect, and a strong increase in the activity of amylase and the content of protein, fucose and hexose in saliva, indicating a strong agonistic effect on noradrenaline transmission. Maprotiline gave an intermediate decrease in saliva secretion rate, and the effect remained after 14 days of treatment. Single doses gave increases in saliva amylase activity and protein content. After 14 days this effect was increased, and there was also a pronounced increase in fucose and hexose content indicating a strong agonistic effect on noradrenaline transmission. Zimelidine gave a low decrease in saliva secretion rate, indicating a mild anticholinergic effect. No certain effect on the saliva composition could be settled after a single dose, but after 14 days there were consistent increases in amylase activity and protein, fucose and hexose content, indicating a facilitation of noradrenaline transmission. Zimelidine as well as its active metabolite, norzimelidine, are selective inhibitors of serotonin reuptake. The action of zimelidine is discussed in view of recent hypotheses about a modulating serotoninergic influence on the noradrenergic system.

Adult

A new individual variation of saliva detected with rabbit anti-human saliva serum.

A rabbit anti-saliva hyperimmune serum was raised against pooled human saliva. After absorption with human serum and semen, the immune serum formed two or three distinct precipitation lines with human saliva by the Ouchterlony method. Based on the presence or absence of a precipitation line, close to the antigen well designated as Pl, all human saliva were divided into two groups; Pl+ and Pl-. The incidence of Pl+ saliva was 114 out of 154 samples (74%). By immunoelectrophoresis with anti-saliva serum preabsorbed with Pl- saliva, Pl+ saliva formed a precipitation line in cathodal area. No amylase activity was found in this precipitation line but it was stained with the Schiff's reagent. Pl protein was isolated by Sephadex G-100 and ion-exchange chromatography. Isoelectric focusing of the Pl protein in polyacrylamide slab gel containing urea revealed that the protein differed from any known parotid salivary proteins such as Pr, Db, Pa and Pm.

Animals

Wetting properties of human saliva and saliva substitutes.

Contact angle measurements were employed so that we could study the wetting properties of human whole saliva and saliva substitutes containing mucins or carboxymethylcellulose (CMC) on human oral mucosa (in vivo) and on ground and polished human enamel (in vitro). A new approach was introduced for measuring contact angles on human oral mucosa in vivo. It was shown that the wetting properties of CMC- and mucin-containing saliva substitutes on human enamel were significantly better than those of human whole saliva. On human oral mucosa, the wetting properties of CMC-containing saliva substitutes and human whole saliva were comparable, but were surpassed by those of the mucin-containing saliva substitutes. Mucin-containing substitutes thus provide good wetting properties on ground and polished enamel as well as on oral mucosa.

Aged

Evaluation of the Q.E.D. Saliva Alcohol Test: a new, rapid, accurate device for measuring ethanol in saliva.

STUDY OBJECTIVE: To evaluate the accuracy of the Q.E.D. A-150 Saliva Alcohol Test, a new device that gives a specific quantitative blood alcohol level by measuring saliva alcohol concentration in the range of 0 to 150 mg/dL. STUDY DESIGN: Forty-two healthy volunteers consumed 4.5 to 6 oz of alcohol in the form of beer, wine, or liquor over a 90-minute period. Blood and saliva samples were obtained for alcohol measurement at 30, 60, 90, and 120 minutes after the last drink. Blood samples were analyzed within 24 hours by gas chromatography at a commercial clinical laboratory. Saliva samples were tested immediately using the new Q.E.D. A-150 Saliva Alcohol Test. RESULTS: Excellent correlation was observed between saliva and blood alcohol levels over the range of 0 to 150 mg/dL (slope = 1.0; intercept = 2.4; r = .98). CONCLUSION: The Q.E.D. Test is an accurate device for specific quantitative measurement of alcohol levels using saliva.

Adult

Fluoride concentrations in plaque, whole saliva, and ductal saliva after application of home-use topical fluorides [published eerratum appears in J Dent Res 1993 Jan;72(1):87].

It is now well-accepted that the primary anti-caries activity of fluoride (F) is via topical action. The retention of F in the mouth after topical fluoride treatment is considered to be an important factor in the clinical efficacy of F. The purpose of this study was to evaluate F levels in ductal saliva, whole saliva, and pooled plaque after treatment with topical F agents intended for home use. Ten consenting adults, mean (SD) age 31.0 (8.2) years, participated in all aspects of the study. Two days before each test, subjects received a professional tooth cleaning and subsequently abstained from all oral hygiene procedures to permit plaque to accumulate, and from the use of F-containing dental products. Treatments consisted of a placebo dentifrice (PD), fluoride dentifrice (FD; 0.24% NaF), fluoride rinse (FR; 0.05% NaF), and fluoride gel (FG; 1.1% NaF). Unstimulated whole saliva and pooled plaque were sampled at multiple points over a 24-hour period. In a separate experimental series, stimulated parotid saliva was sampled over a two-hour period after treatment. Fluoride levels generally followed the same pattern in whole saliva and pooled plaque samples, with FG > FR > FD > PD. Night-time F application resulted in prolonged F retention in whole saliva but not in plaque. Fluoride levels in parotid saliva were only slightly higher after F treatment and returned to baseline levels within two h. The results of this study indicate that the method of F delivery, the F concentration of the agent, and the time of application (daytime vs. night-time) are important factors influencing F levels in the mouth.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent

Comparison between saliva stimulants and saliva substitutes in patients with symptoms related to dry mouth. A multi-centre study.

Five saliva stimulants (Salivin, V6, Mucidan, Ascoxal-T and Nicotinamide) and three saliva substitutes (Saliment, Salisynt and an ex témpore solution) were evaluated in 106 patients with a low salivay flow rate and a long history of dry mouth. The study was carried out as a multi-centre study in collaboration with ten different hospital dental clinics. The participants were interviewed about their complaints related to dry mouth. Each patient was then asked to use the eight saliva stimulants and substitutes for 14 days in a randomized order with one week intervals. After the 14-day-periods, the patients were interviewed about the effect of the various products by a dentist using a standardized questionnaire. Paraffin-stimulated whole saliva samples were collected after each test period. The most serious complaints among the patients were, besides dryness of the mouth, difficulty to talk and difficulty to swallow. The results showed that all eight tested saliva stimulants and substitutes relieved the symptoms of dry mouth to some extent. However, V6 chewing gum and Salivin lozenge were ranked as the two best products by the patients. No long-term effect was found with any of the eight products on the flow rate of paraffin-stimulated whole saliva.

Adolescent

Binding of cloned S-fimbriated E. coli to human buccal epithelial cells--different inhibition of binding by neonatal saliva and adult saliva.

Investigations were carried out on the adhesion of cloned S-fimbriated E. coli, labelled with fluoresceinisothiocyanate (FITC) to human buccal epithelial cells. Fluorescence microscopic analysis revealed binding of bacteria to 75-95% of epithelial cells. Inhibition experiments with fetuin, alpha 1-acid glycoprotein and N-acetyl neuraminic acid confirmed the specificity of bacterial binding to sialoglycoproteins. Further studies using saliva as an inhibitor resulted in a 4-5 times stronger binding inhibition by newborn saliva in comparison to adult saliva coinciding with a 4-5 times higher content of total N-acetyl neuraminic acid in samples of newborn saliva. In Western blot analysis sialoglycoprotein bands with a molecular weight greater than 200 kD reacting with wheat germ agglutinin (WGA), were only identified in samples of newborn saliva. These bands are classified as mucins on account of molecular weight and staining. These data suggest that saliva mucins could represent a major defense mechanism against bacterial infections at a stage of ontogeny where the secretory IgA-system is not yet developed.

Adult

[Acid and basic glycoproteins of human saliva. 2. Investigation of glycoprotein of parotid saliva].

We applied the standard diagnostic electrophoretic method on lyophilized human parotid saliva under the appropriate conditions (pH = 8.6 voltage 90 V and time of 30 seconds). The variation of the essential electrophoretic parameters (volume, time, voltage and pH) gave the best protein separation results in the natural range (pH = 7). Also in all cases, except pH = 11, the catodic side was richer in fractions than the anodic one; this was the qualitative characteristic of the protein component of the parotid saliva. Consequently, the protein content of the parotid saliva was rich in basic elements with the typical electrophoregram and densitogram for human serum and mixed saliva. The Pol-E agarose film method is appropriate for investigation and detection of the protein content in human, especially parotid saliva. It also enables differentiation of samples of mixed and parotid saliva on the basis of appropriate densitograms which are the consequence of different protein and especially glycoprotein components of the content.

Electrophoresis, Agar Gel

Determination of caffeine in saliva by high-performance liquid chromatography: new sampling method for saliva using filter paper.

We report an assay method for the determination of salivary caffeine concentrations by high-performance liquid chromatography. A simple, new method for collecting mixed saliva using a piece of filter paper has been developed. An arbitrary amount of saliva is absorbed and a fixed amount of internal standard is added. The mean coefficient of variation of measurements repeated by changing both volumes absorbed and caffeine concentrations of saliva was 5.6% with this method. The detection limit was determined to be 0.5 microgram/ml. Precision of measurements and recoveries did not depend upon the volume of saliva absorbed. Four volunteers were given a capsule of 230 mg caffeine, and saliva was collected by the ordinary method using chemical stimulation with citric acid and by our filter paper absorption methods; the methods were compared. Salivary caffeine concentrations were always lower than plasma concentrations, and there were no big differences between two methods, although salivary concentrations measured by our method was higher, with a significant difference at 1.5 h after dosing. From the practical viewpoint this method would be convenient and sufficiently accurate.

Adult

Aggregation of group A streptococci by human saliva and effect of saliva on streptococcal adherence to host cells.

The aggregation of group A streptococci by whole, stimulated human saliva (WHS) and the effect of saliva on streptococcal adherence to host cells was investigated. WHS samples from 11 individuals were found to aggregate both M+ and M- group A streptococci to various degrees. The aggregating activity was sensitive to heat, EDTA, EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], sodium dodecyl sulfate, and lipoteichoic acid. None of the simple sugars tested, mercaptoethanol, albumin, or nonionic detergents had any effect on aggregation. The aggregating activity of EDTA-treated saliva was restored by 0.1 mM Ca2+ and 1.0 mM Mn2+ but not by up to 5 mM Mg2+. Only streptococci from the stationary phase were aggregated. Hyaluronidase treatment of streptococci from the exponential phase of growth restored their ability to be aggregated, suggesting that the hyaluronic acid capsule interferes with agglutination. Adsorption of WHS by one strain of Streptococcus pyogenes removed aggregating activity for other strains of S. pyogenes and Streptococcus sanguis but not agglutinins for Escherichia coli, suggesting that the agglutinin is specific for certain gram-positive bacteria. Molecular sieve chromatography of WHS and identification of streptococcus-binding components of saliva suggest that either a glycoprotein of approximately 360 kDa or a mucin of saliva of greater than 1,000 kDa mediates aggregation of streptococci. WHS also inhibited adherence of S. pyogenes to buccal epithelial cells.

Adsorption

[Human mandibular movements during swallowing saliva. 2. Duration of swallowing saliva and that of tooth contact].

The sagittal incisal jaw movement during swallowing saliva was measured on 11 subjects (range: 24-30 years old) with "normal occlusion" by using a two-dimensional system applied infrared sensor. The duration of swallowing saliva and that of tooth contact were calculated with the program developed in our laboratory. The results obtained were as follows: 1. The average of the duration of swallowing saliva (T1) was approx. 3.5 sec on 11 subjects. 2. The average of the duration of tooth contact during swallowing saliva (T2) was approx. 2.1 sec on 11 subjects. Type Ia had the longest T2 (approx. 2.5 sec) among the types of tooth contact. 3. Type Ia had a greater ratio T2/T1 (74.4%) than those of the other types. In conclusion, it was clearly shown that the subject of type Ia had a longer duration of tooth contact during swallowing saliva than those of the other types in the absolute and relative values.

Adult

Lubrication and viscosity features of human saliva and commercially available saliva substitutes.

The lubricating features and viscosity of human saliva and five commercially available saliva substitutes were compared. The results indicate that little correlation exists between these parameters. Saliva substitutes based on carboxymethylcellulose do not appear to lubricate biocompatible hard interfaces well and, therefore, might not protect against the rapid attrition observed in xerostomic individuals. In contrast, a mucin-based substitute proved to be a better lubricant with values comparable to whole human saliva.

Adult

ABO blood grouping of saliva from mixed body fluids by sandwich methods using monoclonal antibodies to tissue specific epitopes on blood group substance in saliva.

In this paper methods for ABO blood grouping of saliva from mixed body fluids have been established. Monoclonal antibodies to tissue specific epitopes on blood group substances in saliva were used as solid phase antibodies to catch the blood group substances. ABO blood grouping of saliva could be performed by these methods without interference from other body fluids (eg. semen, vaginal secretion, urine, sweat and serum). At least 16,000 and 3,000 fold dilutions of secretor saliva were sufficient for ABO blood grouping by sandwich ELISA and sandwich absorption-elution test, respectively.

ABO Blood-Group System