• Mass Spectrometry Letters
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• v.8 no.4
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• pp.114-118
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• 2017

While saliva can be considered as good biological fluid for monitoring biomarkers due to many advantages including its communication with blood and the non-invasive nature during its sampling, its applications to that purpose is still limited. As a part of efforts to expand the applications of saliva to the protein biomarker research, we carried out global absolute quantitation of proteins in human whole saliva (WS) by bottom-up proteomics techniques mainly based on nLC-Q-IMS-TOF employing $MS^E$. From the analyses of a pooled WS sample collected from 22 healthy Korean volunteers, 93 proteins ranging from $5.89{\times}10^1ng/mL$ (immunoglobulin heavy chain) to $1.59{\times}10^4ng/mL$ (${\alpha}-amylase$ 1) were confirmed. For the validation of the present results, human serum albumin in the same sample was quantitated by ELISA and its result was compared with that from the nLC-Q-IMS-TOF study. As a result, there was no significant difference between two results from individual approaches ($1.18{\times}10^4{\pm}0.03{\times}10^4 ng/mL$ from nLC-Q-IMS-TOF experiments vs. $1.23{\times}10^4{\pm}0.07{\times}10^4ng/mL$ from ELISA experiments, n=3, p=0.309). To our knowledge, this is the first global absolute quantitation of proteins in human whole saliva and information from the present study can be widely used as the first level reference for the discovery of new protein biomarkers from human whole saliva as well as for quantitative applications of human whole saliva proteins.

• Journal of Oral Medicine and Pain
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• v.32 no.4
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• pp.365-373
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• 2007

Destruction of oral soft and hard tissues and resulting problems seriously affect the life quality of xerostomic patients. Although artificial saliva is the only regimen for xerostomic patients with totally abolished salivary glands, currently available artificial salivas give restricted satisfaction to patients. The purpose of this study was to contribute to the development of ideal artificial saliva through comparing viscosity and wettability between CMC solutions and human saliva. Commercially-available CMC is dissolved in simulated salivary buffer (SSB) and distilled deionized water (DDW). Various properties of human whole saliva, human glandular saliva, and a CMC-based saliva substitutes known as Salivart and Moi-Stir were compared with those of CMC solutions. Viscosity was measured with a cone-and-plate digital viscometer at six different shear rates, while wettability on acrylic resin and Co-Cr alloy was determined by the contact angle. The obtained results were as follows: 1. The viscosity of CMC solutions was proportional to CMC concentration, with 0.5% CMC solution displaying similar viscosity to stimulated whole saliva. Where as a decrease in contact angle was found with increasing CMC concentration. 2. The viscosity of human saliva was found to be inversely proportional to shear rate, a non-Newtonian (pseudoplastic) trait of biological fluids. The mean viscosity values at various shear rates increased as follows: stimulated parotid saliva, stimulated whole saliva, unstimulated whole saliva, stimulated submandibular-sublingual saliva. 3. Contact angles of human saliva on the tested solid phases were inversely correlated with viscosity, namely decreasing in the order stimulated parotid saliva, stimulated whole saliva, unstimulated whole saliva, stimulated submandibular-sublingual saliva. 4. Boiled CMC dissolved in SSB (CMC-SSB) had a lower viscosity than CMC-SSB (P < 0.01 at shear rate of $90s^{-1}$). 5. For human saliva, contact angles on acrylic resin were significantly lower than those on Co-Cr alloy (P < 0.01). 6. Comparing CMC solutions with human saliva, the contact angles between acrylic resin and human saliva solutions were significantly lower than those between acrylic resin and CMC solutions, including Salivart and Moi-Stir (P <0.01). The effectiveness of CMC solutions in terms of their rheological properties was objectively confirmed, indicating a vital role for CMC in the development of effective salivary substitutes.

• Restorative Dentistry and Endodontics
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• v.23 no.2
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• pp.630-638
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• 1998

The purpose of this study was to evaluate the effect of galvanic current between different metallic restorations on human saliva. The rate of salivary flow and concentrations of IgG, IgM, sIgA and lactoferrin were measured. In this study, unstimulated whole saliva collected before restoration was regarded as control group and unstimulated whole saliva collected 10 minutes, 1 day, 1 week, and 1 month after restoration were regarded as experimental groups. Following results were obtained from this study. 1. There were some differences in values of salivary flow rate between experimental groups, but the changes in values compared to those of the control group were not statistically significant(P>0.05). 2. Measurements of major antibacterial components of saliva showed that while the concentrations of IgG and IgM decreased significantly 1 week and 1 month after restoration(P<0.05), changes in values of sIgA and lactoferrin were not statistically significant(P>0.05). 3. In vitro measurements of galvanic currents decreased sharply in the first 20 seconds and thereafter decreased gradually. Galvanic current values measured in the early stages were greatly varied, but after 2 hours, the values in all groups approximated each other.

• Journal of Oral Medicine and Pain
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• v.31 no.1
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• pp.1-16
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• 2006

The most important progress in diagnostic sciences is the increased sensitivity and specificity in diagnostic procedures due to the development of micromethodologies and increasing availability of immunological and molecular biological reagents. The technological advances led to consider the diagnostic use of saliva for an array of analytes and DNA source. The purpose of the present study was to compare DNA from saliva with those from blood and buccal swab, to evaluate diagnostic and forensic application of saliva, to investigate the changes of genomic DNA in saliva according to the storage temperature and period of saliva samples, and to evaluate the integrity of the DNA from saliva stored under various storage conditions by PCR analysis. Peripheral venous blood, unstimulated whole saliva, stimulated whole saliva, and buccal swab were obtained from healthy 10 subjects (mean age: $29.9{\pm}9.8$ years) and genomic DNA was extracted using commercial kit. For the study of effects of various storage conditions on genomic DNA from saliva, stimulated whole saliva were obtained from healthy 20 subjects (mean age: $32.3{\pm}6.6$ years). After making aliquots from fresh saliva, they were stored at room temperature, $4^{\circ}C$, $-20^{\circ}C$, and $-70^{\circ}C$. Saliva samples after lyophilization and dry-out procedure were stored at room temperature. After 1, 3, and 5 months, the same experiment was performed to investigate the changes in genomic DNA in saliva samples. In case of saliva aliquots stored at room temperature and dry-out samples, the results in 2 weeks were also included. Integrity of DNA from saliva stored under various storage conditions was also evaluated by PCR amplification analysis of $\beta$-globin gene fragments (989-bp). The results were as follows: 1. Concentration of genomic DNA extracted from saliva was lower than that from blood (p<0.05), but there were no significant differences among various types of saliva samples. Purities of genomic DNA extracted from stimulated whole saliva and lyophilized one were significantly higher than that from blood (p<0.05). Purity of genomic DNA extracted from buccal swab was lower than those from various types of saliva samples (p<0.05). 2. Concentration of genomic DNA from saliva stored at room temperature showed gradual reduction after 1 month, and decreased significantly in 3 and 5 months (p<0.05, p<0.01, respectively). Purities of DNA from saliva stored for 3 and 5 months showed significant differences with those of fresh saliva and stored saliva for 1 month (p<0.05). 3. In the case of saliva stored at $4^{\circ}C$ and $-20^{\circ}C$, there were no significant changes of concentration of genomic DNA in 3 months. Concentration of DNA decreased significantly in 5 months (p<0.05). 4. There were no significant differences of concentration of genomic DNA from saliva stored at $-70^{\circ}C$ and from lyophilized one according to storage period. Concentration of DNA showed decreasing tendency in 5 months. 5. Concentration of genomic DNA immediately extracted from saliva dried on Petri dish were 60% compared with that of fresh saliva. Concentration of DNA from saliva stored at room temperature after dry-out showed rapid reduction within 2 weeks (p<0.05). 6. Amplification of $\beta$-globin gene using PCR was successful in all lyophilized saliva stored for 5 months. At the time of 1 month, $\beta$-globin gene was successfully amplified in all saliva samples stored at $-20^{\circ}C$ and $-70^{\circ}C$, and in some saliva samples stored at $4^{\circ}C$. $\beta$-globin gene was failed to amplify in saliva stored at room temperature and dry-out saliva.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.227-235
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• 2009

Many of the protective functions of saliva can be attributed to the biological, physical, structural, and rheological characteristics of salivary glycoproteins. Therefore, the development of ideal saliva substitutes requires understanding of the rheological as well as biological properties of human saliva. In the present study, we investigated the changes of salivary enzymatic activities by saliva substitutes and compared viscosity of saliva substitutes with human saliva. Five kinds of saliva substitutes such as Moi-Stir, Stoppers4, MouthKote, Saliva Orthana, and SNU were used. Lysozyme activity was determined by the turbidimetric method. Peroxidase activity was determined with an NbsSCN assay. $\alpha$-Amylase activity was determined using a chromogenic substrate, 2-chloro-p-nitrophenol linked with maltotriose. The pH values of saliva substitutes were measured and their viscosity values were measured with a cone-and-plate digital viscometer at six different shear rates. Various types of saliva substitutes affected the activities of salivary enzymes in different ways. Stoppers4 enhanced the enzymatic activities of hen egg-white lysozyme, bovine lactoperoxidase (bLP), and $\alpha$-amylase. Saliva Orthana and SNU inhibited bLP activity and enhanced $\alpha$-amylase activity. MouthKote inhibited $\alpha$-amylase activity. Moi-Stir inhibited the enzymatic activities of bLP and $\alpha$-amylase. The pH values were very different according to the types of saliva substitutes. Stoppers4, MouthKote, and Saliva Orthana showed lower values of viscosity at low shear rates and higher values of viscosity at high shear rates compared with unstimulated and stimulated whole saliva. Moi-Stir and SNU displayed much higher values of viscosity than those of natural whole saliva. Collectively, our results indicate that each saliva substitute has its own biological and rheological characteristics. Each saliva substitute affects the enzymatic activity of salivary enzyme and finally oral health in different ways.

• Journal of Oral Medicine and Pain
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• v.30 no.1
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• pp.15-23
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• 2005

Mucin glycoproteins are the primary carriers of the oligosaccharide moieties that constitute the blood group substances in human saliva. The aim of this study was to determine whether or not the conversion of either the A or B blood group antigens to the H antigen can occur during the degradation process of stored saliva samples. Forty subjects (20 subjects in each A and B blood group) identified as secretors were enrolled in this study. Fresh whole saliva samples and their clarified supernatants were stored at room temperature for 1 week. The conversion of the blood group antigens was detected by SDS-PAGE and immunoblotting. Among the subjects showing the conversion in whole saliva, glandular saliva samples were obtained from 8 subjects (4 subjects in each A and B blood group). Submandibular-sublingual saliva (SMSL) and a mixture of SMSL and parotid saliva (PS) were stored at room temperature for 1 week. The conversion of the blood group antigens was detected by the same method. The obtained results were as follows: 1. In the clarified samples of whole saliva, the A antigen was detected as being either intact (5%) or degraded molecules (95%) after the 1 week period. Conversion of the A antigen to the H antigen was detected in 5 subjects (25%). In the unclarified samples, the A antigen was either detected as degraded molecules (90%) or was not detected (10%). Conversion of the antigen had occurred in 4 subjects (20%). 2. In the clarified samples of whole saliva, the B antigen was detected as intact (20%) or as degraded molecules (65%) or was not detected (15%) after the 1 week period. Conversion of the B antigen to the H antigen was detected in 7 subjects (35%). In the unclarified samples, the B antigen was detected as intact (5%) or as degraded molecules (65%), or was not detected (30%). Conversion of the antigen was observed in 2 subjects (10%). 3. In the glandular saliva samples, only one of the four subjects displayed an antigenic conversion from the A to H antigen or from the B to H antigen. The conversion had occurred in both the SMSL samples and the SMSL and PS mixture. No degradation of the antigens was detected in the other three samples of the A or B blood groups, nor was there any conversion. The results demonstrated that conversion of the blood group antigens could occur in saliva, and suggested that the enzymes responsible for the conversion are present in saliva. Further studies on the origin and activity of the specific glycosidases in saliva as well as quantitative measurements of the antigenic conversion will be needed.

• Journal of Periodontal and Implant Science
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• v.46 no.5
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• pp.320-328
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• 2016

Purpose: Human saliva, as a vital part of the immune defense system, contains a number of distinct proteins and peptides. Recently human common salivary protein 1 (CSP1) has been identified as an abundant salivary protein and may play a role in promoting the binding of cariogenic bacteria to salivary pellicles. However, nothing else is known regarding the role of CSP1 in periodontology. The aim of this study was to quantify and compare CSP1 levels between healthy subjects and periodontal patients. Methods: This controlled clinical study was conducted in periodontally healthy individuals and patients with chronic periodontitis Chonbuk National University Hospital, with Institutional Review Board approval. Whole saliva samples were collected from 36 healthy subjects and 33 chronic periodontitis patients and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immune blotting were conducted to ensure that anti-CSP1 monoclonal antibody (mAb) binds to CSP1 in human saliva. A sandwich enzyme-linked immunosorbent assay (ELISA) system was house-fabricated using mAb-hCSP1#14 and mAb-hCSP1#4 as a capture and a detector mAb, respectively. The CSP1 concentrations in saliva from 36 healthy subjects and 33 periodontal patients were quantified using the CSP1 sandwich ELISA system, and the results were analyzed using the Student's t-test. Results: Immunoblot analysis using mAb-hCSP1 as a probe confirmed that CSP1 in human saliva existed as a single band with a molecular weight of approximately 27-kDa. The quantification of CSP1 concentrations by CSP1 ELISA showed that the median values (25th to 75th percentiles) of periodontal patients and healthy subjects were 9,474 ng/mL (range, 8,434.10,139 ng/mL) and 8,598 ng/mL (range, 7,421.9,877 ng/mL), respectively. The Student's t-test indicated the presence of a statistically significant difference between the 2 groups (P=0.024). Conclusions: The presence of a significant difference in CSP1 levels between healthy subjects and periodontal patients suggests that CSP1 may be a potential biomarker for the detection or screening of periodontitis patients.

• Journal of Oral Medicine and Pain
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• v.21 no.1
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• pp.123-131
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• 1996

Bacteremia occurs in a wide variety of clinical procedures in oral cavity. Reduction of the number of causative microorganisms of infective endocarditis in oral cavity by local administration of antimicrobial agents decreases the magnitude of bacteremia and possibility of infective endocarditis. The effects of chlorhexidine on Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus gordonii, Staphylococcus aureus, and Staphylococcus epidermis were investigated by measurement of turbidity. The effects of 0.1% chlorhexidine gargling for 7 days on oral bacterial flora, total streptococci, S. mutans, S. aureus, and S. epidermis in whole saliv a of 7 healthy human subjects, were investigated by measurement of Colony Forming Units (CFU). The obtained results were as follows : 1. Chlorhexidine showed significant antimicrobial effects on Streptococcus snaguis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus gordonii, Staphylococcus aureus, and Staphylococcus epidermis. However, the effects on S. sanguis and S. gordonii were not apparent compared with other microorganisms. 2. Oral gargling of 0.1% chlorhexidine decreased the CFU values of normal oral bacterial flora, total streptococci, S. mutans, S. aureus, and S. epidermis in whole saliva. The antimicrobial effects were significant after 4 days of chlorhexidine gargling. 3. Local antimicrobial administration in addition to systemic antibiotic prophylaxis can be highly recommended as an effective adjunct regimen for prevention of infective endocarditis.

• Journal of Oral Medicine and Pain
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• v.32 no.4
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• pp.347-355
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• 2007

The aim of this study was to evaluate the differences of salivary lead (Pb) and cadmium (Cd) concentrations, using ASV analysis, after various pre-treatment procedures. 10 unstimulated whole saliva samples of non-exposed subjects to Pb and Cd were collected. Each sample was divided into 6 aliquots and centrifugation was performed in only 3 aliquots. After centrifugation, 3 different types of pre-treatment procedures were carried out. Also, these pre-treatment procedures were carried out for another 3 aliquots, without centrifugation. Pre-treated aliquots were analyzed electrochemically, by ASV. The results are as follows: 1. Mean concentration of Pb in saliva after centrifugation was significantly higher than that of non-centrifugation. 2. In the detection sensitivity of Pb in saliva, those of simple dilution technique by HCl and acid digestion technique by nitric acid were significantly higher than that of simple dilution technique by electrolyte. 3. Mean concentration of Cd in saliva after centrifugation was significantly higher than that of non-centrifugation. 4. In the detection sensitivity of Cd in saliva, those of simple dilution technique by HCl and acid digestion technique by nitric acid were higher than that of simple dilution technique by electrolyte. But, there were no significant differences between them.

• Journal of Oral Medicine and Pain
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• v.34 no.4
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• pp.387-395
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• 2009

Nerve growth factor (NGF) and sensory neuropeptides are involved in the process of nociception at peripheral nerve fibers and wide spread in central nervous system. The aims of this study were to investigate NGF and sensory neuropeptides (substance P [SP] and calcitonin gene-related peptide [CGRP]) levels in human plasma and saliva, and the associations between these sensory neuropeptides levels and chronic orofacial pain symptoms. NGF, SP, and CGRP levels in plasma and resting whole saliva samples collected from 67 orofacial pain patients (joint pain, dental or periodontal pain, mucosal pain) and 36 pain free control subjects were measured by enzyme immunoassay. The characteristic pain intensity of each subject was measured using the Graded Chronic Pain Scale and the flow rate of resting whole saliva was measured. Joint pain patients group showed significantly higher plasma NGF level compared to each of dental pain patients (p<0.01), mucosal pain patients (p<0.01), and control group (p<0.01). Plasma NGF level of dental pain patients group was significantly higher than that of control group (p<0.01). Saliva SP level of dental pain patients group (p<0.05) and saliva CGRP level of mucosal pain group (p<0.05) were significantly higher than that of control group. Plasma and saliva SP levels of joint pain patients was significantly associated with pain intensity (plasma: standardized coefficient=0.599, p<0.01, saliva: standardized coefficient=0.504, p=0.05). In dental pain patients group, plasma SP (standardized coefficient=0.559, p<0.01), saliva SP (standardized coefficient=0.520, p<0.01) and saliva CGRP (standardized coefficient=0.599, p<0.01) levels were significantly associated with age. In mucosal pain patients group, plasma SP (standardized coefficient=0.495, p<0.05), saliva SP (standardized coefficient=0.500, p<0.05), and saliva CGRP (standardized coefficient=0.717, p<0.01) levels were significantly associated with age. NGF and neuropeptides may play a role in the maintenance of various orofacial pain symptoms. The examination of those levels in plasma and saliva helps understanding the mechanism of orofacial pain, and furthermore, can be applied to the diagnosis and therapy of orofacial pain.