Mucus glycoprotein of human saliva: differences in the associated and covalently bound lipids with caries.

A high molecular weight mucus glycoprotein has been isolated from submandibular saliva of caries-resistant and caries-susceptible individuals by a procedure involving fractionation on Bio-Gel P-100 and A-50 columns followed by equilibrium density-gradient centrifugation in CsCl. The purified caries-resistant mucus glycoprotein displayed a buoyant density of 1.50 and accounted for 9.5% of the dry weight of caries-resistant saliva. The caries-susceptible mucus glycoprotein represented 14.1% of the dry weight of caries-susceptible saliva and gave a buoyant density of 1.43. Both glycoproteins exhibited similar protein and carbohydrate content, but the caries-resistant mucus glycoprotein contained 28.7% less associated lipids and 3-times less covalently bound fatty acids than the caries-susceptible mucus glycoprotein. The associated lipids were represented by neutral lipids, glycolipids and phospholipids, whereas the covalently bound fatty acids consisted mainly of hexadecanoate, octadecanoate and docosanoate. Extraction of associated lipids caused the caries-resistant glycoprotein to band in CsCl gradient at the density of 1.54 and caused the caries-susceptible glycoprotein to band at the density of 1.52. A further shift in the buoyant densities occurred following removal of the covalently bound fatty acids, and both glycoproteins banded at the density of 1.57. While the intact caries-resistant and caries-susceptible glycoproteins were susceptible to proteolysis by pronase, the lipid-rich caries-susceptible glycoprotein was degraded to a lesser extent. Extraction of associated lipids increased the degradation of both glycoproteins, but the caries-susceptible glycoprotein still remained 25% less susceptible. However, the susceptibility to pronase of the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins was essentially identical. The caries-resistant and caries-susceptible mucus glycoproteins also differed in susceptibility to peptic degradation. The apparent Km values for intact caries-resistant and caries-susceptible glycoproteins were 10.5 X 10(-7) M and 8.1 X 10(-7) M, while the values for the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins were 13.0 X 10(-7) M and 12.4 X 10(-7) M. The results suggest that the differences in the content of associated lipids and covalently bound fatty acids are responsible for the different physiochemical characteristics of caries-resistant and caries-susceptible salivary mucus glycoproteins, which may be determining factors in the resistance to caries.

Immunochemical quantitation of human submandibular-sublingual lysozyme.

A method for quantitating lysozyme by using carbamylated antiserum (commercially available) to human lysozyme in a electroimmunodiffusion technique has been developed. The method measures specific protein not lytic activity as the lyso-plate method does. We have applied this method to tears, parotid saliva, submandibular-sublingual saliva, nasal secretions, serum, and minor salivary gland secretions. We specifically selected submandibular-sublingual saliva for a comparison test with the lyso-plate method because of the known mucin content of the submandibular-sublingual saliva. Our findings indicate that no pretreatment is necessary for the electroimmunodiffusion technique. The lyso-plate method, on the other hand, requires pretreatment with Tris-acetate pH 4.5 to dissociate the mucin-lysozyme complex and give accurate values.

The effect of collection technique on tear composition.

Selected proteins were quantitated after collecting samples of the tears by using two sampling techniques. Tears from the same individual were collected via absorption by Schirmer filter paper strip from the unanesthetized, inferior, conjunctival sac and were compared with tears collected by a capillary tube (taking care not to touch the conjunctiva), after stimulation of tearing by irritation of the nasal mucosa with ammonia vapor. Tear samples were quantitated immunochemically for two typical lacrimal proteins, lysozyme and lactoferrin, and three typical serum proteins, albumin, transferrin, and IgG. Tear analysis of all constituents were performed on a single sample of tears collected by each method from the same individual. Normal subjects without ocular pain or discomfort comprised a sample of 12 subjects ranging in age from 19 to 57 years and consisting of 9 men and 3 women. Concentrations of lysozyme and lactoferrin in samples collected by either method were not significantly different. In contrast, the concentration of albumin, IgG and transferrin collected by Schirmer filter paper technique was significantly higher (P less than 0.01) than the concentration in tears collected by the capillary tube technique. A highly significant increase in serum proteins was seen when the Schirmer filter paper strip was used to collect tears compared to tears collected without mechanical stimulation of the conjunctiva.

The functions of saliva.

Nature's demands on salivary glands are extensive and diverse and range from the reptilian need for a venomous drop to incapacitate its prey to the 100 quarts that ruminants require to digest a day's grazing. Other species depend on saliva not for survival, but for improving the quality of life, using the fluid for functions varying from grooming and cleansing to nest-building. Humans can manage without saliva; its loss is not life-threatening in any immediate sense, but it results in a variety of difficulties and miseries. Oral digestion per se is only of marginal importance in humans, but saliva is important in preparing food for mastication, for swallowing, and for normal taste perception. Without saliva, mealtimes are difficult, uncomfortable, and embarrassing. The complex mix of salivary constituents provides an effective set of systems for lubricating and protecting the soft and hard tissues. Protection of soft tissues is afforded against desiccation, penetration, ulceration, and potential carcinogens by mucin and anti-proteases. Saliva can encourage soft tissue repair by reducing clotting time and accelerating wound contraction. A major protective function results from the salivary role in maintenance of the ecological balance in the oral cavity via: (1) debridement/lavage; (2) aggregation and reduced adherence by both immunological and non-immunological means; and (3) direct antibacterial activity. Saliva also possesses anti-fungal and anti-viral systems. Saliva is effective in maintaining pH in the oral cavity, contributes to the regulation of plaque pH, and helps neutralize reflux acids in the esophagus. Salivary maintenance of tooth integrity is dependent on: (1) mechanical cleansing and carbohydrate clearance; (2) post-eruptive maturation of enamel.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonimmunologic aspects of caries resistance.

A variety of components provide salivary secretions with an array of potentially effective means of combating cariogenic challenges. These defense factors range from a laissez-faire mechanical cleansing to exquisitely controlled production of highly specific antibodies. In between the two extremes are antibacterial systems whose operating characteristics are only beginning to be understood. These systems are well worth our attention. They may be the key to our understanding of variations in individual susceptibility, and could provide valuable leads for development of anticaries agents.

On being a scientist in a rapidly changing world.

The practice of biological science has changed dramatically since mid-century, reshaped not only by a rapid series of landmark discoveries, but also by governmental directives, institutional policies, and public attitudes. Until 1964, the major influences were the mentor, who provided direction and indoctrination into the culture of science, and in dentistry, the newly established NIDR, which fueled the research engine with an expanding research and training program. The 1965-74 period witnessed the advent of the Institutional Review Board, an increased social involvement of biological scientists, and a recognition of the need for biological and physical safeguards in the conduct of research. The most turbulent years were 1975-89, when there was a confluence of animal rights activism and regulation, growing concerns with scientific fraud and publication malpractice, and the stresses and strains (and opportunities) resulting from the rapid expansion of the academic-industrial complex. The current period is characterized by rapid pace, high volume, and an increased depth and breadth of knowledge-a major change in scale in the conduct of science. It is an exciting time but one in which ethical issues are multiplying. Attention must be paid.

A comparative study of salivary lysozyme in caries-resistant and caries-susceptible adults.

Lysozyme concentration was quantitated immunochemically in parotid and submandibular-sublingual saliva of 46 caries-resistant and 17 caries-susceptible adults. There was essentially no difference between the two groups. The concentration of lysozyme was three times higher in the submandibular-sublingual than in the parotid secretion, and was significantly higher in unstimulated submandibular saliva than in secretions stimulated with 1, 2, or 4% citric acid. There were no significant differences in flow rate between caries-resistant and -susceptible subjects. Salivary lysozyme concentration is not a critical determinant of resistance or susceptibility to caries.

Quantitation of human salivary acidic proline-rich proteins in oral diseases.

Acidic proline-rich proteins (APRP) were quantitated immunochemically in salivary secretions from groups of: caries-resistant (CR) and caries-susceptible (CS) subjects; heavy- and light-calculus-formers; and patients with Sjögren's Syndrome, drug-induced xerostomia, and recurrent parotitis. In all groups except the parotitis patients, there were comparable levels of APRP, about 40-50 mg%, with similar values in parotid and submandibular saliva. In chronic recurrent parotitis, the values were somewhat higher (about 60 mg%). There were no differences in the proportion of APRP-A to C in a subset of CR and CS. Taken as a whole, the data support the view that the secretion of APRP is stable and that caries status and propensity to calculus formation are not associated with abnormal levels of these phosphoproteins.

Lactoferrin concentration in human parotid saliva as measured by an enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) was found to be a reliable and accurate method for quantitating lactoferrin in parotid fluid. In saliva from normal glands, the concentration was usually below 1 mg% and was always higher in unstimulated than in stimulated secretion.

Comparative plaque acidogenesis of caries-resistant vs. caries-susceptible adults.

The authors conducted a comparative study of plaque acids in three-day fasting or resting plaque samples from ten pairs of caries-resistant (CR) and caries-susceptible (CS) subjects chewing a sucrose gum for a period of 45 min. The study disclosed two important differences: 1) The amount and rate of production of lactic acid were lower in the CR group, especially at ten min; and 2) in contrast to lactic acid levels, the level of acetic acid was significantly higher in the CR group at zero time (before chewing), and after 20 and 45 min of chewing the sucrose gum. Both the lower levels of lactic acid and the higher levels of acetic acid are (paradoxically) consistent with the higher plaque pH values reported for CR when compared to CS subjects. High pK' acids (such as acetic, as well as propionic and butyric) can provide a buffering system (acetate-acetic acid) capable of countering the pH decrement generated by the low pK' acids (lactic, formic and pyruvic).

The effect of saliva on plaque pH in vivo.

The effect of saliva on plaque acidogenesis was studied in ten caries-resistant and ten caries-susceptible subjects. Plaque pH was measured in vivo following exposure to a sucrose substrate under varying conditions of salivary access. Our findings demonstrate that when there is no salivary access, plaque pH levels are similar in the CR and CS groups. As the access to saliva is increased, the observed pH minima increased to a greater degree in the CR subjects than was noted in the CS subjects. This indicates that saliva (notably stimulated saliva) plays a major role in modifying plaque pH and quantitatively reflects caries status.

Polyamines of dental plaque in caries-resistant vs. caries-susceptible adults.

Utilizing a sensitive liquid chromatographic system, polyamines were quantitated in three-day plaque from 13 caries-resistant (CR) and 35 caries-susceptible (CS) adults after they had fasted for 12 hours. The values for putrescine and cadaverine were significantly higher in the CR group. This may be attributed to a greater availability of salivary substrate precursors of polyamines and/or higher levels of biosynthetic decarboxylase activities in the CR subjects, or both.

The salivary lactoperoxidase system in caries-resistant and -susceptible adults.

In a comparative study of caries-resistant and -susceptible adults, no significant differences were found in the concentrations of lactoperoxidase, thiocyanate, and pre-formed hypothiocyanite in parotid and submandibular saliva, or in hypothiocyanite formed in expectorated saliva over a 60-minute period. The concentrations per se of these components are not critical determinants of caries resistance or susceptibility. Thiocyanate concentration was higher in both unstimulated parotid and submandibular saliva than in the respective stimulated secretions. At comparable flow rates, parotid values were higher than submandibular.

Some characteristics of collagenase activity in gingival crevicular fluid and its relationship to gingival diseases in humans.

Collagenase activity in human gingival fluid was measured using a radioactive collagen fibril assay. The activity was positively correlated with the severity of gingival disease. The fluid collagenase seemed to be controlled by alpha 2-macroglobulin, based on its activation by NaSCN, and to be present solely in the extracellular fraction. Examination of the collagen breakdown products by acrylamide gel electrophoresis indicated that the fluid collagenase was of tissue rather than bacterial origin.

Formation of calcium phosphates in saliva and dental plaque.

This is an X-ray diffraction study of the mineral phases in saliva and early dental plaque. The salivas studied came from patients with cystic fibrosis (CF), those with asthma, and heavy and light calculus formers. One-week old plaque was studied from individuals who are heavy, moderate, and light calculus formers.

Alterations in lactoferrin in salivary gland disease.

During the active phase of chronic recurrent parotitis there is a marked elevation in the parotid concentration of lactoferrin (Lf), and iron-binding glycoprotein with antibacterial properties. The Lf concentration decreases during the recovery period, but still remains above normal levels. The changes of Lf in parotitis parallel recent findings in mastitis and pancreatitis. Elevations in Lf were also noted in five of six subjects with Sjögren's disease, but not in subjects with sarcoidosis, diabetes or "dry mouth" without sialographic changes. The source of the Lf has not been determined; it could arise in part from disrupting polymorphonuclear leucocytes and in part from epithelial cells that synthesize Lf in the salivary glands. Inflammatory stimulation of Lf synthesis would suggest a basic protective mechanism in exocrine glands and should be fully explored.

Association of lipids with proteins and glycoproteins in human saliva.

The distribution of lipids in the fractions of parotid and submandibular saliva following Bio-Gel A-50 column chromatography was measured. Over 50% of the total lipids of submandibular saliva was found in the fraction which contained mainly the high-molecular-weight glycoprotein. This fraction also contained most of the glycolipids, free fatty acids, phospholipids, and cholesterol. In the parotid saliva, the fraction containing the basic glycoprotein (the major glycoprotein fraction of parotid saliva) contained 35% of the total saliva lipids and was enriched in phospholipids ana cholesterol esters.

Lipids of supragingival calculus.

The matrix of supragingival calculus constitutes 15.7% of the calculus dry weight and contains 54.9% protein and 10.2% lipids. Of the total lipids, 61.8% are represented by neutral lipids, 28% by glycolipids, and 10.2% by phospholipids. The neutral lipids exhibit a high content of free fatty acids (63.9%) and triglycerides (15.8%). The glycolipids are comprised of simple glycosphingolipids (17.2%), mainly lactosyl- and glucosylceramides, and of neutral and sulfated glyceroglucolipids (82.8%). The phospholipids contain large quantities of phosphatidylethanolamine (34.2%) and diphosphatidylglycerol (25.5%). Comparison with salivary and submandibular stone lipids indicates that both saliva and bacteria contribute to the lipid content of supragingival calculus.

Lipid composition of human parotid salivary gland stones.

The parotid gland stone matrix constitutes 20.2% of the stone dry weight and contains 8.5% of lipids. Of the total lipids, 74% are represented by neutral lipids, 17% by glycolipids, and 9% by phospholipids. The neutral lipids exhibit a high content of free fatty acids (52.7%) and cholesteryl esters (31.1%). The glycolipids are composed of simple glycosphingolipids (7.1%), and of neutral and sulfated glyceroglucolipids (92.9%). Phosphatidylethanolamine and diphosphatidylglycerol are the principal constituents of the phospholipid fraction.

Comparative growth responses of oral streptococci on mixed saliva or the separate submandibular and parotid secretions from caries-active and caries-free individuals.

Growth of S. mutans on mixed or parotid saliva from CF individuals may be influenced by the availability of growth-supportive proteins or the inhibitory activity present in parotid saliva. A deficiency in growth-supportive proteins may explain the limited growth of S. sanguis on mixed or submandibular saliva from these individuals.