The chemical composition of human saliva.

Thesis (Ph.D.)--Boston University.Whole saliva obtained immediately on arising, before brushing of the teeth, eating, or mouth rinsing was examined for enzyme activity. The enzymes measured were acid phosphatase, alkaline phosphatase, total esterase, pseudocholinesterase, lipase, aryl-sulfatase, beta-D-galactosidase, beta-glucuronidase, hyaluronidase, and lysozyme. Hyaluronidase activities of the saliva samples were determined by a modification of the viscosimetric method. Lysozyme activities were measured turbidimetically. The remaining enzyme activities were determined by the colorimetric methods of Seligman and his co-workers, modified for use with saliva. The measurement of a spectrum of enzymes using various esters and derivatives of a single chromogenic substance, beta-naphthol, afforded a means of comparing the enzyme activities of saliva, while the simultaneous determination of ten enzymes on the same saliva sample permitted evaluation of the possible interrelationship between individual enzyme systems. Sterile parotid saliva, obtained by cannulation with a parotid cap, was shown to contain acid phosphatase, esterase, pseudocholinesterase, lipase, beta-glucuronidase, and lysozyme. The acid phosphatase activity of parotid saliva was 1O% of that found in whole saliva. Cholinesterase represented 60% of the activity of whole saliva. Parotid saliva contributed about 1% to the whole saliva total esterase activity, while parotid lipase was approximately 1O% of the whole saliva lipase titer. Parotid beta-glucuronidase was from 5-20% of the amount found in whole saliva. Only with lysozyme was the activity of parotid saliva higher than that found in whole saliva. Broth cultures of whole saliva indicated that all but sulfatase and lysozyme could be produced by the microorganisms normally inhabiting the oral cavity. These findings indicated that the parotid gland secretion contributed a part of six of the ten enzymes present in whole saliva, with the remaining share of these six enzymes apparently derived from the oral flora, cellular debris, or the other salivary glands. In order to determine further the part-whole relationship of the parotid secretion to whole saliva, various inorganic, organic, nitrogenous and enzymic components of stimulated whole and parotid saliva were measured. An aqueous mouth rinse was employed to minimize the effect of oral microorganisms and cellular debris. One enzyme, beta-glucuronidase, was determined in whole saliva both before and after the washing process as an indicator for the effectiveness of the mouth rinse. Blood obtained from the test subjects was examined for enzyme titer so that serum and saliva enzyme levels could be compared using the same substrate for both, while the saliva levels for calcium, sodium, potassium, chloride, bicarbonate, phosphorus, lactic acid, and non-protein nitrogen, total proteins, albumins, and globulins could be compared to established normal serum values. In measuring the effect of mouth rinsing on the whole saliva enzyme levels it was found that the beta-glucuronidase level decreased 40% after washing. Similarly, acid phosphatase values showed a 29% decrease from the values found in the group which did not have the preliminary mouth washing. Two tests, the chi square goodness of fit and a plot of the distribution, of each of the saliva and serum components measured, were used together in a combined judgment in order to gauge the normality of the distribution. Seven (total esterase, lipase, cholinesterase, total proteins, globulins, organic phosphorus, and lactate) of the twenty-one factors measured in whole saliva were abnormally distributed; six (total esterase, lipase, total proteins, albumins, globulins, and potassium) of the twenty factors determined in parotid saliva were found to have an abnormal distribution; while one of the serum enzymes, beta-glucuronidase, proved to be abnormal. The remaining components for whole saliva, parotid saliva, and serum could thus be considered normally distributed populations. Measurement of the various salivary components indicated that whole saliva was higher than parotid saliva in regard to the calcium, inorganic phosphorus, albumins, non-protein nitrogen and hydroxyl ion contents; while the parotid secretion contained greater amounts of sodium, potassium, chloride, bicarbonate, organic phosphorus, lactic acid, total proteins and globulins. An analysis of the difference between the means of each variable in parotid saliva versus its counterpart in whole saliva was made. With the exception of chloride, inorganic phosphorus, organic phosphorus, albumins, and lactic acid a significant difference existed between the mean whole saliva level and the mean parotid saliva level. Human serum normally maintains an anionic and cationic balance of approximately 155 milli-equivalents per liter, with little variation except during marked acidosis, alkalois, or excessive salt excretion. Whole saliva had a mean total anion content of 37.7 +/- 12.0 mEq/liter and a mean total cation content of 40.7 +/- 12.8 mEq/liter. The mean total anion content of parotid saliva was 47.4 +/- 19. 2 mEq/liter, while the mean total cation content was 43. 9 +/- 17. 1 mEq/L. Saliva thus contained approximately 25% of the ionic content of serum. When the individual salivary components were compared to their serum counterparts it was found that only the potassium and inorganic phosphorus levels of whole and parotid saliva and the calcium level of whole saliva were greater than normal serum values. It was noted that parotid saliva contained relatively high levels of acid phosphatase and total esterases. The acid phosphatase levels of the parotid secretion were found to be similar in titer to those present in human serum. It was these findings which led to the study of the properties of the parotid saliva phosphomonoesterase and total esterase. Characterization of these enzymes thus permitted comparison with similar enzymes present in the other body tissues and aided in their identification. The pH activity curve for the phosphomonoesterase was determined over the range pH 2.70-5.98 in 1.2 M acetate buffer at a substrate concentration of 8.9 x 10^-4 M. Results indicated that the pH optimum for parotid saliva acid phosphatase was 4.57 with beta-naphthyl phosphate as substrate. The reaction rate followed zero order when the substrate concentration was sufficiently high. When the initial substrate concentration was lowered there was a deviation from zero order as the reaction proceeded. If the initial substrate was further decreased and increased amounts of enzyme employed the reaction tended to follow those of a monomolecular reaction, or first order, Using the method of Lineweaver and Burk a plot of S (substrate concentration) against S/V (substrate concentration/ velocity of reaction) the Michaelis constant, Km, was found to have a value of 2 x 10^-4 M. The energies of activation and inactivation were determined for the salivary phosphomonoesterase at the pH optimum. Enzyme activity was measured at nine temperatures over 25 and 55°C. The energy of activation was between 6,600 and 7,600 cal./mole., while the energy of inactivation was found to be between 32,900 and 35,900 cal. /mole. The temperature coefficient (Q10) was 1.48. Maximum activity occurred at 47°C. Although the substrate employed for the investigation of the parotid total esterases was attacked most readily by the nonspecific esterases of liver; cholinesterase and lipase were also able to catalyze its hydrolysis. Thus it may be assumed that the cholinesterase and lipase observed in saliva, as well as any nonspecific esterase present contributed to the hydrolysis of beta-naphthyl acetate by parotid saliva. In dealing with the multiple effects caused by the simultaneous action of several enzymes on a single substrate, each enzyme having its own particular characteristics, certain deviations of the resulting data from ideal were to be expected. However, this did not occur with the total esterase activity of parotid saliva, which acted as a single enzyme. Results of the investigation indicated an optimum of pH 8.57 for parotid saliva total esterases. When a sufficiently high initial substrate concentration was employed the reaction was zero order up to six hours. Longer periods of incubations could not be employed due to the high degree of spontaneous hydrolysis encountered. The Michaelis-Menten constant was calculated to be 14.48 x 10^-4 M. A linear relationship between saliva volume and the amount of beta-naphthyl acetate hydrolyzed was observed. The energy of activation was 5,860 calories/mole. Maximum activity occurred at 60°C. The temperature coefficient (Q10) of the interval 25-35 degrees C was 1.69, while a QlO of 1.38 was observed for the interval 35-45 degrees C