| Summary: | 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.
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