A COMPARATIVE STUDY OP CERTAIN ENZYMES OF THE SALIVA AND OP THE ORAL MICROPLORA OF THE HUNT-HOPPERT CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS By Norman Paul Willett AN ABSTRACT Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1955 Approved by v / • Norman Paul Willett 1 ABSTRACT A study was made of certain enzymatic activities of the saliva and of broth cultures of oral microflora of the HuntHoppert caries-resistant and caries-susceptible rats. Saliva was obtained by stimulating the salivary glands of anesthetized rats with pilocarpine. Broth cultures of the oral microflora were obtained by brushing the lower right molars with swabs and placing the swabs In brain-heart infusion broth. Preliminary work showed that acid and alkaline phospha­ tases, and aryl-sulfatase were detected in saliva and in broth cultures of the oral microflora. Lysozyme and protease were found in the saliva, but not from the oral microflora, whereas hyaluronidase was shown to be of bacterial origin. Urease was not detected from either source. Three of the enzymes studied exhibited differences of various magnitudes between susceptible and resistant animals. The aryl-sulfatase activity of both salivary and microbial origin was slightly higher in resistant animals than in susceptibles. Acid and alkaline phosphatases of saliva showed extremely variable results. The data obtained for salivary protease showed the widest differences between resistant and susceptible animals, and further experiments involving this enzyme were carried out. Norman Paul Willett 2 The saliva of mature susceptible males displayed about three times as much protease activity as that of resistant males. Mature susceptible females showed approximately two and one-half times the activity of resistant females. Susceptible males exhibited about one and one-half times as much activity as susceptible females in rats 88 to 125 days old. However, these differences were not observed in rats prior to puberty or in rats older than 125 days. Activity increased sharply with age in the case of sus­ ceptible animals, whereas much smaller changes were found in resistants. Experiments conducted to determine whether the protease was inherent in saliva or produced by the cellular materials indicated that part of the activity was contained in the saliva. A COMPARATIVE STUDY OF CERTAIN ENZYMES OF THE SALIVA AND OF THE ORAL MICROFLORA OF THE HUNT-HOPPERT CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS By Norman Paul Willett A THESIS Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture ana Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1955 ProQuest Number: 10008670 Alt rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008670 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 VITA Norman Paul Willett candidate for the degree of Doctor of Philosophy Pinal examination, September 28, 1955, 10:00 A«M., 101 Giltner Hall Dissertation: A Comparative Study of Certain Enzymes of the Saliva and of the Oral Microflora of the Hunt-Hoppert Caries-Resistant and Caries-Susceptible Rats Outline of Studies Major Subject: Minor Subject: Microbiology Biochemistry Biographical Items Born, April 13, 1928, Paterson, N. J* Undergraduate Studies, Rutgers University, B. S* 1914-5-U9} New York University, 19l|-9-50. Graduate Studies, Syracuse University, M* S*, 1950-52; Michigan State University, 1952-55* Experience, American Can Company, 1952 Member of Society of American Bacteriologists, American Association for Advancement of Science ACKNOWLEDGMENTS The author wishes to extend, sincere appreciation to Dr. H. J. Stafseth under whose guidance this thesis was carried out. Grateful acknowledgment Is due Dr. Samuel Rosen under whose direction this research was conceived and completed. Sincere thanks Is also accorded to Dr. H. R. Hunt for the statistical analysis of the data* Deep appreciation is also extended to Dr. Carl A. Hoppert and Dr. Hans Lillivik for their helpful sugges­ tions and criticisms. TABLE OF CONTENTS Page INTRODUCTION..... ..................................... 1 GENERAL SURVEY OFL I T E R A T U R E ......................... 3 GENERAL METHODS ANDMATERIALS ......................... 6 HYALURONIDASE.... ..................................... 8 A* B. C# Survey of Literature • • • • • ............ 8 9 Methods and Materials...... ........ .. Results and Discussion .............. 10 LYSOZYME............................................... 12 A. B* C. Survey of Literature ............ • . • • • Methods and M a t e r i a l s . * Results and Discussion ...... 12 13 13 SULFATASE........ ..................................... 16 A. B# C. UREASE A. B. C. Survey of Literature •• • • • • ........... Methods and Materials • • • • • ........... Results and D i s c u s s i o n .............. . . . . 16 17 17 ............................ '.................. 20 Survey of Literature . . . . . . .......... 20 Methods and M a t e r i a l s ....................... 21 Results and Discussion . • • • • • • • • • . 21 ACID AND ALKALINE P H O S P H A T A S E S ....................... 2k A. B. C. Survey of Literature ................... 2k Methods and Materials.... • • ................. 26 Results and Discussion .................. 27 P R O T E A S E ............................................... 33 A. B* C* Survey of Literature ..................... 33 Methods and Materials ♦ ♦ • • • * 3^ Results and Discussion ........ . . . . . . 37 ii TABLE OP CONTENTS (Cont.) Page GENERAL DISCUSSION SUMMARY .................................... 5l ................................................. 55 BIBLIOGRAPHY ........................................... ill 57 LIST OF TABLES TABLE I* II# III# IV# V# VI# VII# VIII# IX# PAGE Hyaluronid&se activity of saliva of cariesresistant and caries-susceptible rats expressed as percent loss in viscosity • • • 11 Hyaluronidase activity of broth cultures of oral microflora of caries-resistant and caries-susceptible rats expressed as percent loss in viscosity ................. .. . 11 Lysozyme activity of saliva of caries-resistant and caries-susceptible rats expressed as ............... percent transmission II4. Lysozyme activity of broth cultures of oral microflora of caries-resistant and cariessusceptible rats expressed as percent transmission • * • • • < 14 Sulfatase activity of saliva of caries-resistant and caries-susceptible rats expressed as percent transmission » • • • • • • • » * • * « » • • 16 Sulfatase activity of broth cultures of oral microflora of caries-resistant and cariessusceptible rats expressed as percent transmission « • • « • • • • • # 18 Urease activity of saliva of caries-resistant and caries-susceptible rats expressed as percent transmission • • • • • • 23 Urease activity of broth cultures of oral microflora of caries-resistant and cariessusceptible rats expressed as percent ..................... transmission 23 Alkaline phosphatase activity of saliva (diluted 1-5 ) of caries-resistant and caries-susceptible rats expressed as percent transmission (Method of Seligman) • • # « • • 28 iv LIST OP TABLES (Cont.) fag: Alkaline phosphatase activity of broth cultures of oral microflora of caries-resistant and caries-susceptible rats expressed as percent transmission (Method of Seligman) • • * • • • » 28 Acid phosphatase activity of saliva (diluted 1-5) of caries-resistant and caries-susceptible rats expressed as percent transmission (Method of Seligman) « • ............... • • • • • • • 29 Acid phosphatase activity of broth cultures (diluted 1-5) of oral microflora of cariesresistant and caries-susceptible rats expressed as percent transmission (Method of Seligman) • 29 Alkaline phosphatase activity of saliva (diluted 1-5) of caries-resistant and caries-susceptible rats expressed as percent transmission (Method of Gomcri) ..................... 31 Acid phosphatase activity of saliva of cariesresistant and caries-susceptible rats expressed as percent transmission (Method of Gomori) * . 31 Autoproteolytic activity of saliva of cariesresistant and caries-susceptible rats expressed as percent transmission ............. 38 Protease activity of saliva of caries-resistant and caries-susceptible rats 88-125 days of age expressed as ug of tyrosine liberated /ml of saliva • • • • • • ........................... 39 Significance of results on protease activity obtained from rats 88-125 days of age ........ 39 Protease activity of saliva of caries-resistant and caries-susceptible rats 125-li+O days of age expressed as ug of tyrosine liberated /ml of saliva ....................... ............ 1+1 Significance of results on protease activity obtained from rats 125*140 days of age . . • « kl v LIST OF TABLES (Cont*) TABLE XX. Protease activity of saliva of caries-resistant and caries-susceptible rats I4.9-55 days of age expressed as ug of tyrosine liberated /ml of saliva • ........... ......... XXI. Significance of results on protease activity obtained from rats 1^9-55 days of age . . . . XXII. Protease activity of saliva of caries-resistant and caries-susceptible rats 88-92 days of age expressed as ug of tyrosine liberated /ml of saliva • • • • . . « • ............. • • • • XXIII. Significance of results on protease activity obtained from rats 88-92 days of age • • . . XXIV. Protease activity of broth-cultures of oral microflora of caries-resistant and cariessusceptible rats expressed as ug of tyrosine liberated /ml"of saliva XXV. Effect of filtering on trypsin activity and on protease activity of saliva from male susceptible rats expressed as ug of tyrosine ............... .. liberated /ml of saliva XXVI. Effect of activators on protease activity in saliva from male susceptible rats expressed as percent transmission ........... . « • • • 1 INTRODUCTION Hunt and Hoppert (27-32) succeeded in evolving two dis­ tinct strains of rats, one caries-resistant and the other caries-susceptible* In the case of the susceptible animals, caries develops in approximately 70 days and in the resistants, 585 days* The development of these strains affords investi­ gators a unique opportunity for experimentation, since each group of animals can serve as controls for the other. Since the terms resistant and susceptible Imply inherent differences, it seemed logical to investigate the biological and chemical differences existing in the oral cavities of these rats so as to study the factor or factors which predisposes one group to dental caries while protecting the other. A secondary pur­ pose of this investigation was to seek a quick test for resis­ tance and susceptibility. It also seemed desirable to determine the degree of resistance possessed by individual resistant rats because of the variability of this strain in the develop­ ment of caries. Rosen &t al (5^4-), investigating possible differences in the oral microflora of the two strains found that lacto- bacilli and Streptococcus salivarius were recovered more fre­ quently and In higher numbers from caries-susceptible than from caries-resistant rats. Another study which suggested 2 itself was investigation of differences in the saliva since it is constantly in contact with the teeth. Benarde (6) began the study of the saliva and serum of these animals and found that the saliva of the susceptible animals had a lower relative viscosity than that of resistant animals* An extension of this study would be to explore biochemical differences existing in saliva. Inasmuch as enzymes are im­ portant In degrading various solid and semi-solid materials present to end products which might affect the carious process, it was thought that a comparison between various enzymatic activities of the saliva of resistant and susceptible animals might be fruitful* The enzymes chosen were selected on the basis of being the most pertinent to this study as judged by the investiga­ tor. A preliminary investigation was undertaken at first to see where the greatest differences in enzymatic activities between resistant and susceptible strains occurred. If dif­ ferences were found, the study would be extended where the widest differences occurred. 3 GENERAL SURVEY OP LITERATURE Numerous enzymes have been reported in human saliva, but no agreement has been reached regarding their role in various pathological conditions of the oral cavity* Pigman and Reid (lj.5) mentioned some of these enzymes in their review of the organic compounds and enzymes of human saliva* Other than the studies on amylase, the earliest were those of oxidases. Prinz (50) mentioned that as early as 1899 Slowtzow discussed the nature and function of oxidase in saliva. He identified amylase, maltase, catalase, and oxidase in saliva. In 1909, McDonald and Smith (39) also detected an en­ zyme f*distinct from ptyalin1* in human saliva which exhibited the properties of an oxidase and peroxidase. Included also among enzymes of this type, detected by later investigators, were peroxidase (l|.l), found in the saliva of man, horse, cow, sheep, dog, and cat, and catalase (10 ), which was demonstrated in human saliva. Rapp (51) detected the presence of carbonic anhydrase in human saliva and suggested that this enzyme could speed the removal of carbon dioxide from saliva near the salivary ducts. Carbon dioxide loss would be accompanied by alkalini- zation of the saliva which would allow the precipitation of k calcium phosphate on tooth surfaces giving rise to oral calculi. Fosdick, Hansen and Wessinger (17) postulated that pyruvic acid may play a part in decalcification and found that typical oral microorganisms reduced pyruvic acid to lac­ tic acid in a substrate of sterile human saliva containing insoluble calcium salts. Pincus (lp6 ) investigated the reducing properties of saliva toward some dyes and demonstrated a pyruvic dehydrogen­ ase and possibly a lactic dehydrogenase. In an analogous investigation, Kesel, 0 ’Donne1 and Kirch (3*4-) discovered that saliva of caries-immune individuals have enzyme systems capable of forming ammonia from at least six amino acids, i.e. arginine, alanine, asparagine, isoleucine, glutamic acid and serine. Caries-susceptible individuals had a wider range of enzyme systems capable of forming ammonia from amino-acids but lacked the ability to act on glutamic acid. The importance of the degradation of monosaccharides and disaccharides in the oral cavity has led several workers to investigate the enzyme systems involved. Fosdick (18 ) proved that the fermentation products of the oral microflora in a medium containing saliva, glucose and calcium phosphate, were similar to those formed in tissue during alcoholic fer­ mentation. He suggested that the degradation of carbohydrates 5 to lactic acid by means of bacteria, yeast and tissue enzymes followed similar paths which would indicate that acids such as phosphoglyceric, pyruvic, acetic, butyric and lactic played a part in dental caries* Sreebny* Kirch and Kesel (65) passed saliva through a Seitz filter and noted that the ability to ferment glucose was decreased* They concluded that the acids formed by salivary action on carbohydrates occurred mainly as a result of microbial fermentation. Enzymes in the oral cavity may originate from several sources: the salivary glands, bacteria, oral tissue, and ingested substances* Chauncey et al (9) determined the ranges, means and origin of the following ten enzymes found in native saliva: acid and alkaline phosphatase, total esterases, cholinesterase, lipase, sulfatase, beta-glucuronidase, betaD-galactosidase, lysozyme, and hyaluronidase* The presence of six of these was established in parotid saliva: acid phosphatase, total esterases, cholinesterase, lipase, betaglucuronidase and lysozyme. In addition, broth cultures used in this study indicated that all but sulfatase and lysozyme were produced by the oral flora. Amylase was not included in this study since Rosen e_t al (55) found that there was no difference in activity of this enzyme between Hunt-Hoppert caries-resistant and caries^ susceptible rats. 6 GENERAL METHODS AND MATERIALS The method of Benarde jet al (5) was used to collect rat saliva* This involves anesthesia by intraperitoneal injection of nembutal followed by a small dose of pilocarpine given subcutaneously. Prior to injection with pilocarpine, the teeth and oral cavity were vigorously brushed and cleaned with a stencil brush dipped in distilled water so as to reduce con­ tamination by fecal matter, sawdust and retained food materials. All tests were performed on unfiltered saliva. Saliva was obtained from individual rats whenever possible but when pooled salivas were used, they were collected from rats of the the same litter. All determinations were begun within one hour after collection of the saliva. Testing for enzymes of bacterial origin was carried out by swabbing the lower right molars of each rat prior to cleaning. The swabs were then placed into brain heart infusion broth which was incubated at 37 C for 2l± hours. Enzymatic determinations were then made on the supernatant fluid obtained after centrifugation. Controls were run on uninoculated broth. It must be noted that this method of determining the enzymes of the oral microflora is somewhat limited since studies were made only on the bacteria growing in these particular broth cultures. However, it was believed that this would 7 give an indication as to which enzymes originated from bac­ teria and which were contained in the saliva obtained by stimulating the salivary glands# In each experiment in the preliminary screening phase, an equal number of animals, usually two, were selected from each sex and strain. The rats were from 80 to 130 days of age in all the preliminary work. When there was a possibility of a significant difference in enzymatic activity of the saliva or broth cultures between resistant and susceptible animals, determinations were made using larger numbers of ani­ mals from a succeeding generation. The procedure used in determining individual enzymes will be reported in the section devoted to each enzyme# 8 HYALURQNIDASE A* Survey of Literature The presence of hyaluronidase in human saliva was demon­ strated by Lisanti (38 ) in 1950. In a subsequent study, Mahler and Lisanti (39) showed that this enzyme originated in the oral flora and was not present In parotid saliva obtained by cannulation. Since salivary mucoids provide a source of fermentable carbohydrates, any enzyme depolymerizing this complex is of considerable interest* Knox and Still (36) demonstrated the presence of an enzyme complex active against salivary mucoid which contained amylase and hyaluronidase* Hyaluronidase was not capable of decreasing the viscosity of salivary mucoid, although it would liberate reducing sugars from trypsindepolymerized mucoid, which suggested that before the hydroly­ sis of the glycosidic bonds could take place, a masking peptide had to be removed. In a previous investigation (35)> the same authors isolated several microorganisms with mucolytic activity from saliva and demonstrated the presence of an extracellular mucolytic enzyme. Simmons (60) found evidence of a mucolytic enzyme in parotid secretions and Rogers (53) demonstrated the presence 9 of organisms in saliva capable of breaking down mucin to glucosamine. B. Methods and Materials The technique for testing hyaluronidase activity in saliva was modified slightly from that described by Mahler and Lisanti (Ij-O). The principle involves measuring the change in viscosity of a buffered hyaluronic acid-saliva mixture. A dropping pipette was constructed from a one ml volu­ metric pipette by heating it and drawing it out to a point where it took distilled water 15 to 20 seconds to pass between two given marks on the pipette. The buffer employed was a.G5M N&C1-phosphate solution of pH 6.3 (23)* Two substrates were used. One was a commer­ cial hyaluronic acid preparation received from General Bio­ chemicals Inc. which was diluted with the buffer to a concen­ tration of .93 mg/ml. The other was received through the courtesy of Dr. V. P. Lisanti of Tufts College Dental School. The concentration of this substrate was .69 mg/mf. It took approximately 60 seconds for each substrate to fall between two marks on the pipette. Equal volumes of buffered substrate and saliva, usually 1.5 ml of each, were mixed and an initial viscosity obtained by measuring with a stopwatch the time In seconds for the mixture to drop between two marks on the pipette. The final 10 viscosity was obtained in the same manner after l\5 minutes incubation at room temperature. Hesuits in all cases were expressed as percent loss in viscosity. The testing for enzymes of bacterial origin has been described previously* After the cultures were centrifuged, the supernatant fluid was tested in the manner described above. C. Results and Discussion The results given in Tables X and II show that little or no activity was found in resistant or susceptible saliva, whereas activity was demonstrated by broth cultures of the oral microflora. The results parallel those of Mahler and Lisanti (IpO) who were unable to detect hyaluronidase in human parotid saliva obtained by cannul&tion, but observed activity from microorganisms isolated from saliva. IX TABLE I HYALURONIDASE ACTIVITY OP SALIVA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT LOSS IN VISCOSITY Trial No. Resistant Males o • o 1 2 .13 Average <•1 . Resistant Females . Susceptible Males 0.0 .33 0.0 •16 0.0 .2? Susceptible Females 0.0 .15 ^.1 TABLE II HYALURONIDASE ACTIVITY OF BROTH CULTURES OF ORAL MICROFLORA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT LOSS IN VISCOSITY Trial No. Resistant Males 16.8 Resistant Females Hi-.7 Susceptible Males 23.7 Susceptible Females U-.9 12 LYSOZYME A. Survey of Literature Skrotakii, Makhlinovskil and Stutskaia (62) have showed lysozyme to he present in the saliva of 95 to 98 percent of 336 patients without any relation to the condition of the mouth. Chauncey ert al (9) noted that of 58 persons, all showed lysozyme activity to some degree although the activity of the parotid saliva was considerably higher than in whole saliva. Roberts ,et al (52) obtained an active lysozyme prepara* tion from human saliva which was serologically distinct from egg white and cat salivary lysozyme. On the other hand, Nernes and Wheatcraft (if.2) proved by electron and phase microscopy that the mode of action of salivary lysozyme was similar to that of crystalline enzyme. They also noted that organisms of oral origin susceptible to the action of the enzyme included neiserriae, micrococci, sarcinae, streptococci, staphylococci and mycobacteria. Rudino (57) tested 220 subjects and found higher lysozyme concentrations in healthy males than in female subjects. also noted more activity in pregnant women regardless of their caries picture. He 13 Simmons (61 ) obtained evidence that lysozyme can be inhibited by forming complexes with salivary mucoids# B# Methods and Materials Lysozyme in saliva and from broth cultures was assayed turbidimetrically (6i|.)# This involves the action of the enzyme on a standard suspension of Micrococcus lysodeikticus with subsequent lysing of the organism and decrease in tur­ bidity# Lysozyme substrate (Difco) was suspended in re­ hydrated lysozyme buffer (Difco) in a concentration (approxi­ mately 50 mg/100 ml), so that 10 percentlight transmission at a wave length of 530 mu was obtained* Equal amounts of buffered substrate and saliva or broth were mixed and incu­ bated at room temperature for 20 minutes# Transmission readings were made at 530 mu at the beginning and at the end of this period on a Hellige colorimeter* C# Results and Discussion As noted in the footnote under Table III, the average initial transmission reading of the substrate when diluted with an equal amount of saliva was 19#6 percent# The increase in percent light transmission over the initial reading as a consequence of decreasing turbidity is a measure of enzyme activity. As shown in Table III, both resistant and susceptible 114- TABLE III LYSOZYME ACTIVITY OP SALIVA OP CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Resistant Males Resistant Females Susceptible Males 1 29*0 36.0 22.5 36.0 2 28.3 29.5 20.2 26.0 Average 28.7 32.8 21.1* 31.0 Trial No. Note: Susceptible Females Average initial transmission of substrate-saliva mixture = 19.8. TABLE IV LYSOZYME ACTIVITY OP BROTH CULTURES OF ORAL MICROFLORA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Resistant Males Resistant Females Susceptible Males Susceptible Females 1 17.0 15.3 18.0 15.0 2 18.5 16.3 Average 17.8 l£.8 Trial No. Note: 15.8 18.0 15.ij. Average initial transmission of substrate-broth mixture = 15*?* 15 animals showed activity. However, there were only slight differences between strains and sexes* The average initial transmission reading of the substratebroth mixtures as noted in the footnote under Table IV, was 15*5 percent. Since there was little or no increase in transmission after 20 minutes, it can be assumed that lysozyme was not produced by the oral microflora in the medium used* These results agree with Chauncey and his co-workers (9) who found that lysozyme in human saliva was not produced by the oral microflora. 16 SULFATASE A. Survey of Literature In I9I4.8 Pincus (1+7) postulated an enzymatic attack on enamel protein* He confirmed the presence of a sulfatase in certain gram negative organisms and suggested that certain of these organisms, found in the carious lesion, will attack the enamel protein with release of sulfuric acid* The cal­ cium phosphate which forms the inorganic part of enamel will then be changed to calcium sulfate* In a subsequent investi­ gation (148), Pincus demonstrated the enzymatic action of a dried preparation of a gram negative bacillus and a living culture of Penicillium spinulosum on a sulphated mucopoly­ saccharide from human dentin. Finally in 1950 (i+9) he an­ nounced that he had Isolated sulfatase containing bacteria in pure culture from carious material. He then applied these facts to his original theory of enzymatic attack on enamel protein. He suggested that since enamel protein resembles mucoprotein, which contains mucoitin sulfate, a enzymatic hydrolysis might release sulfate from enamel in a manner simi­ lar to that by which sulfate is formed from sulfated mucopoly­ saccharides in dentin. 17 Candeli and Tioneri (7) claimed that the sulfatase of Proteus and Micrococcus aureus attacked the glycoproteins of enamel and dentin in vitro. Chauncey et a^l (9) found aryl-sulfatase in whole saliva, but not in parotid saliva and no sulfatase activity was ex­ hibited by the oral microflora. Since mammalian tissues are known to contain aryl-sulfatase, he suggested that the pres­ ence of the enzyme was due to either the cellular debris in saliva or to other glandular secretions. B. Methods and Materials The method of Rutenberg, Cohen and Seligman (58) was adapted for the determination of aryl-sulfatase in saliva by Chauncey et al (9). The principle of the method involves the enzymatic release of a chromogenic product, beta-napthol, which when combined with a diazo compound produces a water insoluble dye. The dye is extracted with ethyl acetate and measured colorimetrically. C. Results and Discussion The results in Tables V and VI indicate that activity was present in saliva and in broth cultures. Although the activity in the saliva may have originated from cellular debris or may have been present in the saliva per .se, the 18 TABLE V SULFATASE ACTIVITY OF SALIVA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Resistant Males Resistant Females Susceptible Males Susceptible Female s 1 76.5 78.0 89.5 90.8 2 89.0 66.5 90.5 86*5 Average 82.8 72.3 90.0 88.7 Trial No# TABLE VI SULFATASE ACTIVITY OF BROTH CULTURES OF ORAL KLCROFLORA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Trial No. Resistant Males Resistant Female s Susceptible Males Susceptible Females 1 1+5.0 61.3 63.5 71.0 2 $6.8 1+1.3 69.3 59.0 Average 50.9 51.3 66.1+ 65.0 Note: Average uninoculated broth control = 72*0 19 possibility also exists that it may have originated as a result of bacterial proliferation in the reaction mixture during the 2lj.-hour incubation period* Chauncey ej; al (9) found the enzyme in whole human saliva, but were unable to find activity of microbial origin* Barber, Brookbank and Kuper (I4.) however, indicated that certain bacteria can elabor­ ate the enzyme. Saliva from resistant rats exhibited slightly greater activity than that of susceptibles* The enzymatic activity produced by the oral microflora showed the same degree of difference as produced by the saliva* Further investigations, using larger numbers of animals, may indicate a real differ­ ence in the aryl-sulfatase activity in the saliva and of the oral microflora between resistant and susceptible rats* Additional studies on this enzyme were not carried out be­ cause greater differences were found subsequently in other enzyme activities of the saliva of resistant and susceptible rats . 20 UREASE A. Survey of Literature Vladesco (72) reported urease in saliva as early as 1932* Stephan (70) noted that the urea in saliva was converted to ammonium carbonate locally on the tooth surface by the action of urease-c©ntaining bacteria such as Micrococcus aureus and Micrococcus albus which grew on the tooth surface in the the bacterial plaque* The ammonium carbonate neutralized some of the acids produced in the oral cavity* On the other hand, certain strains of lactobacilli contained no urease* Thus, these bacteria increased caries activity not only by fermentation of carbohydrate to lactic acid, but also by failing to retard caries activity by conversion of urea to ammonium carbonate# Ballantyne e_t jal (2) observed an ammonia producing mechanism in saliva which produced five to ten times the amount of ammonia present originally* Urea added to saliva increased ammonia production which indicated the presence of a urease* Following this up, Ballantyne, Rae and Lawford (3) showed that the amount of ammonia present in saliva appeared to be unrelated to caries activity as measured by lactobacillus 21 counts* There also was no direct relationship between urease content of saliva and lactobacillus counts# B. Methods and Materials Urease was determined by the Permutit method This involves the absorption of the ammonia released by enzymatic action onto an ^exchange silicate.” The ammonia is set free from the silicate by treating it with alkali# The mixture is then nesslerized and the resulting color measured with a color­ imeter# Determinations were made using a three percent urea-buffer solution as substrate at a pH of 6.5 (71)* Prior to mixing with the substrate, the saliva or broth was shaken with about two grams of Permutit to remove preformed ammonia# In the case of the broth, it was extremely difficult to remove all the preformed ammonia so initial determinations were carried out to determine the increase in ammonia at the end of the in­ cubation period. The reaction mixture consisted of two ml of saliva or broth and £> ml of buffered substrate. C. Results and Discussion The results in Tables VII and VIII indicated no activity present in saliva and little or no activity produced by the oral microflora. 22 The failure to detect urease by the methods used gives rise to speculation whether the rat possesses an ammoniaproducing mechanism in saliva* If one is present, it could possibly by the deamination of amino-acids as mentioned by Kesel, 0 !Donnel and Kirch (34)* 23 TABLE VII UREASE ACTIVITY OF SALIVA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Resistant Males Resistant Females 1 98.8 100.0 99.7 100.0 2 100.0 100.0 100.0 99.9 99*ii 100.0 99.9 Trial No. Average Susceptible Males Susceptible Females 100.0 TABLE VIII UREASE ACTIVITY OF BROTH CULTURES OF ORAL MICROFLORA OF \ CARIES-RESISTANT AND CARIES'-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Susceptible Males Susceptible Females Trial No Resistant Males Resistant Females 1 92.5 92.5 86.0 96.0 2 87.0 914— S - 91.2 Average 89.8 93.5 86.0 93.6 Note: Average initial transmission = 2h ACID AND ALKALINE PHOSPHATASES A* Survey of Literature Demuth (12) claimed in 192f> that he had found small quantities of phosphatase in saliva, but his evidence was poor. More positive results were obtained by Adamson (1) who detected an enzyme capable of hydrolyzing glycerophosphoric ester in human gum tissue and in the oral tissue of the dog* He suggested that since the saliva contains phosphate complexes, the interaction of the enzyme with these complexes would result in the liberation of inorganic phosphate giving rise to dental calculi* The presence of this enzyme in saliva was confirmed by Smith (63 ) who maintained that it was contained in the desqua­ mated epithelial cells found in saliva. The optimum pH for the enzyme was 5*5* Another early investigator, Giri (19), detected a phos­ phatase in saliva which he claimed was identical with urinary phosphatase by virtue of their common optimum pH. Deane (J.1) surveyed the phosphatases present in mammalian liver, pancreas, and salivary glands and found an alkaline phosphatase with an optimum pH of 9*5 which was active toward glycerophosphate, glucose 1-phosphate, fructose diphosphate, and yeast nucleic acid* 25 Noback and Montagna (i+3) showed that acid and alkaline phosphatase were present in the parotid, submaxillary, and sublingual glands of the rat and mouse. In an investigation concerning the possible hormonal influence on salivary glands, Junquiera ejt al (33) found differences between the acid phosphatase contents of adult male and female rats. There appears to be several sources of phosphatases in the oral cavity. Eggers-Lura (lip) detected both acid and alkaline phosphatase distributed in all oral tissue with the strongest activity in the sublingual tissue. Submaxillary, parotid, and whole mixed saliva also exhibited enzyme activity which was distributed in the sediment and supernatant fluid after centrifugation. The optimum pH was 5«5* but there was appreciable activity at 9*4* he also concluded that the ac­ tivity of phosphatase was extremely variable depending on pH and substances contained in saliva. Dentay and Rae (13) showed that the enzyme activity of pooled saliva was reduced almost to zero by passage through a Seitz filter and concluded that the phosphatase in saliva was derived mainly from cellular debris and food residue. Fitzgerald (16) extended this by including bacteria as a source of enzyme. He suggested that a parallel might exist between acid phosphatase content and lactobacillus counts but Helman and Mitchell (25) were unable to confirm this. 26 Chauncey ejt al (9) demonstrated acid and alkaline phos­ phatase in whole saliva, but were unable to show an alkaline phosphatase in parotid saliva* The acid phosphatase activity of parotid saliva was approximately one percent of that found in whole saliva, indicating that the enzyme was either of exogenous origin or due to the secretion of other glands* Clock and his co-workers (20) found that Penicillium spinulosum. Rhodotorula rubrum, and Actinomyces buccalis* all common to the oral cavity, were able to produce extracellular phosphatase* Kroncke and Haujoks (37) failed to find differences in acid phosphatase content between caries-resistant and cariessusceptible individuals, but detected differences in the alka­ line phosphatase content* They also found significant dif­ ferences in the ratio of acid to alkaline phosphatase between caries-resistant and caries-susceptible individuals* B* Methods and Materials Acid and alkaline phosphatase were determined by the method of Seligman et al (59)* The principle depends on the enzymatic release of a chromogenic product, beta-napthol, which when combined with a diazo compound produces a water insoluble dye which can be extracted with ethyl acetate and measured in a colorimeter at 5^0 mu* Since Helman and Mitchell (2$) found the optimum pH of acid phosphatase to be 5*5, 27 a 0*2 M acetate buffer was used for the determination of tills enzyme rather than the buffer cited in the original method. For the determination of alkaline phosphatase 0,1 M carbonate buffer was substituted for the veronal buffer used in the original method. Gomori (22) stated that veronal buffer does not have an adequate buffering capacity at the desired pH. Dilutions used for the saliva were 1-5 for both acid and alkaline phosphatases. The broth cultures were diluted 1-5 only in the case of the acid phosphatase, whereas alkaline phosphatase was undiluted. An additional experiment was also performed using Gomori*s (21) modification of Seligman's (59) technique. This modifica­ tion avoids the time consuming extraction with ethyl acetate by keeping the azo-dye in a clear colloidal solution by use of sodium lauryl sulfate. In addition, sodium lauryl sulfate had the added advantage of stopping the enzymatic reaction. The transmission used In this case was 5&5 rou. C. Results and Discussion As noted in Tables IX and XI little or no activity in saliva was detected using Seligitian*s technique with the ex­ ception of alkaline phosphatase of one resistant male. On the other hand, Tables X and XII show that a high degree of activity was produced in the broth cultures although there was no differ­ ence between resistant and susceptible animals. 28 TABLE IX ALKALINE PHOSPHATASE ACTIVITY OP SALIVA (diluted 1-5) OP CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION (Method of Seligman) Resistant Males Resistant Females Susceptible Males Susceptible Females 1 27.0 89,2 87.0 100.0 2 92.8 98.5 98.8 100.0 Trial No. TABLE X ALKALINE PHOSPHATASE ACTIVITY OF BROTH CULTURES OP ORAL MICROFLORA OP CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION (Method of Seligman) Trial No. Resistant Males Resistant Females Susceptible Males Susceptible Females 1 19.3 22.3 27.7 21.6 2 10.8 17.8 19.6 14.5 Average 15.2 19.1 23.6 18.1 Note: Average uninoculated broth control = 76.2 29 TABLE X I ACID PHOSPHATASE ACTIVITY OF SALIVA (diluted 1-5) OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION (Method of Seligman) Resistant Males Resistant Female s Susceptible Males Susceptible Females 1 99.0 99.3 99.5 100.0 2 99.3 99.5 100.0 100.0 Trial No* TABLE XII ACID PHOSPHATASE ACTIVITY OF BROTH CULTURES (diluted 1-5) OF ORAL MICROFLORA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION (Method of Seligman) Trial No. Resistant Males Resistant Females Susceptible Males Susceptible Females 1 9.8 6.3 h- 8.0 2 15.3 5*k 11.5 26.0 Average 12.6 5.9 13.0 17.0 Note: Average uninoculated broth control = 95 30 In the second experiment using G o m o r i ^ modification, extreme variability in activity by saliva from resistant and susceptible animals was noted as shown in Tables XIII and XIV* Determinations for acid and alkaline phosphatase were carried out simultaneously on the same sample of saliva and when one result varied, the other varied in exactly the same manner• Two puzzling points arise as a result of these experi­ ments* These are the failure to demonstrate the presence of both acid and alkaline phosphatase in saliva in the first experiment, with the exception of one animal, and the extreme variation obtained in the second experiment using Gomori!s modification. Gomori (21) determined the esterase content of human blood serum and several organ extracts but did not examine saliva for enzyme activity. Since rat saliva varies in tur­ bidity, this might have affected the final transmission readings on the colorimeter. However, as far as the naked eye could detect, the colloidal solutions obtained as a re­ sult of the detergent-dye complex were crystal clear. The most likely explanation of the absence of the phos­ phatases in the first experiment and their variability in the second experiment, lies in the nature of saliva. Saliva is an extremely complex fluid differing in composition from day to day and even depending on the time of day. It seems 31 TABLE XIII ALKALINE PHOSPHATASE ACTIVITY OP SALIVA (diluted 1-5) OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION (Method of Goraori) Trial No* R®sistant Males Resistant Females 1 50*14. 5.3 2 80.8 73.0 Susceptible Males .9 83 .ij. Susceptible Females 32.3 23.7 TABLE XIV ACID PHOSPHATASE ACTIVITY OF SALIVA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION (Method of Gomori) m . N iriai imo* Resistant Males Resistant Females Susceptible Males Susceptible Females X 59.0 23.0 7.8 72.1 2 79.0 62.5 76.0 2^.5 26.2 " 3 . " 32 logical that the composition of saliva would depend largely on when the rat last ingested food and drank water# In addition, bacteria, introduced with the saliva, may elaborate enzymes. Therefore, the phosphatase content of saliva might depend on the type and quantity of bacteria present in the saliva-substrate mixture. It has already been shown that both acid and alkaline phosphatase were pro­ duced by the oral microflora. It is not known, however, whether the enzyme was a result of lysing of bacterial cells or was actually a product of intact cells. 33 PROTEASE A* Survey of Literature The early literature concerning proteolytic activity of saliva was mainly of a descriptive nature. As early as 1911* Warfield (710 noted a substance in saliva which had the power to split glycyl-tryptophan. Willstatter, Bamen and Rhodewald (76) reported that the salivary glands of man, the dog, horse and pig, possessed proteinases and peptidases. The proteinases consisted of activated trypsin and cathepsin. They concluded that in hu­ man saliva, the proteolytic action was due to the presence of epithelial cells, leukocytes, and bacteria in suspension although some activity did exist in the supernatant fluid of centrifuged saliva* Voss (73) provided evidence of a trypsin-like enzyme in parotid saliva with an optimum at pH 8, but found no activity in sublingual or submaxillary saliva. In a demonstration of the proteolytic enzymes of whole saliva, Chauncey, Johnson and Lisanti (8), found activity at acid and alkaline pH levels. The active principle was shown to be associated with the debris normally present in saliva* In addition, a kinase activated proteolytic enzyme 34 was found in the supernatant fluid of centrifuged whole saliva which appeared similar to the serum protease, plas­ minogen. Collagen&ses have been demonstrated by several investi­ gators in the peridontal pocket (56)* and in saliva (66). Sreebny (67) characterized a proteolytic enzyme in the submaxillary gland of the white rat which acted on casein and azocoll. The optimum pH for casein breakdown was 9*6. In a subsequent Investigation (68) he found that submaxillary glands of male rats had a higher degree of proteolytic activity than those of female rats* The difference in enzymatic activity appeared at the onset of puberty and Increased steadily as the animal grew older. He concluded that the proteolytic activity of the submaxillary gland was under hormonal control. In still another experiment, Sreebny (69) found that submaxil­ lary glands of caries-susceptible male rats of the Hunt-Hoppert strain had approximately five times as much activity as those of resistant males. A relationship between proteolytic activity and caries susceptibility was demonstrated by Weinman (75) who claimed that saliva from caries-resistant individuals had more of an autoproteolytic action than that from caries-susceptible in­ dividuals. He stated that the enzymatic activity originated with the leukocytes. 35 B. Methods and Materials The preliminary experiments for the determination of protease were carried out using the casein-digestion method of Kunitz « It is based on the principle that the enzy­ matic breakdown of casein will release acid—soluble products such as tyrosine* The unreacted casein is precipitated with trichloroacetic acid and the precipitate filtered or centri­ fuged* The supernatant liquid may be measured either directly with a spectrophotometer at 280 mu or colorIraetrically at 600 mu using Polinfs phenol reagent* Little or no protease activity was detected by this method in rat saliva or in human saliva. Investigations were undertaken to modify the technique so as to prove conclusively the presence or absence of the enzyme* Since Sreebny (67) had found that the maximum protease activity of rat submax­ illary gland extracts occurred at pH 9*6, a borate buffer at this pH was substituted for the phosphate buffer at pH 7*6 in the original procedure of Kunitz. As protease activity in saliva was evidently of a low order, it was thought feasible to increase the incubation period of the saliva-substrate mixture. An experiment was set up to determine the optimum time of incubation. Activity was measured at various time Intervals in human and rat saliva using trypsin as a control. It was determined that at least four hours Incubation was necessary In order to obtain a measureable amount of protease 36 activity. Junquiera et al (33) had previously used the same incubation period to demonstrate protease activity in the submaxillary glands of rats. Therefore, a four hour incuba­ tion period was used for all subsequent determinations. The following procedure was finally adopted for the determination of protease activity in rat saliva. One percent casein solution was prepared by dissolving Hammersten casein in the borate buffer. The solution was then heated in a water bath at 95-98 C to destroy any enzymes present in the casein. This solution was stable for at least three weeks when kept at L|_ C. Equal amounts of saliva and substrate, usually one ml, were mixed and incubated for four hours at 37 C. A one ml portion of the mixture was withdrawn immediately after mixing and added to 3 ml of five percent trichloroacetic acid for the zero time reading. The precipitate formed was fil­ tered through #lpO filter paper after standing one hour or longer at room temperature. At the end of the incubation period, three ml of five percent trichloroacetic acid was added to the remaining milliliter of saliva-substrate mixture, and the precipitate formed was filtered after standing at room temperature for an hour or longer. The concentration of split products in the supernatant solution, measured as ug of tyrosine liberated per ml of saliva, was determined colorimetrically at 600 mu by Herriot's method using a Beckman model B spectrophotometer, a Hellige colorimeter 37 or a Bauech and Lomb "Spectronic 20" colorimeter* Sine© most of the data were obtained from the latter two instruments, standard curves were constructed for only these colorimeters using various dilutions of pure L-tyrosine to calculate the ug of tyrosine liberated by enzymatic action* Controls using casein and buffer only were included in each experimental trial to eliminate the possibility of non-enzymatic breakdown of the substrate* Saliva controls, i.e., saliva incubated with buffer rather than substrate, exhibited slight autoproteolytic activity as shown in Table XV. It was felt, however, that in the presence of the large amount of casein substrate, the breakdown of salivary protein would b© small and have little effect on the activity values* C* Results and Discussion The results of the first experiment involving protease activity in saliva are listed in Tables XVI and XVII. The experiment was originally only part of the preliminary screening phase, but was enlarged to include more animals when differences between resistant and susceptible animals were detected. This group of animals was 88-125 days of age. The average amount of tyrosine liberated per ml of saliva by enzymatic action from saliva of resistant males was 58.2 + 9 »3 US» whereas the average value for saliva from susceptible males was 178.2 + 18.1 ug. Thus, the susceptible males 38 TABLE XV AUTOPROTEOLYTIC ACTIVITY OF SALIVA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Strain and Sex Saliva + Substrate Saliva + Buffer Resistant male 8I|..0 100.0 Resistant male 73.8 100.0 Resistant female 87.8 90.8 Resistant female 78.8 82.0 Resistant female 82.0 6i|..8 Susceptible male 51.3 89.5 Susceptible male 60.0 9^.0 Susceptible female 6l.lj. 86.2 Susceptible female 77.8 83.4 39 TABLE X V I PROTEASE ACTIVITY OF SALIVA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS 88-125 DAYS OF AGE EXPRESSED AS ug OF TYROSINE LIBERATED /ML OF SALIVA Trial No* Resistant Male s Resistant Females Susceptible Males Susceptible Females 1 1 2 .0 3 8.0 2 1 2 .0 9 6 .0 2 31;. o 9 6 .0 2 5 8 .0 144.0 3 92.0 5 8 .0 1 2 2 .0 1 9 2 .0 k 8 6 .0 3 3.0 9 8 .0 1 9 6 .0 5 4 2 .0 28.0 2 6 2 .0 1 5 6 .0 6 1 0 0 .0 4 0 .0 1 3 8 .0 1 1 8 .0 7 4 0 .0 6 2 .0 144.0 124.. 0 8 5 2 .0 6 6 .0 1 8 0 .0 6 8 .0 9 4 2 .0 4 8 .0 14.8 . 0 2 0 8 .0 10 8 2 .0 67.0 2 2 0 .0 24..0 m* 11 Average and Standard Error 5 8 .2 + 9 .3 — — 53*6+6.5 1 7 8 . 2 + 1 8 .1 6 4 .O 1 2 6 . 4 + 1 8 .0 TABLE XVII SIGNIFICANCE OF RESULTS ON PROTEASE ACTIVITY 88-125 days OF AGE OBTAINED FROM RATS Level of Significance (percent) Comparison t value So* with R ^ 5.9 < .1 S$ 3*8 < *1 with R $ R <5* wi th R %■ with S $ •36 2*0 None 5 1*0 showed an activity approximately three times that of resis­ tant males* The t value for this comparison was 5.9, which was significant at the < . 1 percent level. Similarly, a smaller difference was obtained when comparing susceptible to resistant females. The susceptible females showed approxi­ mately two and a half times the activity of resistant females. The average value for susceptible females was 126 £ 18.0, whereas the average value for resistant females was 53-6 + 6.5. The t value for this comparison was 3*8. which is significant at the < .1 percent level. Susceptible males showed greater activity than females, but there were no dif­ ferences between sexes of resistant animals. The second experiment was used to verify the results of the first. Previously untested rats of 125-11*0 days of age were used in this phase. The choice of age group had no particular significance at the time they were tested. The results as listed in Tables XVIII and XIX again brought out significant differences between resistant and susceptible strains of both sexes. As in the previous experiment, there was also approximately a threefold difference between re­ sistant and susceptible males. The average values were 95.0 + 16.8 for resistant males and for susceptible males 21*2.3 + 21*.3. The t value for this comparison was 5.0 which was significant at the < .1 percent level. Resistant females exhibited a value of 86.3 £ 16.7 as compared to susceptible ll.l TABLE XVIII PROTEASE ACTIVITY OP SALIVA OP CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS 125-H|-0 DAYS OF AGE EXPRESSED AS ug OP TYROSINE LIBERATED / ML OF SALIVA Resistant Male s Trial No# Resistant Females Susceptible Males Susceptible Females 1 124.0 72.0 270.0 194.0 2 142.0 82.0 140.0 100.0 3 30.0 72.0 284.0 100.0 k 142 .O 126.0 I64 .O 108.0 5 144.0 162.0 196.0 214.0 6 62.0 28.0 3kb*o 308.0 7 50.0 62.0 268.0 336.0 8 66.0 — 272.0 14.16•0 242.2+24.3 220.0+42.6 Average and Standard Error 95.0+16.8 86.3+16.7 TABLE XIX SIGNIFICANCE OP RESULTS ON PROTEASE ACTIVITY OBTAINED FROM RATS 125-lij.O DAYS OF AGE Comparison S& with R ^ S-£ R with R-? with R ¥■ with S $ t value Le ve 1 of Significance (percent) 5.0 3.0 .37 -1 1. None J-l-1 None 1+2 females with, a value of 220.0 ♦ 42 .6 , and gave a t value 3«0, which was significant at the 1 percent level. Sig­ nificant differences, however, were not obtained between sexes of the same strain* It should be pointed out that the average value for the 125-11+0 day old rats was higher in all cases than those obtained with 88-125 day old rats sug^ gesting an increase of activity with age. As a result of the previous experiments, it was decided to investigate the age level at which differences in protease activity appeared. An experiment was designed to determine protease activity at the pre-caries level. selected were from 1+9-55 days of age. The animals This experiment was extended to measure the enzyme activity in the saliva of the same rats at a later age. Prom the results in Tables XX and XXI, it can be seen that even with rats only 49-55 days old, the salivary protease activity for susceptible males was approximately one and a half times that of resistant males. The t value for this comparison was 3.7, which was significant at the ^ .1 percent level. Too much emphasis should not be placed on this dif­ ference as the average values obtained for protease activity were low, and resistant females showed almost as much activity as susceptible males, whereas in all other experiments there always was a wide difference between these two groups. Sig­ nificant differences were not obtained between susceptible i+3 TABLE XX ACTIV*TY OP SALIVA OP CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS ij.9-55 DAYS OP AGE EXPRESSED AS ug OF TYROSINE LIBERATED /ML OF SALIVA Trial No# R 0 3tstant Males Resistant Females 1 2 0 .0 268.0 5 2 .0 81+.0 2 2 8 .0 2 6 .0 12 I4..O 6 8 .0 3 I4.6 .O U4.O.O 8 0 .0 5 0 .0 k 2 8 .0 l(-3 . 0 8 0 .0 6 I4.O 5 514-. 0 3 8 .0 7 6 .0 614..0 6 14.6 . 0 I4.2 .O 514-.0 72.0 7 14.8 . 0 6 6 .0 7 2.0 0 .0 8 514..0 3 2 .0 I4.O.O 70.0 9 5 8 .0 1+1+. 0 I4.6 .O 8 2 .0 10 I4.2 .O 5 0 .0 3 6 .0 1514-. 0 11 14.2 . 0 1U4-.0 8 1 .0 0 .0 12 I4.2 .O 1+8 .0 6 0 .0 5 5 .0 13 M4-*0 3 8 .0 51+.0 6 8 .0 14 2 8 .0 1(.2 .0 76.0 14.8 .0 13 — 5 6 .0 — — 16 __ i).2 .0 —— -- Average and Standard Error ij.l#!i>3#0 Susceptible Males Susceptible Females 63*7*15.1 66#5+6#l 62#8+9#9 TABLE XXI SIGNIFICANCE OP RESULTS ON PROTEASE ACTIVITY OBTAINED FROM RATS R9-55 DAYS OP AGE Comparison Serf S$ Rcr? S d* with. with with with t value__________ Significance (percent) R<5^ R£ R$ S^ 3*7 *08 1.5 *32 *1 None None None hh and resistant females* In addition, there were no differences between sexes of the same strain in this experiment* The rats used in the previous experiment were tested again for salivary protease activity when they were 88-92 days of age* Tables XXII and XXIII show that resistant males had an average value of $ 6 £ l(.«7 as compared with 178*7 £ llf.*5 for susceptible males. Significant differences were also obtained between susceptible and resistant females. Values obtained are $3*3 £ resistants as compared to lll(..5 £ 17*5 for the susceptibles* Significant differ­ ences were not found between sexes of resistant animals. However, there was a difference in activity between male and female susceptible animals. was 178.7 £ 1 111^.5 £ 17*5* The value for susceptible males whereas that for susceptible females was The occurrence of a difference in activity between sexes of susceptible animals confirmed the results In the 88-12$ day-old group. It should be noted in Table XXII that the average pro­ tease activity of susceptible rats of the 88-92 day old group increased about two to three fold over the ij.9-55 day old animals (Table XX). In the same period of time there were only slight changes in the proteolytic activity of the re­ sistant animals. In addition to the main investigation which noted dif­ ferences between resistant and susceptible animals, several U5 TABLE X X I I PROTEASE ACTIVITY OP SALIVA OF CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS 88-92 DAYS OF AGE EXPRESSED AS ug OF TYROSINE LIBERATED /ML OF SALIVA Trial No. Resistant Males 1 6 2 .0 7 0.0 251+. 0 2 3 6 .0 2 1+7.0 7 6.0 2 6 8 .0 2 0 6 .0 3 8 0 .0 7 6.0 1 3 2 .0 1 8 6 .0 k 3^. 0 51+.0 1 3 6 .0 1 3 2 .0 5 1+2 . 0 3 8 .0 161+.0 6 0 .0 6 8 1 .0 1+7.0 1 9 2 .0 1+9 . 0 7 i+JU-.o 1+8 . 0 9 6 .0 1 0 0 .0 8 3 8 .0 31+.0 11+0 . 0 1 3 0 .0 9 7 6 .0 l+l+.o 1 7 6 .0 1 0 0 .0 10 6 2 .0 3 8 .0 2 8 0 .0 5 6 .0 11 5 2 .0 1+0 . 0 2 2 0 .0 6 6 .0 12 51+ .0 1+2 .0 196.0 1+0.0 13 “ 2 8 .0 1 3 6 .0 1 2 8 .0 Hi- — 9 6 .0 1 2 0 .0 — 15 — 1+8 .0 170.0 — 5 3 . 3 +1+.8 178.7+11+.? Average and Standard Error 5 6 .0 il+ .7 Resistant Females Susceptible Males Susceptible Females 1H+.5+17.5 TABLE XXIII SIGNIFICANCE OF RESULTS ON PROTEASE ACTIVITY OBTAINED FROM RATS 8 8 - 92 DAYS OF AGE t value Comparison with with Rtf’1 with with R67 R? R? S2 8 .1 3 .^ .IfO 2 .8 Level of Significance (percent) 2L .1 .1 None 1. 2+6 experiments were carried out in an attempt to establish the source or protease activity in saliva* The enzyme may be con­ tained in saliva per s c . bacteria, leukocytes, epithelial cells, or secretions other than saliva that may have been collected with the sample. An attempt to determine the protease activity of the oral microflora was made using the technique previously des­ cribed under general methods and materials. broth of the following composition was used: A casein-lactose casein - 1 percent, lactose - .5 percent, NaCl - .5 percent. activity was obtained as noted in Table XXIV. Negligible However, sine© casein precipitated in the tubes due to acid production, it was possible that protease activity was inhibited by the low pH. It was thought that filtration of the saliva might pro­ vide a clue to the source of the enzyme. The filtration was accomplished by attaching a Swinney filter to a five ml syringe. Individual samples of saliva were filtered and com­ pared with unfiltered samples from the same animal. The data In Table XXV demonstrated that protease activity was reduced In all cases ranging from 10-9^ percent. In comparison, trypsin activity was reduced 100 percent by passage through the filter. The loss of protease activity could have been due to loss of cellular debris and/or the absorption of the enzyme by the filter pad. The results of this experiment, although inconclusive, suggest that at least part of the protease activity may be inherent in the saliva* k7 TABLE XXIV PROTEASE ACTIVITY OP BRQTH-CULTURES OF ORAL MICROPLORA OP CARIES-RESISTANT AND CARIES-SUSCEPTIBLE RATS EXPRESSED AS ug OF TYROSINE LIBERATED /ML SALIVA Trial No. Resistant Males 1 16 8 28 2 0 32 0 Resistant Females Susceptible Males Susceptible Females 36.0 k TABLE XXV EFFECT OF FILTERING- ON TRYPSIN ACTIVITY AND ON PROTEASE ACTIVITY OF SALIVA FROM MALE SUSCEPTIBLE RATS EXPRESSED AS ug-OF TYROSINE LIBERATED /ML OF SALIVA Unfiltered Saliva Filtered .Saliva Unfiltered Percent Reduction Trypsin in Activity 1 276.0 214-8.0 10 S 8I4..O 2 314-8*0 20.0 9b 70I4..O 3 228.0 200.0 12 k 2I|.0.0 138.0 27 5 260.0 178.0 32 Filtered Trypsin Percent Reduction in Act! vity 6.0 99 0 • 0 Trial No. 100 U8 A more critical experiment to detect the source of the enzyme was carried out with the assistance of Dr. Leo M. Sreebny of the University of Illinois Dental School, The submaxillary gland was cannulated and saliva obtained by stimulation from this source was compared with saliva ob­ tained by stimulation from the same animal collected via the oral cavity by methods previously described. The protease activity, expressed as ug of tyrosine liberated/ml of saliva was 198,0 for the sample obtained via the oral cavity as compared to 108.0 for the sample collected by cannulation. Although only one trial was performed, it indicated that at least a fraction of protease activity is inherent in saliva. The results also indicate that protease activity must be contained in salivary glands other than the submaxillary and/ or cellular debris. An attempt was also made to detect protease activity in the blood serum of male susceptible rats. Activity was tested at pH values of 7.6 and 9.6 by methods previously described, but none was detected. The report of Chauneey, Johnson, and Lisanti (8) that a kinase activated protease was present in centrifuged whole saliva led to the thought that protease activity in rat saliva might possibly be increased by the addition of an activator. Two activating agents were tested: cysteine at a concentration of .025M, and streptokinase at a concentration k9 of 500 units/ml. The Incubation period for enzyme action was shortened to one hour* As can be seen in Table XXVIf the sample with added cysteine exhibited slightly greater activity, whereas addition of streptokinase had no effect* 50 TABLE XXVI EFFECT OF ACTIVATORS ON PROTEASE ACTIVITY IN SALIVA FROM MALE SUSCEPTIBLE RATS EXPRESSED AS PERCENT TRANSMISSION Activator Tested Percent Transmission Control No Cysteine 88*5 Cysteine 71*..5 Control No Streptokinase 89.0 Streptokinase 87.0 51 GENERAL DISCUSSION On© of the primary objects of this work was to detect possible differences in salivary and microbial enzyme ac­ tivity between caries-resistant and caries-susceptible rats* Among the enzymes studied for possible differences, salivary protease showed the greatest potentiality• This does not preclude the possibility that significant differences might be found with other enzymes such as aryl-sulfatase or the phosphatases provided the experimental techniques are im­ proved in sensitivity and accuracy, and more animals tested* This is especially true in the case of the phosphatases where large variations were noted* One reason for the present investigation is that Sreebny (68) had previously shown that submaxillary gland extracts of Hunt-Hoppert susceptible male rats had five and one-half times more protease activity than those of resistant males* The results of the present work extend this observation not only by showing that similar differences exist in the saliva, but that significant differences also occur between resistant and susceptible females* Several interesting parallels may be drawn to an earlier investigation carried out by Sreebny (69) comparing submaxil­ lary protease activity between sexes of Sprague-uawley rats* 52 He noted that males exhibited more activity than females, but that these differences did not occur until after puberty and that activity increased with age* In this investigation, susceptible males also exhibited more activity* Moreover, the protease activity was slight until after puberty and activity increased with age. The differences in protease activity between susceptible and resistant rats should be considered from two aspects* Why do they exist, and what is their relationship, if any, to caries? A possible correlation between protease activity and caries is suggested since the average caries time and the appearances of these differences approximate each other rather closely* If a relationship does exist, one must speculate as to which is cause and which is effect* The data suggest that protease activity may not contri­ bute too much to the development of caries. The average caries time for susceptible animals is about 75 days* this is the time at which gross lesions in tected. In reality, the However, the teeth are de­ carious lesion starts much earlier. If protease activity is a major 1 actor in the cause of caries, the differences obtained at the i|9-55 day age level would have been greater in the case of the males and also would have appeared in the females. On the contrary, the values ob­ tained did not show differences of any magnitude until the animals were about 90 days old. Intervening examinations 53 were not made* By the time the animals were 90 days old, the susceptibles had already developed caries* These facts, however, do not preclude the possibility that protease may accelerate the carious process since protease activity in­ creases sharply as susceptible rats develop caries* There are several ways that protease may accelerate caries* The enzyme may possibly, but not likely, act on the small portion of protein in the enamel to initiate a carious lesion* Protease may also attack dentinal protein after the initial lesion has been established. It has been observed in the Hunt-Hoppert rats that caries develop rapidly in sus­ ceptible animals after the Initial lesion, whereas, caries develops slowly in resistants after the initial lesion* Another way in which protease may accelerate caries is in the break­ down of food material In the mouth to provide amino acids for cariogenic bacteria. Bernarde (6) observed that the saliva of susceptible animals contained three to four times as many leukocytes as resistant animals. It has been established that leukocytes possess some proteolytic activity (75)* The question arises as to whether some of the protease activity originates from these leukocytes since susceptible animals showed three times as much protease activity as resistant animals. This correla­ tion suggests that leukocytes in saliva may contribute to the protease content* 5k The association between susceptibility to caries and high protease content of the saliva does not prove that the latter is one of the causes of the former* Both processes might be effects of the same gene or a gene for high pro­ tease might be closely linked (on the same chromosome) with a gene for caries suceptibility. The lines of rats under investigation are highly inbred. One of the important effects of inbreeding is to render the stock more and more homozygous for all genes. depend However, the alleles which become homozygous on those which were present in the foundation stock and chance unions of gametes. Thus,the high protease con­ tent of the saliva of susceptibles does not prove that the protease is one of the causes of susceptibility. 55 SUMMARY A screening study was made to compare various enzymatic activities of the saliva and of the oral microflora of the Hunt—Hoppert caries—resistant and caries— susceptible rats* Acid and alkaline phosphatases and aryl—sulfatase were detected in saliva and in broth cultures of oral microflora* Lysozyme and protease were found in the saliva, but not from the oral microflora, whereas hyaluronidase was shown to be of bacterial origin* Urease was not detected from either source• Differences between the two strains were found only in three of the enzymes studied. Resistant animals possessed slightly more aryl-sulfatase of both salivary and microbial origin than susceptible animals* Extremely variable results were obtained for acid and alkaline phosphatases of saliva* The data obtained for salivary protease showed the widest dif­ ferences between resistant and susceptible animals, and further experiments involving this enzyme were carried out* In mature animals, the saliva of susceptible males dis­ played about three times as much protease activity as that of resistant males* Mature susceptible females showed approxi­ mately two and one-half times the activity of resistant female s * 56 Susceptible males exhibited about one and one-half times as much activity as susceptible females in rats 88-125 days old. However, these differences were not observed in rats prior to puberty or in rats older than 125 days. Activity increased sharply with age in the case of sus­ ceptible animals, whereas much smaller changes were found in resistants of comparable ages. An experiment was conducted to determine whether protease was inherent in saliva or was produced by the cellular material obtained with the collection of saliva. Cannulation of the submaxillary gland and subsequent detection of protease ac­ tivity indicated that saliva contributed part of the activity. Further evidence that protease was contained in saliva was that activity was retained in saliva after filtration. The possible relationship of differences in protease activity to caries was discussed. 58 12. Demuth, Fritz; Uber Phosphatstoffwechsel, I. Uber Hexasephosph&tasen im Menschliehen Organen und Korper flussigkeiten; Biochem. Z.; 159: 1+15; (1925). 13. Dentay, J. T. and J. J. Rae; Phosphatase in Saliva; J. Dent. Res.; 28: 68; (191+9). llw Bggers-Lura, H . ; Investigations on the Salivary Phos­ phates and Phosphatases; J. Dent* Res.; 26: 203; (191+7). 15. Engel, Milton B*; The Softening and Solution of the Dentin in Caries; J. Dent. Res.; 28: 61+6; (191+9). 16. Fitzgerald, Robert J.; Preliminary Studies on the Presence of Acid Phosphatase In Certain Oral Micro­ organisms; J. Dent. Res.; 31: 189; (1952). 17. Fosdick, Leonard S., Harold L. Hansen, and George D. Wessinger; The Reductase Activity of Various Mouth Organisms; J. A. D. A.; 2l+: 11+1+5; (1937). 18 . 19. Fosdick, L. S.; Carbohydrate Degradation by Mouth Micro­ organisms; J. A. D. A.; 26: 1+15; (1939) • Giri, K. V.; (1936). 20 . Glock, and 21 G.; . Gomori, 1+2: 1+1+5; 22 . Salivary Phosphatase; Biochem. Z. 285: 306; G. E., M. M. Murray, and P. Pincus; The Origin Significance of Salivary Phosphatase; Biochem. J. 32: 2096; (1938). Human Esterases; J. Lab* and Clinical Med.; (1953). Gomori, G.; Methods for Quantitative Estimation of Hydrolytic Enzymes in Serum and Other Biologic Fluids; Am. J. Clin. Path.; 2l+: 99; (1951+) - 23. Hadidian, Z., and N. W. Pirie; The Preparation and Some Properties of Hyaluronic Acid from Human Umbilical Cord; Biochem. J. 1+2: 260; (191+8). 21+• Hawk, Phillip, Bernard Oser, and William Summerson; Practical Physiological Chemistry; 12th ed; Blakiston Company, New York; p. 830; (I95l). 25. Helman, Edith Z., and David F. Mitchell; Phosphatase In Human Saliva: Its Relationship to Calculus and Lactobacillus Counts; J. Dent. Res.; 33: 385; (195U- 57 BIBLIOGRAPHY 1# Adamson, Kenneth T.; The Role of Enzyme Action in the Formation of Dental Calculi; Australian J. of Exptl. Biol, and Med. Sci. 6: 215; (1929)* 2. Ballantyne, R. M . , C. T. Clegg, J. J. Rae; and F. H. Lawford; Ammonia Production in Saliva; J. Dent. Res.; 30: 385; (1951). 3. Ballantyne, R* M. , J. J. Rae, and F. H* Lawford; Ammonia Production and Urease Activity; J. Dent. Res. 31: 281; (1952). 4. Barber, Mary, B. W. L. Brookbank, and I. W. A. Kuper; Staphlococcal Phosphatase, Glucuronidase and Sulfatase; J. Path, and Bact.; 63 : 57; (1951). 5. Benarde, Melvin A., F. W. Fabian, and S. Rosen; A Method for the Collection of Large Amounts of Rat Saliva; (Submitted for Publication). 6. Benarde, Melvin A.; A Comparative Study of the Saliva and of the Blood of the Caries-Susceptible and CariesResistant Strains of Hunt-Hoppert Rats; Ph. D. Thesis, Michigan State College; (1954); 97 numb, leaves. 7. Candeli, A. and A. Tioneri; Bacterial Sulfatase and Dental Caries; Boll. Soc. Ital. Biol. Sper.; 27: 651; (1951). Chem. Abst. 46: 6l85c. 8. Chauncey, H. H., V. M. Johnson, and V. F. Lisanti; Proteolytic Enzymes of Human Saliva; J. Dent. Res.; 33 : 652; (1951+). 9. Chauncey, H. H., Fabian Lionetti, Richard A. Winer, and Vincent F. Lisanti; Enzymes of Human Saliva; J. Dent. Res.; 33*- 321; (195U). 10. Deakins, M.; Effect of Storage Temperature on Precipitable Protein Content and Catalase Content of Whole Saliva; J. Dent. Res.; 20: 129; (194D* 11. Deane, H. W.; Cytochemical Survey of Phosphatases in Mammalian Liver, Pancreas, and Salivary Glands; Am. J. Anat.; 80: 321; (1947). 59 26. Herriot, R. M.; Reaction of Folin's Phenol Reagent with Proteins and Biuret Compounds in the Presence of Cupric Ion; Proc. Soc. Exptl. Biol.; 46: 31+2; (191+1). 27. Hunt, H. R* and C. A. Hoppert; Genetics; 2i+: 76; (1939). 28 . Inheritance in Rat Caries; Hunt, H. R. and C. A. Hoppert; Inheritance in Rat Caries; Year Book of the American Philosophical Society 1315 (191+1 ). 29. Hunt, H. R. and C. A. Hoppert; Inheritance of Suscepti­ bility and Resistance to Caries in Albino Rats (Mus norvegicus); J. Am. Col. Dent.; H i 33; (191+1+). 30. Hunt, H. R., C. A. Hoppert, and Braunschneider, G. E.; Heredity and Environmental Factors Influencing Tooth Decay in the Albino Rat (Rattus norvegicus)j Records Genetics Soc. Am. 16: 37; (191+7). 31. Hunt, H. R., C. A. Hoppert, and W. G. Erwin; Inheritance of Susceptibility to Caries in Albino Rats (Mus norve­ gicus); J. Dent. Res.; 23* 385; (191+14-) • 32. Hunt, H. R., and C. A. Hoppert. Occlusion as a Factor in Dental Caries of Albino Rats (Rattus norvegicus); J. Dent. Res.; 27: 553; (191+8). 33. Junquiera, L. C., A. Fajer, M. Rabinovitch, and L. Franken thal; Biochemical and Histochemical Observations on the Sexual Dimorphism of Mice Submaxillary Glands; J. Cell, and Comp. Physiol.; 3l+: 129; (191+9). 3k-• Kesel, Robert G., Joseph F. 0*Donnel, and Ernst R. Kirch; Deamination of Amino Acids by the Human Oral Flora; Science; 105* 230; (191+5). 35. Knox, K. W. and J. L. Still; Observations on the Salivary Mucolytic Enzymes; J. Dent. Res.; 32: 367; (1953). 36. Knox, K. W. and J. L. Still; Observations on the Action of Mucolytic Enzymes on Salivary Mucoid; J. Dent. Res.; 32 : 371+; (1953). 37. Kroncke, Adolph and R. Nsu joks; Die Speichel phosphatasen und ihre Beziehung zur Zahnkaries; Deut. Zahnarzt.; 8, Suppl.: 71; (1953). 38. Lisanti, Vincent F.; Hyaluronidase in Human Saliva; J. Dent. Res*; 29* 392; (1950). 60 39. MacDonald, C. Franklin, Jr., and H. Carlton Smith; Oxidase in Saliva; J# Allied Societies; I12 346; (1909). Lj-0# Mahler, I, R., and V. F. Lisanti; Hyaluronidase Producing Microogranisms from Human Saliva; Oral, Surg., Oral Med., and Oral Path.; 5: 1235; (1952). 41. Moisman, Walter, and James B. Sumner; Salivary Peroxidase; Arch. Biochem. and Biophys.; 33: 4^7; (1951). 1+2. Nernes, J. L . , and M. G. Wheatcraft; Action of Salivary Lysozyme on Micrococcus lysodeikticus; Oral Surg., Oral Med., and Oral Path.; 5s 653; Tl952). 4-3. Noback, C. R. and W. Montagna; Histochemical Studies of Basophilia Li pase and Phosphatase in the Mammalian Pancreas and Salivary Glands; Am. J. Anat.; 81: 31+3> (191+7). J4I*. Northrup, John H., M. Kunitz, and Roger Herriot; Crystalline Enzymes, 2nd Ed.; Columbia University Press, New York; p. 309; (1948). 45. Pigman, Ward, and A. Jane Reid; Organic Compounds and Enzymes of Human Saliva; J. A, D. A.; 1+5* 325; (1952). 1+6 . Pincus, Paul; The Reducing Properties of Saliva; Brit. Dent. J. 72: 181; (19l+2>. 47. Pincus, Paul; Relation of Enamel Protein to Dental Caries; Nature; l6l: 1014; (1946). 1+3« Pincus, P.; Sulfated Mucopolysaccharides in Human Dentin; Nature; 166: 187; (1950J. i+9- Pincus, P.; Sulphatase in Dental Caries; Experimental Med. and Surg. 8: 309; (1950). 50. Prinz, Herman; Relationship of Oral Secretions to Dental Caries: III. Enzymes of Human Saliva Other Than Amylase; Dental Cosmos; 60: 287; (1918). 51. Rapp, Gustave W . ; The Biochemistry of Oral Calculus: II. The Presence of Carbonic Anhydrase in Human Saliva; J. A. D. A.; 33-* 191; (1946), 61 52. Roberts* E. B., B* G. M&egraith, and H. W. Florey; A Comparison of Lysozyme Preparations from Egg White, Cat and Human Saliva; <4uart. J. Exper, Physiol.; 27: 381 ; (1936). 53. Rogers, H. J.; Bacterial Hydrolysis and Utilization of Polysaccharide Like Substances, (Mucin), in Saliva: Nature; 16X: 815; (1948). 514-. Rosen, S., M. A. Benarde, H. R. Hunt, and C. A* Hoppert; Microbiologic Differences in the Oral Cavities of the Hunt-Hoppert Caries-Resistant and Caries-Susceptible Rats; J. Dent. Res.; 34: 113; (1955). 55. Rosen, S., M. A. Bernard©, F. W. Fabian, H. R. Hunt, and C. A. Hoppert; Several Properties of Saliva from Hunt-Hoppert Caries-Resistant and Caries-Susceptible Rats; (Submitted for Publication). 56. Roth, G. D., and H. I. Meyers; Identification of a Pro­ teolytic Enzyme of the Peridental Pocket; J. Dent. Res.; 31: 18; (1952). 57. Rudino, Ivaldo; Lysozyme in Saliva; Hass. moda Sarda; $1 67; (1954). Chem. Abs. 48 : 13938 g. 5 8. Rutenberg, A. M., R. B. Cohen, and A. H. Seligman; Histochemical Demonstration of Aryl-Sulfatase; Science; 116: 539; (1952). 59. Seligman, Arnold M., Howard H. Chauncey, Marvin M. Nachlas, Leon H. Manheimer, and Herbert A. Ravin; The Colorimetric Determination of Phosphatases in Human Serum; J. Biol. Chem.; 190: 7; (1951). 60 • Simmons, N. S.; The Existance of a Parotid Salivary Mucinose; J. Dent. Res.; 20: 255; (1941). 61. Simmons, N. S.; Studies on the Defense Mechanisms of the Mouth; J. Dent. Res.; 30: 494; (195l). 62 . 63. Skrotskii, E. B., L. I. Makhlinovskii, and M. M. Slutskaia; Lysozyme and Inhibin in Human Saliva; Annaly. Mechnikovskogo Instituta; 6 : 91; (1937). Biol. Abst. art. 907; p. 93. (1939). Smith, Geoffry H.; Factors Affecting the Deposition of Dental Calculus; Australian J• of Exptl. Biel, and Med. Sci.; 7: 45; (1930). 62 61*. Smolelis, A. N., and S. E. Hartsell; The Determination of Lysozyme; J. of Bact.; 58: 731; (191*9). 65. Sreebny, Leo M., E. R. Kirch, and R. G. Kesel; Location of the Glycolytic Enzymes in Saliva; J. Dent. Res.; 29: 506; (1950). 66 . 67. 68 . Sreebny, Leo M., and M. B. Engel; Collagenase Activity in a Salivary Fraction; J. Dent. Res. 30: 1*93; (1951). Sreebny, Leo M . ; Characterization of a Proteolytic Enzyme in the Submaxillary Gland of the White Rat; J. Dent. Res.; 33: 685; (1951*). Sreebny, Leo M., natal Changes Morphology of Female Albino Julia Meyer, and Erica Bachman; Post­ in Proteolytic Activity and in the the Submiaxillary Gland in Male and Rats; (Growth, In Press). 69. Sreebny, Leo M., Julia Meyer, and Erica Bachman; Morphologic and Enzymatic Differences in CariesResistant and Caries-Susceptible Rats; (Submitted for Publication, J. A. D. A.). 70. Stephan, Robert M.j Two Factors of Possible Importance in Relation to the Etiology and Treatment of Dental Caries and Other Dental Diseases; Science; 92: 578; (191*0 ). 71. Voss, Otto; Ueber eine Trypsinartige Protease im Sekret der Menslichen Parotisdruse; Z. for Physiol. Chem. 197; 1*2; (1931). ^ 72. Van Slyke, Donald D. and Reginald M. Archibald; Manometric Titrimetric, and Colorimetric Methods for Measurements of Urease Activity; J. Biol. Chem.; 151*: 623; (191*1*). 73. Vladesco, R.j The Hydrolysis of Urea by Human Saliva; Comp. rend. soc. Biol.; 109: 1001; (1932). 71*.. Warfield, Louis M.; A Peptid-Splitting Ferment In Saliva; Johns Hopkins Hosp. Bull. 22: 150; (1911). 75. Weinman, Josef; Zur Atiologie der Zahnkaries Weitere Untersuchungen uber die Eigenproteolyse im Speichel; Z. fur Stomatologie; 36 : 95; (1938). 76. Willstatter, Richard, Eugen Bamen, and Margarite Rhodewald; Enzymes of the Leukocytes; III. Enzymes of the Salivary Glands; Z. fur Physiol. Chem.; 186: 85; (1929).