DOCTORAL DISSERTATION SERIES A Study cf the Influence c f Certain _ Dietary Constituents on the development c f TITLE. Denial Caries in 'Rats AUTHOR IhomasLesier Canniff fay DATE /9V3 Michigan Stale Colleqte % 1 ) 7 J K H k dA_________ PUBLICATION NO. DEGREE UNIVERSITY II. U mu 1 M I INI UNIVERSITY MICROFILMS ANN ARBOR MICHIGAN iJ STUDY OF THE INFLUENCE OF CERTAIN DIETARY CONSTITUENTS ON THE DEVELOPMENT OF DENTAL CARIES IN RATS by Thomas Lester Canniff A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 19U ACKNOWLEDGMENTS The writer wishes to express his sincere appreciation to Dr. C. A. Hoppert, Professor of Chemistry and Chairman of the Guidance Committee, for his patient cooperation, friendship, and inspiration! also to other members of the committee: Dr. D. T. Ewing, Professor of Physical Chemis­ try; Professor B. B. Roseboom, Head of the Department of Physiology and Pharmacology; and, Dr. R. C. Huston, Dean of the Division of Applied Science. The author wishes to express appreciation to Professor A. J. Clark, Head of the Department of Chemistry, for his interest and cooperation. Acknowledgment should be'made of assistance given by help­ ers in the rodent laboratory. The author also wishes to express his appreciation for suggestions and information by Dr. J. E. Hecko, D. D. S.; Dr. D. C. Mosher, D. D. M . ; and Dr. 0. L. Ricker, D. D. S. It would only be fitting to express gratitude for the inspiration and suggestions derived from associations with Dr. R. W. Bunting, and co­ workers in the Michigan Group Research on Dental Caries, from the School of Dentistry, University of Michigan. May, 1945. TABLE OF CONTENTS TABLE OF CONTENTS INTRODUCTION . 1 HISTORICAL 4 ETIOLOGY OF DEMEAL CARIES 5 REVIEW OF LITERATURE PART I PART II Part III 10 Development of the Caries producing Li et. " Experiment A Experiment.B_ Experiment C 20 25 SO 55 Tables end Charts for Part I 39 The Effect of Vitamin D on the Experimental Production of Caries Experiment 1 Exp oriment 2 Experiment S 48 49 49 50 Taolea and Charts for Part II 56 The Effect of Feeding Various Carbo­ hydrate n on the Development of Carles Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Exporiment 6 Experiment 7 Experiments 8 & 9 62 62 66 69 70 72 75 77 80 Tallies and Charts for Part III BIBLIOGRAPHY AnD LITERATURE CITED 84 102 PLATES A. Diagram of Molars in Man and hat B. Diagram of Molars Shoving Scoring Areas C. Photograph of Carious and Lon-Carious Molars D. Photograph of Graded Series of Mandibles E. Photograph of Graded Series of Mandibles F. Photograph of Graded Series of Mandibles 5a 25a 24a 245 24c 24d INTRODUCTION Introduction Any study involving: human beings and the control of their dietary habits presents at the very beginning cer­ tain difficulties and complications that are often insur­ mountable. One of the very significant factors is that the life span of the individual Is too lengthy to be able to include the complete picture desired in some experi­ mental studies. Control of dietary habits is extremely difficult unless the individuals participating are con­ fined to an institution where there is little opportunity for indulging: in food preferences. The cooperation of the subjects is not always what might be desired. Consequently, when feeding experiments are to be conducted, workers usu­ ally try to substitute experimental animals in which these difficulties can be eliminated. However, such selection must be made keeping in mind the fact that response to treatment should be reasonably comparable to the human re­ sponse. Such a task is not always easy, for, differences in dietary habits, physiological structure, environment, and psychological factors_.may vary widely from one species to another. The white, or albino rat has been a most satisfactory substitute for the human in many feeding experiments. are prolific, They thrive on a great variety of diets, have a short life span, and are small and easily handled. In the study of dental caries, all these factors are favorable.- 2 The physiological structure of the rat molar cannot be said to be identical, but at least is comparable, and de­ velops lesions that are very characteristic, and from an histological standpoint seem to be quite similar to carious lesions in human beings. The purpose of the investigation was to study the ex­ perimental production of dental caries, and the influence of certain dietary factors on the development of such- le­ sions in the albino rat* In this study, the work has bean divided into three parts: I. Development of the Caries Producing Diet. This part of the work sought to establish an adequate diet (one on which the animals would have normal de­ velopment, reproduction, and lactation), which would cause the development of dental caries in a certain definite period of time. Mechanical features were also considered. II. The Effect of Feeding Certain Vitamin Supple­ ments. This part of the work sought to determine mainly whe­ ther a supplement of these vitamins had any appreci­ able effect in preventing or retarding dental caries. III. The Effect of Feeding a Diet Containing High Levels of Certain Carbohydrates. \ This part of the work was done to observe the effect of a high carbohydrate content of the diet on the production and development of carious lesions. . Carbohydrate was supplied in the form of Su­ crose, Glucose, Lactose, and Corn Syrup, HISTORICAL and REVIEW OF THE LITERATURE 4 Historical and Review of the Literature To write a discussion of the history of dental caries would practically be to write the history of the develop­ ment of man. Dental caries is one of the oldest and com­ monest diseases of man. References to its existence have been traced back as far as the 22nd Century B.C.. The skull of a mummy in the British Museum, dating 2800 B.C., exhibits well marked signs of caries and other dental dis­ eases. In the various records of m a n ’s progress and de­ velopment, one finds quite frequent reference to the fact that dental caries occurred, no matter what the degree of civilization provided their diet included cooked starchy foods. Attempts to determine the cause of dental caries, or to find a method by which it might be prevented have, until recently, met with little success. Some of the ear­ lier methods had to do with feeding certain naturally oc­ curring substances, which would indicate that a few of the earlier scholars felt the disease to be one caused by a de­ ficiency of some of the elements required by the body for the production of good bones and teeth. It was not until the latter part of the 19th Century that any orderly method of study was suggested, although relatively little was accomplished beyond the accumulation of theories. Starting about 1920, several groups in this country and abroad began a series of studies on the eausitive factors in dental caries. Since that time, the field 5 . has proved to be a most prolific one, and much has been done in the way of clarifying the causes and treatment of the disease. One of the pioneer groups of this field was the Michigan Group Research on Dental Carles, headed by Dr. Russell W. Bunting (7). standing, Their work has been most out­ Other groups such as the one headed by Drs. Klein and McCollum at Johns Hopkins University, -by Dr. Rosebury and co-workers at Columbia University, and Dr. Hankers group in the Chicago Dental Research Club of Chicago, Illinois, have done very noteworthy work in this field. In addition to such groups, there have been many outstanding individu­ als whose contributions have added materially to our know­ ledge and understanding of the disease. Among these workers, the names of Mellanby, the Agnews, Bodecker, Boyd, McBeath, and Macy should be mentioned, and there are many others. It is significant to the progress of research that this problem brought together people from many fields such as dentistry, physiological chemistry, nutrition, and bacteriology. With such a widely diversified group, much ..more thorough and successful work could be expected. A great deal has been accomplished but there is still much to be done before the problem begins to assume the nature of completion. The Etiology of Dental Caries: In discussing the eti­ ology and causes of dental caries, one should first consider the structure of the tooth and how a carious lesion develops. In the studies carried on with rats and reported in this fs/oLAiis M olars o f A!a a / T^Ftr PLATE A Diagrammatic Representation of Molars in ManJ arul Rats. A-- Enamel; B-- Dentin; 0 — - Pulp. Note the enamel covers entire crown in Man, while in Rats the Dentin is exposed at the molar cusps. 6 paper, we were mainly concerned with the structure and make-up of the molars. Caries of the incisors in rats is quite rare, and the experimental production of caries in­ volves for the greater part the lower molars, and to a slight extent the upper molars. The structure of the tooth is divided into three main sections: tin, and the Pulp. the Enamel, the Den­ The enamel layer normally covers the outer exposed portion of the tooth, next comes the dentin, with the pulp comprising the innermost portion. A diagram­ matic drawing of the lower molars of man and rat is shown in plate A. This schematic comparison shows quite clearly the striking similarities and the significant differences in the structure of the molars of these tw7o species. Note that the enamel layer in the molars of man covers the entire crown of the molar. In the rat, the dentin is exposed at the summit of the molar cusps. Occlusal fissures in the rat molar are relatively deeper and wider (actually they are similar in size in the two species). Rat molars approximate over a surface, rather than at a point as in human molars. This last feature may he held accountable for the relative infrequency of interproximal caries in rats (36)• ’In the development of a carious lesion, it is generally accepted that the process of excitation begins with the de­ velopment of a bacterial colony on the surface of the tootli, which is frequently referred to as a plaque. This plaque is a thick, felt-like mass in which the micro-organisms are I 7 imbedded. The bacteria in the plaque require food, and obtain it from the carbohydrate and albuminous materials which come in contact with them. Lactic acid is one of the conspicuous products formed in the bacterial fermenta­ tion of carbohydrates. The plaque prevents the excreted acid from being washed away, and, as a result, erosion of the tooth by acid begins to take place. The acid attacks the inorganic matter of the enamel, first the interprismatic cement substance between the enamel prisms or rods, later dissolving the transverse cement substance between the enamel globules. Thus, as the interprismatic cement substance is dissolved, crevices are formed by which the bacteria eventually reach the dentin. When the entire thickness of the enamel has been penetrated and the dentin is attacked, there is a change in the mode of progress of the decalcification. The process procedes along the junc­ tion of the enamel and dentin, as well as directly into the dentin. The enamel may now be attacked from its dentinal side as well. When the dentin is attacked, we find a different chemi­ cal and anatomical structure to be acted upon. Beneath the enamel, the first layer of dentin is of such nature as to permit the bacteria to rapidly spread laterally in this zone. They also enter the dentinal tubules and penetrate by multiplication toward the pulp. is produced. A wedge shaped area of decay In most cases, decalcification precedes these 8 . invasions. In the rat molar, the dental caries developed are of the type known as fissure caries. They are the typical dental caries of molar fissures and are histologically simi­ lar to fissure caries in man, and establish the suscepti­ bility of rats to dental caries. Four stages in the pro­ gress of these lesions have been described: (l) Penetra­ tion of the enamel through the enamel lamellae, or diffusely in the absence of lamellae; (*2) spread at the dentinoenamel junction; (s) penetration of the dentin by infiltration of the tubules and disintegration of the dentin matrix; and (4) loss of surface continuity and formation of the cavity. These lesions can be distinguished from other destructive processes in rat teeth, one of which seems to result from the impact of dense food particles in teeth weakened by a rachitic condition, producing fracture through the cuspal dentin primarily, followed by secondary caries-like changes* The latter is not comparable to dental caries in man (35). From the above discussion, one would be lead to believe that the formation of a bacterial plaque was essential for the production of dental caries. However, lodgement or im­ paction of food may serve equally well to provide conditions favorable for Initiating tooth decay. Breese (5) says food lodgement is an indispensable factor in the production of caries. Sweets in their many forms, owing to their sticky nature, are the chief causes of food lodgement. Haber and co-workers (14) found in research in the Youth Movements in 9 * Germany, that the incidence and progress of caries, also film and tartar formation, increase as the degree of masti­ cation decreases. However, Rosebury and co-workers (32) found that fissure caries was caused primarily by food par­ ticles rich in carbohydrate that were impacted under strong masticating pressure, and that the particles not being sub­ sequently dislodged furnished a pabulum for acid producing bacteria. These workers also found that the primary agent in fissure caries in rats (impaction) also causes the proxi­ mal type caries, so'it may be that the etiology of both types follows an analogous pattern. Klein and McCollum (22) believe the cause of macro­ scopic caries in rats to be due to (a) fracture of the molar cusps; (b) decay at the bottom of molar sulci (which the writer believes most frequently follows the impaction of food particles); (c) interproximal caries where food impac­ tion has been frequently noted; and three factors. (a) a combination of all They found that the ingestion of a rachito- genic diet containing coarse corn meal gave carious lesions, whereas the same diet containing finely ground c o m showed absence of caries. Mellanby (31) questions whether or not caries begins as a process of decalcification, or as disintegration of pro­ tein, and states that there is no sound evidence as to the immediate cause of caries. This latter remark seems somewhat questionable in the light of all the work that has been done and the evidence that has been submitted* The importance of heredity should probably be men­ tioned. Bunting and co-workers (7) believe inherited ten­ dencies, or inherent individual characteristics in some cases are more important determining factors in caries than ordinary dietary conditions. Hunt and Hoppert (19) in their work on the role of inheritance in rat caries have been able to develop a resistant line and a susceptible line. Although the strains are probably not homozygous, they have quite definitely shown that the characteristics of resistance or susceptibility are inherited. Review of the Literature: In reviewing the literature on Dental Caries, the first publication that should be men­ tioned is the review compiled for the Research Commission of the American Dental Association by the Advisory Committee on Research in Dental Caries. The theories on the cause of dental caries are numerous, but when stripped of the modifi­ cations elaborated by the many workers, they may be grouped into three principal categories: (1) Theories based on the structure of the teeth; (S) theories based on the dietary picture; and (3) theories based on the bacteriological pic­ ture. With this classification as a basis for the review, we may greatly simplify the mass of research that has been done. For many years, caries was associated primarily with two factors— the tooth itself, and the hygienic condition of the mouth. It was felt that if, during the period of growth and development of the teeth, the supply of calcium and phosphorous was adequate a good tooth structure was certain to follow. A sound, well calcified tooth was not supposed to decay if kept clean. However, we now find that our thinking must he somewhat revised. (31) Mellanby says that the type of tooth structure is related to diet during the development, and that there is a certain defi­ nite association between tooth structure and susceptibility to caries. Hypoplasia certainly does involve an inferior tooth structure, but this is far from necessarily a prime factor in caries. Cox (8) believes that enamel structure is a dominant factor in preventing initiation of caries. Gottlieb (13) feels that the degree of calcification is of some importance in the control and incidence of caries. Rosebury and co-workers (36) report that they found caries in rats that were healthy, well grown, and in teeth without appreciable structural defects. In considering the hygienic condition of the mouth,1 one of the most important factors is the oral fluid or sa­ liva. flora. Certainly, the saliva would normally contain varied For the bacteriological picture, it is advisable to wait until later in the review. Branson (4) states that the basic property underlying both immunity and susceptibility to caries is rendered comparatively simple when two attending local physiological factors, in addition to the bacteriologi­ cal aspect, are clearly understood. One of these is the . 12 ability of saliva under normal conditions to protect teeth effectively against caries. Koehne and Bunting (23) found that on a fine diet impacted food removed from the rat mo­ lars was cement-like and dry. If the impacted food was impermeable to the oral fluids, its bacterial decomposition could not take place. This substantiates Hoppertfs theory as to why no caries resulted when rats were fed a fine diet (20). Further, Bunting and co-workers (?) found there was no consistent relationship between hardness or perfec­ tion and the state of mouth hygiene and the activity of caries. Florestano (10) postulates that the diastatic ac­ tivity of the saliva, carried to all dental surfaces, is the main factor in the prevention of caries. Hanke (15) says the difference in caries-immunes and caries-susceptibles is due locally to the quality of their salivas. The buffering of saliva is thought by Hanke to be highly im­ portant. The Ann Arbor Group (7) has found no correlation demonstrable between amounts of salivary calcium, phosphorus, chlorides, pH, carbon dioxide capacity, total alkalinity, total solids, or ash and the activity of caries. Hanke (17) was unable to show any correlation between the pH of vari­ ous regions of the mouth and dental caries. The idea that dental caries was a disease of dietary deficiency is an old one. It was thought that certain in1 organic constituents were required in order to lay down the necessary deposits in tooth and l?one structure that made 1 ' ' them well formed and hard# Such elements as calcium and phosphorus were believed the most important because of the role they play in bone structure. To attribute this idea to any individual worker or group would be impossible— ra­ ther was it the opinion of the majority of workers. Natu­ rally, then, these were two factors that were first stu­ died. Fieisch (9) says caries is a deficiency disease. Forshufvud ( n ) says caries is ordinarily an expression of disturbed metabolism, often due to inferior diet, but more frequently to excessive irregularity in diet and mode of living. However, there has been much work done that re­ futes the idea of caries as a deficiency disease. Rosebury from his work with rats (36) believes that dietary defici­ ency is not a determining factor in the etiology of fissure caries. Hanke (17) finds there is no apparent correlation between total calcium and soluble phosphate content of the blood serum and the incidence of dental caries. Further, he has stated that a superior diet of the average American child will not protect completely against caries, and that the juice of citrus fruits contains something (Vitamin C) required by a child to maintain oral health. LeFevre (26) has found that there is little difference in composition between fresh, human carious teeth, and sound ones, except in moisture content. Lilly (27) believes that caries in rats is not 14 definitely related to a deficiency of any known food fac­ tor. McCollum and co-workers (30) think it not yet possi­ ble to name any one dietary deficiency as the specific cause of dental or oral disease, but do suggest that it may be a multiple or complex deficiency. They feel that any slight variation in the average American diet— which always so dangerously approaches the level of deficiency— might become active at any period of lowered resistance, or of physical or of nervous stress. McClendon (29) has shown that sound teeth contain more fluorine than carious teeth, and that the addition of from 5 to 22 parts per million of sodium fluoride to a fluorine deficient diet de­ layed the onset of caries. Bunting and co-workers (7) found there was no relationship between the intake of cal­ cium and phosphorus, or acid/base dietary values and the activity of caries. They feel that there is no evidence that caries is primarily produced by malnutrition, or may be prevented by an adequate diet. The subject of the in­ fluence of carbohydrates should undoubtedly be mentioned as a dietary factor, but because of the mechanism through which it acts, mention will be made of it in the bacterio­ logical phase of the discussion. Bacteria have been definitely proved an important factor in the production of dental caries. co-workers of the Ann Arbor Group work on this phase of the problem. (7) have Dr. Jay and done outstanding They have found that the relative numbers of lactobacillus acidophilus in the . 15 , mouths of individuals with caries is relatively greater \ than in caries free individuals. Belding and Beldlng (2) consider caries to be a bacteriological disease subject to interpretation in terms of the laws of bacteriology and immunology. They view caries as a relatively specific dis­ ease occuring in those who partake of modern diets and have oral food retention areas. Rosebury and Karshan (S3) have found aciduric bacteria, similar to L. acidophilus, to be normal inhabitants of the rat mouth, their presence being constant and independent of the feeding of human lactobacilli. Mellanby (31) suggests that certain bacteria play a part in the progress of caries, but regards questionably whether or not they initiate caries. Wessinger (37) be­ lieves caries due to localized action of various organic acids produced by mouth organisms from carbohydrates. He feels, however, that no single organism can be regarded as the sole etiologic factor, but that it is probably a sym­ biosis among many organisms that may account for the ex­ tremely rapid acid formation in the mouth. All the trork on this phase of the problem has led to the use of the term B. odontolyticus in referring to those organisms which cause or contribute to the breakdown of dental tissue that is referred to as dental caries. The role of carbohydrates then becomes quite apparent. Rosebury and co-workers (32) believe that carbohydrates in general enrich the acid yielding substrate, and that the addition of sugar to a caries producing diet tended ir­ regularly to increase the incidence of caries. Lilly (27) showed that high carbohydrate diets containing finely ground corn or corn starch would not initiate caries. Hanke (15) states that oral bacteria convert cane sugar and other carbohydrates into acid. Cox (8) found that fermentable carbohydrates promoted the enlargement of ex­ isting carious lesions, but that they had no effect with diets that initiate caries. Florestano (10) postulates that complex carbohydrates, under the salivary diastatic action, form simpler sugars required by aciduric micro-or­ ganisms for acid production. Branson (4) suggests that the perverting influence of refined carbohydrates— sugar especially— in breaking down the salivary defense thereby promotes fermentive processes, bacterial activity and propagation, thus virtually inviting caries development. Belding and Belding (2) state that increase in suscepti­ bility to caries is due to a relative or absolute Increase in the pathogenicity of oral flora and ensuing accelerated formation of acid, rather than by any change in the affected tooth that makes it less resistant to the action of caries producing acids. Sucrose seems to favor a shift in the na­ ture of the normal oral flora resulting in a strain that will cause practically any of the carbohydrates consumed to be rapidly converted Into acid. Cereals are rapidly fer­ mented producing large amounts of acid. Thus, the simul­ taneous consumption of sugar and cereals, particularly in 17 refined forms, causes an increase in the relative and abso­ lute streptococcic pathogenicity, 'fhis is probably the chief cause of the excessively high carie®, rates of civi-^ lized man. The sugar establishes the flora, whereas the cereals provide the chief source of acid for enamel decalclfication. Bunting and co-workers (7) have found sugar to be a very important causitive factor in caries. A remark­ ably low degree of caries was found in children living on a diet low in sugar, even though the diet was deficientin calcium, phosphorus, and vitamin D. Active caries was in­ duced by increasing the sugar intake while receiving a diet nutritionally adequate. Ingestion of a low sugar diet by children is conducive, as a rule, to freedom from caries. If, as would be indicated from the amount of study, and as would seem very plausible, the incidence of caries is due to a bacterial activity, then the use of certain types of inhibitors might be valuable. Blayney (S) tested the effect of specific inhibitors of bacterial metabolic activities of bacteria on tooth surfaces. It was presumed these inhibitors would reduce the incidence of caries. This conclusion was tested using fluoride and iodo-acetate on the development of caries in the albino rat. Using a coarse particle diet which Induces a high rate of caries, it was found that those animals whose diet contained the inhibitors showed a marked reduction in the incidence of carious lesions. Cultures from a small series of carious lesions showed both L. acidophilus and acid producing streptococci with the latter in marked preponderance. Jay (7) states that an immunologic principle related to L. acidophilus has been demonstrated in the blood of caries free individuals, in whose mouth as a rule L. acidophilus does not exist, and when planted therein promptly disappears. It seems very probable that some of the most promising fu­ ture work might well lie in the field of bacteriology. In connection with the nutritional aspects of dental caries, the vitamins C and D seem to present the greatest interest. Vitamin D has long been felt to exert considerable influence on oral health, while vitamin D and its association with bone development, rickets, and calcification make it of importance. Brodsky and co-workers (6) found that massive doses of vitamin D, in addition to a well balanced hospital diet, resulted in marked diminution in the incidence of new carious lesions in some 200 children. Cox (8) and co-wor­ kers found increased caries immunity in young rats resulted after feeding haliver oil to mothers during pregnancy and lactation, in excess of amounts necessary to prevent ric­ kets. Hanke (15, 18) found that vitamin C in preventing gingivitis prevents regions of stagnation which in turn lowers the incidence of caries. Lilly (27) reported that rats on vitamin A and B deficient diets developed no cari­ ous lesions, and that the addition of vitamin D had no ef­ fect on the incidence of caries. Rosebury and co-workers (32) report that the feeding of vitamin D reduced the inci­ dence of caries without preventing it. Hoppert et al (20) found that liberal additions of vitamins A, C, or D did not appreciably retard the decay of the teeth. This would point to a lack of direct relationship between these fac­ tors and the caries producing process. EXPERIMENTAL PART I DEVELOPMENT OF THE CARIES PRODUCING DIET Part I: Development of the Caries Producing Diet. In the development of a caries producing diet, the first and most important factor to he considered is the nutritional value of the diet. In order that the picture of the cause of dental caries he not confused with a poor nutritional background, it was essential that the diet used should produce normal growth, reproduction, and lactation— the latter heing one of the principal criteria in judging the adequacy of a diet, ^he second factor is of a mechani­ cal nature, for it was found that apparently one of the es­ sential requirements of a caries producing diet was that im­ paction of food particles in the teeth of the rat must pre­ cede the development of the lesions. This heing the case, it was indicated that the physical nature of the diet— that is, the size of various particles in the diet— might contribute to the success of the diet in producing carious lesions. The first factor was quite fully demonstrated in the work of a previous investigator, Dr. P. A. Weber, whose work along with some preliminary work of this study has al­ ready been published (20). A controversial factor appeared at this point concerning the cereal portion of the diet. The cereal portion of the diet was made up of yellow corn ground in a Wiley .Mill using the coarse sieve. The ground corn was a composite mixture as far as particle size was concerned. As the cereal came from the mill, it was composed of particles ranging in size and texture from that of flour fineness to coarse sand. A sample of this ground corn was passed through a series of graded sieves. 250 grams of this ground corn was found to grade as fol­ lows: Portion remaining on 20 mesh sieve... 66 grams 26.4$ « » « 40 * « ...110 » 44 $ « * « 60 » « . . . 51 * 20.4$ n passing through 60 « «... 15 * 6 $ « lost (retained in sieves) ... 8 « S.2$ TOTAL 250 grams100.0$ Some investigators felt that in feeding, the rats se­ lected the finer portions of the ground corn, and discarded the coarser particles. In so doing, they were apt to upset the mineral "balance found naturally occurring in the whole kernel of the corn. However, in the addendum to the publi­ cation by Hoppert, Webber, and Canniff (20), the possibility of caries being due to a deficiency of phosphorus was shown unlikely. The incidence of caries quite clearly appears to be independent of the phosphorus content of'the diet. (See Table 1, taken from publication of Hoppert et al (20).) To demonstrate the possible effect of particle size as a factor, a series of experiments was set up in which the cereal portion of the diet was supplied by the various frac­ tions obtained by sifting the corn (ground in the Wiley Mill using the coarse sieve) through sieves of graded mesh as above mentioned. Another point to be considered was the variability of the corn used. The corn kernel was found to vary widely, TABLE I Data showing lack of relationship between phosphorus content of the diet and incidence of caries. Diet Parts I. Cornneal 69 Wheat gluten 20 Whole-milk powder 5 Crisco 5 NaCl 1 100 Same diet, with 1% CaCO, o II.Oatmeal 69 Wheat gluten 20 Whole-milk powder 5 Crisco 5 Ns?.Cl 1 100 Same diet, with 156 CaCOv O III. Stock Rati on-:;coarse cornmeal P Ca Ca/P ratio Caries gram grain 0.2001 0 .”0096 0.0400 0.0156 0.2 63 0 .0420 0.0490 per cent 90-100 43.5 90-100 50.4 Average of Femurs & Humeri 0.2621 0.0742 0.2821 0.4742 1.68 0 .2994 0.0702 0.0400 0.0156 0.0420 0.0490 0.41 0 45.5 0 51.9 90-100 56 .2 0.3436 0.1408 0 .3436 0.5408 1.57 0 .4302 0.3424 0.7 * Values given by Klein and McCollum. 22. from time to time, in moisture content, hardness, and other features. Thus, it seemed desirable to substitute for the c o m some other cereal that would be more constant in nature, thus eliminating such variations. Rice seemed to possess the qualities desired, and a series of experi­ ments was conducted to determine the effectiveness of this grain in the experimental production of caries. Rosebury and others had used rice and found it quite effective. With these things in mind, the series of experiments in Part I was set up to clarify the following points: A. The effect of particle size of the cereals used on the incidence of carious lesions. B. The period in which the impaction of food particles was at a maximum, and so produced more caries. C. The effectiveness of rice as compared with corn in the experimental production of caries in rats. The feeding experiments inPart I, as well as Parts and II III, are all established onthe same general plan. Young animals were used when weaned, at 21 to 28 days, having attained a weight of from 50 to 60 grams. They were placed on the experimental diets immediately Qr at such time as the plan of the experiment directed. The period of experimental feeding was from five to ten weeks depending on the infor­ mation desired. At the end of the period, the animal was destroyed, the mandibles removed, cleaned, and filed for 23. % scoring. If the upper molars showed any carious lesions, the maxillary plate was clipped from the skull and filed with the mandibles. The caries score for each animal was computed by as­ signing a value to each lesion found, the score consisting of the sum total of these values in each animal. A rather arbitrary means of scoring was worked out as follows: Since caries seldom occur in the incisors, the plan of scoring was based on the molars (lower) which are six in number, three on the right and three on the left. side was scored separately and recorded. Each The uppers were scored together since there was seldom more than one cavity. The molars were numbered 1, 2, and 3 beginning at the front. The molar was divided into several areas as suggested by the frequency with which the lesions appeared in the molars. Plate B shows a diagram of the three molars and the desig­ nated areas in each molar. A pin point cavity was scored 1, a well defined medium cavity 2, a large cavity 3, this latter score being given as the cavity involved practically the entire area. Plates C, D, E, and F show a series of right mandibles with cavities of graded severity. Although such a method might lead to slightly different scores by various operators, the over-all pictures would be comparable and the averages would make the same conclusions possible. The method is simple, fairly rapid, and gives significant values. Examinations during the feeding period were made —a — a ■ -b PLATE B Diagram for Method of Scoring, Lesions are indicated by the red shaded a reas. Score would be: l:a-x,b -xx,bQ-xx; 2:b-xxx. Total = 8. with the aid of a nasal speculum ground off at the tips. Such an instrument greatly facilitates the examination of the lower molar teeth of rats. The animals can be made to submit to such a gross examination without injury, and after learning to hold the animal properly, the examinations may be done quite rapidly. Two workers are most effective here, one to hold the animal, the other to make the examination. PLATE D .ght Mandibles Showing Carious Molars, mhers denote score of encn Lianciiole. PLATE 1 ; Ight Mandibles Showing Carious Molars, umbers denote score of each mandible. A. The Effect of Particle Size of the Cereal on the Production of Carious Lesions. Having determined quite conclusively that the cari­ ous lesions were due to the impactions of fairly coarse particles of corn in the sulci of the teeth, the following experiment was set up in which the cereal portion of the diet was made up of ground corn of varying degrees of par­ ticle size. It was felt advisable to learn something about the effectiveness of the relative particle size in the initiation of the carious lesion. The corn used in the preparation of the rations was first ground in the Wiley Mill using the coarse sieve. This corn was then screened through a series of three graded sieves: 20 mesh, 40 mesh, and 60 mesh. This screen­ ing gave four fractions: Fraction Fraction Fraction Fraction 1, the portion retained on the 20 mesh screen 2, » * * ® 11 40 ® « 3, » ® " * 60 « « 4, w ” passing through the 60 mesh screen The ration used was based on the following formula: 60$ Cereal, 50$ Whole Milk Powder, 6$ Flax-seed Meal, 3$ Alfalfa Meal, and 1$ Sodium Chloride. Using the above formula, six modifications were produced as follows: 1. The corn used was that as it came from the Wiley Mill 2. The corn used was from Fraction 1 in the above table. 3. The corn used was from Fraction 2 in the above table. 4. The corn used was from Fraction 3 in the above table. 5. The corn used was from Fraction 4 in the above table. 6, The corn used was 2/3 Fraction 2 plus 1/3 Fraction 3 above. 7. The corn was replaced with ground oatmeal as the cereal. A group of fourteen animals v/as selected at weaning, and two animals were placed on each of the above diets. The animals were fed the experimental ration for eight weeks. At the beginning of the fifth week, and each week thereafter, the animals were examined and the development and progress of the lesions was observed. At the end of eight weeks, the animals were destroyed, mandibles removed, cleaned, and scored. The scores appear in Table I—1, and the comparison of the scores is shown on Chart I-l. Results From the chart and table of data, the following ob­ servations were made: The effectiveness of the diet in the production of dental caries is dependent on the particle size of the cereal. The group fed corn from Fraction 1 showed an average score that was lower than the controls fed the composite mixture, but higher than those fed diets containing the finer corn. The highest score was attained with diets containing corn retained by the 40 mesh sieve (Fraction 2), or with a mixture of the corn from Fractions 2 and 3 as' described in modification 6. Animals fed on corn from Fraction 3, or 4 gave the lowest scores. The animals fed on the diet containing the oatmeal showed no caries whatever. Discussion It is interesting to note that the largest particles are not the most effective in initiating caries, hut rather the corn which passed through the 20 mesh sieve but was re­ tained on the 40 mesh sieve (Fraction 2). This appears en­ tirely reasonable for it has been shown that impaction must precede the formation of a carious lesion. The particles retained on the 20 mesh sieve were so large that the chance of impaction was materially reduced. That there was some impaction and resulting caries is probably due to the fact that as the molars performed their crushing action on the large particles, the particles were fractured, giving frag­ ments of a size suitable for impaction in the sulci of the molars. That the most severe caries should be produced with the corn of Fraction 2, or a mixture of Fraction 2 and 3, also seems reasonable. Particles of Fraction 2 were of a size that made impaction in the sulci much easier, much more frequent, and in all probability much faster. Since impactions were more numerous,’ there was a greater inci­ dence of caries thus giving a higher score. In the case of rat no. 4x was found five distinct lesions, while in 14x, there were four. The mixture of Fractions 2 and 3, in the ratio of 2 to 1 respectively, showed the highest score and the most extensive impaction with nine lesions showing in rat 9x and five in lOx. From this it would logically be concluded that the particles more nearly 2S. approaching in size the dimensions of the sulci of the teeth are the ones that are most effective in initiating the development of the carious lesions. In the case of those animals whose diet contained only the fine corn impaction assumed an entirely different na­ ture. This brings us to the diet containing the oatmeal which gives a comparable picture although a bit more at the extreme end of the scale. When a diet contains much fine material, we do have impaction but the effect obtained is very different from that obtained with the coarser cereals. Here the material is so fine that it packs into the sulci so solidly as to be practically impermeable to the saliva, whereas the coarse particles permit the free access of the saliva. In the review published by Koehne, Bunting, and Hadley (23) their findings corroborate those of this work. Thus if the material packed into the sulci of the lower molars is impermeable to the oral fluids, its bacterial de­ composition will be impossible because of the lack of mois­ ture, The impaction behaves much in the nature of a filling, keeping out the saliva and further impaction of the coarser particles. The impacted material is very dense and so firmly imbedded in the sulci that it may be removed only with diffi­ culty with :an explorer or sharp probe. For experimental production of caries, then, it would seem that the composite mixture as it comes from the mill makes the most effective form of the cereal that may be used. While the tot al score may not be as great, it gives a truer 29 picture, and relieves one of the arduous task of sifting that is necessary in grading the cereal. Further, if there should be any possible disturbance in the distribution of the nutritional constituents brought about by sifting, the use of the composite mixture as it comes from the mill would for the greater part eliminate this factor. It must be kept in mind that there has been no attempt to establish any gra­ dation in the caries producing effect of the diet. While ultimately it would be desirable to produce several diets whose caries producing effect might be established as slight, medium, and severe, in these and other preliminary experiments we have only sought a ration with high capacity for the pro­ duction of caries. Summary and Conclusions To summarize it might be stated that the particle size of the cereal portion of the diet is of definite significance in the experimental production of dental caries. The particles should not be too large nor yet too small, but somewhere within the range comparable to the dimensions of the sulci of the molars. The composite mixture of particle sizes as is obtained in a sample of corn coming directly from the Wiley Mill using the coarse sieve, seems to give the best over-a.ll results. B. To Determine the Period During Which Time Impaction is at a Maximum. In this phase of the study, it was attempted to ascer­ tain whether or not there was some particular period during the development of the animal when a maximum amount of im­ paction might take place. In this way, it was hoped that it could be determined at what age the animals shox'ved maxi­ mal response to a caries producing diet, or whether there was sometime in the development of the teeth when their structure was particularly sesceptible or resistant to the impaction of food particles which caused the' formation of carious lesions. With this in mind a series of three experiments was conducted on the following plan. Litters of six or seven animals were selected, and at weaning were placed on the following diet: 60c Jo Ground Oatmeal, JQfo Whole Milk Powder, 6fo Flax-seed Meal, 3/0 Alfalfa Mead, and 1 Sodium Chloride One animal from each litter was placed on the caries pro­ ducing diet which is the same as the above ration except that coarsely ground corn or rice was substituted for the oatmeal. At two week intervals, one animal from each litter was trans­ ferred to the caries producing diet for a period of eight weeks. At the end of the eight weeks of experimental ration feeding, the animals were destroyed, mandibles removed, cleaned, and filed for scoring. In case of lesions in the upper molars, the maxillary plate was also removed. At the completion of the experiment, the "caries score" for each animal was calculated and recorded, and the results of the entire group compared. The distribution of animals used was as follows: Experiment 1: Experiment 2: Experiment 3: 3 litters of 7 4 litters of 7 4 litters of 6 animals each; Corn animals each; Corn animals each; Rice as cereal as cereal as cereal The scores of the group in Experiment 1 are found in Table III-3, with the comparison of the scores shown in Chart III-l. The scores of the group in Experiment 2 are found in Table III-2, with the comparison of the scores shown in Chart III-l. The scores of the group in Experiment 3 are found in Table III-l, with the comparison of the scores shown in Chart III-l. Average of Tables III-l, 2 & 3 are shown on Chart III-2. Results From the data and comparison of the scores of the two series on corn, one finds that as the age of the animal in­ creased, the caries score decreased. In the case of the two series on corn, the decrease seemed fairly consistent, throughout. It will be noticed that in the last group there was a slight increase in score. The series on the rice diet in general showed a higher score with more severe caries, and there was one less group than on the corn diet. This brings in a point which was further studied in the next ex­ periments, the relative effectiveness of corn as compared with rice in the caries producing diet. In all cases, whe­ ther the diet was corn or rice, the older animals showed distinct wearing down of the molars Discussion From the caries scores it would seem that impaction takes place to a greater extent during the first two to four weeks after weaning. Further, as the animal matures and grows older the score decreases enough to lead one to believe that impaction is less. However, the fact that the last group in each series showed an increase in score would lead one to believe that there is a point reached when impaction again increases with a resulting increase in carious lesions. In view of a lack of a comparable group on the rice diet, no conclusions for that cereal are possible. It would be extremely easy to leave the discussion at this point and draw only the simple conclusions stated above. There are, however, two significant points that must not be overlooked. The first point involves imp ac­ tion of the fine diet that might have preceded the feeding of the coarse rice and corn diets. It will be recalled that in an earlier discussion on the nature of impactions on a fine diet, it was found that the fine material was very firmly packed into the sulci of the molars. This fac­ tor seems most likely to play an influential role in the present experiment. It is highly possible that as the peri­ od of feeding on the noh-caries producing diet becomes longer, the sulci are eventually fairly well filled with the 33 impacted fine diet and thus reduces the frequency of im­ paction of coarse particles when the animal is placed on the caries producing diet. To say, then, that as the ani­ mal matures, the structure of the tooth changes making im­ paction less frequent may not be true. An attempt was made to examine carefully the teeth and see that there had been no impaction or development of caries on the oatmeal diet. However, in view of the difficulties encountered in re­ moving impacted fine diet from molars, even after the man­ dibles had been removed, cleaned and dried, such attempts as were made to remove impacted material from the molars in situ in the live animal would not always be particularly effective. The second point that should be considered is that in the older animals a marked erosion of the molars was notice­ able. This erosion would tend to decrease the depth of the fissure, and at the same time expose more of the dentin of the tooth. The nature of caries in the last group of ani­ mals on the rice diet, and the last two groups on the corn diet appear to be of slightly different nature than in the earlier grotips. It appears rather doubtful whether or not true fissure caries developed in these last groups. The work of Rosebury et al (36) most clearly defines the true fissure caries, and was identified most effectively by i means of ground sections. Gross examination leads one to believe that in the last groups the type of caries was different due to the larger exposed area of dentin. To establish this unquestionably ground sections would have to be made at fairly carefully chosen intervals. After the breakdown of the tooth structure has progressed far enough, it is practically impossible to differentiate between true fissure caries and other types of carious lesions. Summary and Conclusions As the animal matures, a change from the non-caries producing diet to the caries producing one shows a decrease in the caries score. The decrease in the caries a decrease score can be attributed to in frequency of impaction, but the decrease in the frequency of impaction may not necessarily be due to any change in tooth structure. Other fluence on contributing factors are cited and their in­ the results obtained have been discussed. The last groups show a slight increase in caries score, which may or may not be due to a type of caries other than true fissure caries. C. The Effectiveness of Rice as Compared with C o m in the Production of Experimental Caries. As was mentioned previously, the use of corn in the caries producing diet did not always give consistent re­ sults— particularly as to the period of time required to develop carious lesions. This was thought to be due pri­ marily to varying degrees of hardness and moisture content. For that reason, it was deemed advisable to try some of the other cereal grains. Both wheat and rice had been tried, and rice gave promise of being the best selection. Other workers, notably Rosebury, had used rice quite successfully. Accordingly, a comparison was made between the caries pro­ ducing ration compounded with coarsely ground corn and the same ration compounded with coarsely ground rice. Both grains were ground in the Wiley Mill using the coarse sieve. As the work progressed further, and after it had been demon­ strated thait rice was a more suitable cereal, the grinding was done on a Hobart Feed Mill setting the adjustments of the burrs at 2 jr. This gave a much more uniform grind, and an average particle size somewhat smaller than from the Wiley Mill. In this experiment, several groups were used but there is presented here only one of the more typical ones. A group of three litters giving a total of 19 animals was used. weaning, a selection of 8 animals (4- males and At females) was made and these were placed on the caries producing diet con­ taining the coarsely ground corn. The remaining 11 animals 36. were placed on the same diet in which the coarsely ground rice was used. The feeding was continued for five weeks, at which time 2 animals from the corn group and 3 from the rice group were destroyed. cleaned, dried, and scored. The mandibles were removed, Each week thereafter (i.e. at the 6th, 7th, and 8th weeks) a similar number was terminated and the caries score computed for each animal. were then compared. The scores The scores are found in Table the comparison of the scores is shown on Chart II-l and .tl-l. Results In the case of the rice group, the score shows that a more rapid production ox caries was noticed at the end of the first five weeks than in the same period with corn. The scores for the entire rice group increase from an average of 2,5 up to 9 at the end of eight weeks. In the case of the corn, the scores run from 0 up to 6.5 at the end of eight weeks. The rice showed higher scores at the end of the 5th, 6th, and 6th weeks. The slight difference in the 7th week may have been due to differences in the animals. Discuss ion The use of rice as the cereal portion of the caries producing diet instead of corn showed that the effects ob­ tained were equally as reliable, and, in most cases, the response came earlier in the period of feeding. Animals fed the rice thrived and. developed just as well, and the diet showed itself to be as adequate nutritionally as when the corn was used. Growth curves were not included in any of 37. the tables or charts of this paper as they contributed nothing of specific interest to the work, and when an ani­ mal failed to gain normally as shown by weight, records, it was discarded. In preliminary work on the various diets, the animals were weighed each week to be sure that a nor­ mal gain was being made on the diet. Where diets failed to give the normal gain in weight, they were modified to remedy the deficiency, and the proved ration was the one used in the experiments reported here, There were very few instances where this happened, and specific mention will be made of such cases. The main object of the portion of the work was to es­ tablish definitely that rice was as good or better than corn in the diet for production of experimental caries. It was found that rice produced caries more rapidly than corn; that is, that it caused the development of caries quite con­ sistently in the 5*^ ^<3- 6th weeks while the corn produced caries about a week later. This was probably due to the fact that the average particle size of the rice was somewhat smaller than the corn, and thus impaction took place more readily and with greater frequency. Further, the physical characteristics of the rice kernel are apparently much less variable than with the corn kernel which would contribute greater consistency of results both with respect to time and severity of the lesions produced. On this basis, rice was used instead of corn in the subsequent experiments. Summary and Conclusions Rations containing coarsely ground rice werefound to produce experimental caries readily. Caries were found to develop consistently and about a week earlier than with corn and were slightly more se­ vere . The particle size of the ground rice, as well as the more consistent physical characteristics of the rice, were undoubtedly responsible for the more rapid and somewhat more severe nature of the lesions produced. It was concluded that rice was superior caries producing diet. tocorn in the TABLE 1-1 CARIES SCORE RAT NO. Sex Upoer Lower Lower Total Average Remarks Rt. Lft. lx F -------- 3 2 5 6 13x F 2x 3 ^ M 7 1 1 4-.5 3x F 1 5 2 S 4-x F —— k- k $ l4-x M Rx F 6x F --- 7* m --- gx F 9x F lOx. F -- 3 ^ 2 --- 1 -— Fraction III; Corn 1.5--------------------------- r mesh sieve 3 Fraction IV; Corn passed through 60 mesh sieve 6 2/3 Fraction II plus 1/3 Fraction III 0 Oatmeal Control 6 6 3 9 7 1 lx F ----- 0 12x F. ----- 0 Fraction I; Corn retained on 20 mesh sieve Fraction II; Corn 7.5---------------------------mesh sieve 0 4 - 2 3 3 0 ■ 3 1 7 Stock Diet with coarse corn S T O C ./O 7& A T / o r t f^tf/rcr/OA/Z JL ■' 2Z " JZ 2L&M Jvj/xEO O a r/YEAAj r~| ZG/Zo Z/r/xz CHART 1-1 Prom Table 1-1; Graded Corn Series. TABLE II-l CARIES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 M 0 5 weeks 3 M ------ 1 1 2 1.5 F 2 5; M ^ if g 6 F --- 2 2 4- 7 M --- k- 2 6 g F --- 3 10 F 11 M 13 F 14 F 15 - If ----- 1 M 3 l 3 1 6 2 F ----- 3 17 F ----- 5 is m ----- 6 7 weeks O ,5 ^ weeks 2.3 5 weekB ^ 6 weeics 10 1 16 6 1+ 4- — - Coarse Corn 7 2 1 ----- ^ £ xveeka 1 3 ^ Coarse Rice 6 5*3 7 weeks 9 7 13 & weeks 19 F 3 2 5 »riY Coe/J ■tsMEettrS g/ce Sweeps CHART II--2 Prom Table II-l; Comparative Action 43 TABLE III-l CARIES SCORE RAT NO. Sex Upuer Lower Lower Total Average Remarks Rt. , Lft. Place on Caries Producing Diet at beginning of: 4 5 3 3 5 4 3 4 9 9 6 7 7,8 3rd- week 4 Ll♦ 2 3 3 9 3 2 7 9 5 3 6.5 5th week 4 4 1 4 5 3 3 4 9 7 6 8 7.5 7th week 2 —— 4 3 3 2 4 3 2 2 S S 7 4 6.8 9th week 11 M F F j l -----—* 3 1 5 3 3 , s' 0 - 4 6 1 11 7 6.2 11th \veek Ivl M F F —----- 2 2 2 1 1 ? 4 3 3 3 4 . 3*8 13th week 1 2 ? 4 M M F F 5 6 7 8 F F M M ---, 9 10 11 12 M M F F --2 — — 14 16 16 F F M M 17 IS 19 20 21 22 26 24 -- . --——— ' ; M — --- — Ex f t 3 /s/ Z ZnJ 1/ 3 r4 n 4H, Sfi « &1k » A/o Q-ftoi/p neRe £:.xrT Zj lot z ween* ■ ;.'Zftd.- ♦ 3t~J 4ft ■3th (rth .. “ „ *l * ® m .m rfth £xfi>f- f 1st, Z V'Jt&KS Zfid « <3rJ 4ti sb ytl " 'Jitt Z b £ c>L/ajb CHART III-l P r o m TabD.es 1 1 1 -1,2, & 5 Periods *5. TABLE Ill-g CARIES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt Lft. Place on Caries Producing Diet at beginning of: 1 llSPi MM liss 2 2 M F F 1 9 10 11 12 f\y M M F F M M 17 18 19 20 M M F F 21 22 2^ 24 F M J M F F n M 1S’ b 0 4 2.8 5th week 2 ip 2 --- 3 3 4- 5 7 g 0 5*0 7th week 3 1 1 3 4 7 1 4 4 . 4.0 9th week 0 1 44- _ „ 2 3 1 2.8 13th week 3 61 0 3 , 2 “° 15th week 1 1 2 2 1 1 x 3^d week 1 1 2 S 6.2 6 3 -----F P F --- 13 14 15 16 25 26 27 28 3 5 3 F F M M 7 8 4 6 9 2 1 1 1 2 3 1 1 1 3 3 3 11th week 3 TABLE XII-S RAT WO* CARIES SCORE Sex Upper Lower Ldwer Total Average Remarks Rt. Lft. Place on Caries Producing Diet at beginning of: 1 2 3 M M M 4 5 6 F F F 4 5 7 s 9 M M ‘M 10 11 12 F F F 13 14 15 F F M 16 17 IS F M M 19 20 21 F M M --- ___ —— —— I 3 5 2 6 10 5 7.0 3rd week 3 4 ~i 3 s s 6.3 5tii week 2 4 3 2 3 4 4 7 7 6.0 7th week 4 3 3 4 3 3 s 6 6 6.6 9th week 1 1 4 2 5 3 o 2*6 11th week ___ ___ --- 0 2 0 3 5 3 2 3 1 --- T _____ . 1 1 3 2 1 .66 2.0 13th week 15th week hi 2.Vdezxs 2nd BB, 3rd - B 41* " B " ‘ Sli ” B* CHART ITI-2 Average of Tables 111-1,2, 8: 3. Periods Of Impaction. PART II THE EFFECT OF VITAMIN D ON THE EXPERIMENTAL PRODUCTION OF CARIES 4 g. Part II: The Effect of Vitamin D on the Experimental Production of Dents,1 Caries The importance of Vitamin D in a diet has been quite conclusively demonstrated and its function as a regulator of calcium-phosphorus metabolism is well known. It controls the deposition of these minerals in the tissues, hence it may be (Considered an essential in the formation of normal bone and should play an important role in tooth formation and the maintainance of a normal tooth structure. Studies of blood calcium and phosphorus values in normal animals and in animals on experimental diets that Vitamin D effects the absorption and excretion of these two minerals as well as their utilization and deposition. It would appear, there­ fore, that Vitamin D is needed throughout life, the require­ ment being greatest d u r i n g infancy, and next greatest during pregnancy and lactation. Calcium storage in the human fetus begins at about the fourth month, increasing thereafter, and becoming most rapid toward the end of pregnancy. If the calcium and phosphorus stored by the mother falls below the fetal demand, a severe drain on the tissue reserves of the mother will take place, which may account for the old adage, ”for every child a tooth”. The work of E. V. McCollum and of May Mellanby (*3&) indicated that Vitamin D played, a protective role in the development of caries, and while it did not prevent completely, it did reduce the incidence of caries. There has been considerable controversy as to the role and importance of this vitamin in the picture of dental caries. It was thought advisable to determine its effects, if possible, in the development or prevention of caries as produced in rats. Many workers are of the opinion that the Vitamin D re­ quirement of the rat is relatively much less than in the case of the human. To study the effect of this factor, a series of experiments was set up as follovrs: Experiment 1. To study the effect of high Vitamin D backgrounds, the offspring of four virgin females were used. The females were started on the stock diet containing 3*J> ir­ radiated dry yeast as a source of the vitamin. Feeding the diet was continued through pregnancy and lactation, til the young were 5 weeks old. and un­ The young v/ere than placed on the caries producing diet containing coarse rice as a cereal. The control group was composed of young animals of the seme age selected from the stock colony. For the first five weeks, these controls were fed the stock diet contain­ ing y!o ordina.ry dry yeast. caries producing diet. They too were then planed on the Both groups were fed the experimental diet for a period of ten weeks. At the end of this period, the animals were destroyed, the mandibles removed, cleaned, scored, and filed, Scores of this group will be found, in Table IV-1 and the comparison of the scores is shown on Chart IV-1. Experiment 2. A second group of young from the females used above were placed on a duplicate experiment. Results of 50. this second experiment will be found in Table IV-S and the comparison of the scores is found on Chart IV-1, with the combined comparison of the two groups on Chart IV-1. Experiment 3* The third experiment was set up to de­ termine the possible significance, if any, of the time at which the Vitamin D supplement was fed. The animals used in this experiment were offspring of 12 virgin females. Of the 12, only 10 produced litters which ivere reduced to 6 animals each in all cases but one, where 7 were used. Ir­ radiated dry yeast was used as a source of Vitamin D. The supplement was fed each group for a certain period. The feeding plan was as follows: Groups A &. B: 2 females were fed the stock diet con­ taining 3$ irradiated yeast through pregnancy and lactation and until the young were five weeks old.. At the beginning of the 6th week, the young, which made up Groups A and B, were placed on the coarse rice diet for 9 weeks. The period of feeding on the coarse rice diet was shortened here, as the lesions in Experiments 1 and 2 were rather severe for good scoring. The breakdown after the end of the eighth to ninth week seemed to be greatly accelerated possibly due to fracture of the shell-like structure that remained. Groups 0 & D: 2 females were fed stock diet containing 3% irradiated veast through pregnancy and lactation and until the young were 17 days old. Then, the stock diet containing plain dry yeast was substituted until the young were 3 weeks old. At the beginning of the 6th week, the young, which make 51. up Groups C and D, were placed on the coarse rice diet for 9 weeks. Groups E & F: 2 feraales were fed the stock diet with 3% irradiated yeast through pregnancy and until the young were born. At birth of the young, the diet was changed to the stock diet with plain dry yeast. This was fed until the young were five weeks old, then changed to. the coarse rice diet for 9 weeks. One of the feraales failed to pro­ duce a litter, so that this group is made up of young frorn the one female and is Group E. Groups G & H: 2 feraales were fed the stock diet wj. th plain dry yeast through pregnancy and until the young were born. The rad ion was then changed to the stock diet con­ taining yjo irradiated yeast and fed until the group was 5 weeks old. The young, which make up Groups G and H, were then placed on the coarse rice diet for 9 weeks. Groups I & J: 2 females were fed the stock diet con­ taining plain yeast through pregnancy and until the young were 1J days old (this is about the earliest age at which the young will begin to eat the stock ration). The ration was then changed to stock diet containing 3fi irradiated yeast and was fed until the young were 5 weeks old. At the beginning of the sixth week, the young were placed on the coarse rice diet which was fed for 9 weeks. Groups K & hi 2 females fed stock diet with plain yeast through pregnancy, lactation, and until the young were 5 weeks old. Only one female produced a litter, the young 52. of which make up Group K, and which were placed on the coarse rice diet at the beginning of the sixth week, and continued for 9 weeks. The results of this third experiment are found in Table IV~3» and the comparison of the scores is shown on Chart IV-S. Results The results of Experiments 1 and 2 quite closely parallel ea.ch other. The average score for the group with the high Vitamin D background in Experiment 1 is 1.S4, in Experiment 2, the score is 2.0. The control group in Ex­ periment 1 shows a score of 9.7> while Experiment 2 is 11.4. This would definitely point toward a protective ac­ tion when Vitamin D was fed prenatally at liberal levels. In Experiment 3> "the average scores run from 2.3 to 8.4-. They are quite consistent for all except two groups, which when eliminated give an average range of 6.3 to S. These two groups, i.e., Group E and Group K, gave abnormally low values and since there was only one litter in each they should probably be disregarded. Discussion From the results of these experiments, the feeding of high levels of Vitamin D show some fairly significant results In the case of Experiments 1 and 2, we see that there was a definite decrease both in incidence and in severity of caries The controls on this group gave scores that were quite comparable with results obtained in other similar experi­ ments, and the wide difference in the two scores would certainly indicate that the Vitamin D does act as a pro­ tective factor in the development of caries. In Experiment 3> no such clear cut evidence is found. The groups on the high Vitamin D levels, in fact, show a higher caries score than do the control groups. The pur­ pose of the experiment was to determine, if possible, what period of feeding of the Vitamin D supplement produced the most effective protection against caries. The data are too limited to warrant the drawing of any definite conclu­ sions. In view of the results obtained with the first two experiments, it might be that a general statement could be made to the effect that high Vitamin D supplements fed the mother during“periods of pregnancy and lactation would pro­ duce a protective action in the teeth of the young. The rapidity with which the teeth develop in the young rat is almost startling, and it is easily conceivable that any di­ etary imbalance might easily cause a hypoplasia that would go unnoticed under macro examination. It is certainly logical to believe that a well developed tooth of sound, structure and calcification should be more resistant to caries than one which is the opposite in nature. In all probability, the pattern for the development of a carious lesion is the same in any tooth, regardless of its struc­ ture, development, or calcification. The difference would 5^. surely be found in the rate of breakdown, the poorer struc­ ture going to pieces much more rapidly. From that stand­ point then, Vitamin D can be of considerable value in the development of teeth of superior structure and calcifica­ tion. This would seem to be the manner in which the vita­ min exerts its protective action. Further, if such is the case the most important period for the feeding of a supple­ ment would be that during which the teeth are being formed, that is, to the mother during pregnancy and lactation and to the young during the first five weeks after birth. Some exploratory work was also done with Vitamin 0 supplements but the data gave no evidence to encourage fur­ ther investigation. In studying such dietary factors as the vitamins, it must be Kept in mind that the requirements of the rat are undoubtedly different than for humans, and further that the vitamin picture in the dietary is one of many factors and not of a single factor. Sumrnary and Conclusions Vitamin D appears to exert a definite protective action against dental caries when fed prenatally. There was little or no evidence of any protective ac­ tion when the Vitamin D was fed post-natally to the mother or to the offspring. The most effective period for feeding supplements of the Vitamin D is to the female during pregnancy and lactation, and to the young during the first five weeks. The protective effect is undoubtedly due to the fact that the Vitamin D permits the development of teeth of superior structure and calcification. 56. TABLE IV-1 CARIES SCORE RAT NO, Sex Upper Lower Lower Total Average Remarks Rt. Lft. — 1 M --- — — 2 M --- ------- ------- 3 M —— ------- — ^i- M 3 ------- ------- ** M —— — 6 M --- 7 F 6 F --- 9 F --- 1 2 3 10 F --- 5 2 7 11 F --- 4- 3 7 12 F --- 1 ------- l 13 F --- 1 -------, l l4 M --- 6 6 12 15 F --- 6 5 11 6 5 11 - - --- 0 0 0 3 0 1 ------- 1 1 --- 1 ------- 0 ------- Experimental Gr oup §I8S 16 9.7 » §1I11P sip 17 F "--- 6 3 9 18 F --- 5 2 7 19 M — '4 8 Control Group ft/Qrt V/T £> m GOA/7-/L0 ij m /■j/GH !//r £? Co/v 7-/zots A V£Z/iG£ 0£ E^rr. Ifijz /-//GE V/7- jy COAJ-T7Z.Ol~ CcoAKSE fc/CEj CiiAhT IV-1 Prom Tables IV-1, & 2? High Vitamin D Background. TABLE IV-2 CARIES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 2 3 4 5 6 7 F F F F F F F S 9 10 11 M M M M 12 13 14 15 16 17 IS M M M M M M 19 20 21 22 97 M F F F F F --_ 1 5 — — --~— ---— 4 2 3 3 7 2 4 4 ^ ^ •2p 3 — ------- ------- 1 1 ------- 2 — — „ ------- ------- — _ ------- ------- _ _ — — — ------- — _ 1 1 2 . — ------- — 2 ------- 1 _ — — — . 1 2 ------- — — — _ _ _ ------- . 1.7 ___ 1 0 4 6 0 0 0 2 — ___ --1 < —+ _ ___ 3 1 — 3 Experimental Group 2 0 0 0 0 X s 1 3 1 3 0 4 2.2 Average for all above 24 25 £6 27 2S 29 30 31 M M F F M M F F ------------— — - — — . — ------- 4 5 5 4 6 6 7 S 12 5 5 3 7 6 5 3 16 10 10 7 13 12 12 11 Average for all above - 11,4- Control Group TABLE IV-3 CARIES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 2 3 4 5 '6 M M F F 6 5 5 1 5 5 4 ^ -------- 5 5 F F 7 6 9 10 11 12 ----------------------------- 11 10 9 5 3 3 10 M M M F F * Group A l1 -------3 -------5 -------6 F 3 ----5 3 5 6 2 5 6 10 12 5 10 Group B Average of Group A & B 7*3 M 15 16 17 16 19 20 21 22 23 24- ----------------- m F f F u M M — --------- M F 1 4 5 5 7 0 7 12 10 15 7-3 Group 0 6 R 6 4 2 2 4 - 5 3 3 5 2 11 10 4 9 6 7 7.3 Group D 3 1 1 5 1 1 F ----— - M M F F ------— -— -------- 1 4 2 -----— 5 2 11 £ ::: i. 35 35 F F 6 5 3 3 7 5 3 Average of Group C & D 7.3 M 31 32 M F M --— 2 - 25 26 ?7 26 29 30 - 4 3 3 3 I 6 2 13.5 Group E 6 5 3 5 5 12 7 Continued on next page 7-5 Gr oup G 6o. TABLE IV—3 (cont*d.) CARIES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. 37 3S 39 4-0 41 42 43 ----- 5 5 4 45 3 4 4 9 5 10 4 s R 9 4 9 3 6 4 g F 5 3 3 3 4 3 5 3 2 R 5 3 M 3 4 - 7 5 4 M M M M F F F ---------------- S.4 Group H Average of Group G & H & M M M f 44 45 46 47 4S 49 f 50 — - — - 51 52 m ---- m --p 54 55 F F ----- 2 1 5 10 6 5 g 10 6 - , 7-5 Or oup I _ . 5*1 T Group J 9 0 2 3 10 2 5 Average of Group I & J 6.3 56 57 56 59 60 61 M M 2 M m F F 1 2 1 1 2 1 3 1 3 2 2 5 1 1 0 , «roup ^ A Ve/LA C,£ \5cOHE of Cjfzoup A C & ■ C ^ ' ' I i» CHART I V -2 Prom. Table IV-3; Host Pffective Peri od of Feeding V i ta mi n D. PART III THE EFFECT OF FEEDING VARIOUS CARBOHYDRATES ON THE DEVELOPMENT OF DENTAL CARIES Part III: The Effect of Feeding Various Carbohydrates on the Development of Dental Caries The literature is replete with references to the sig­ nificance of carbohydrates in the picture of dental caries* Practically every article written emphasizes the role of carbohydrates. Bunting and his group (?), .Bosebury and co-workers (32), Belding and Belding (2), Hanke and co-wor­ kers (3), to mention only a few, have all pointed out the significance of carbohydrates. In only one instance, the work of Lilly (27), was it reported that there was no ap­ preciable effect of carbohydrates on the development of caries. The possible explanation in this case may be that a fine diet was used which alone would fail to initiate carious lesions. Moderate use of sweets has long been the advice of the dental fraternity, and the work of Koehne (25) with a group of children has amply shown the effect of su­ gar on the incidence of caries. Against this background, a series of experiments was planned to answer, if possible, several questions such as: What types of carbohydrates are most active in their in­ fluence on caries? Do all carbohydrates initiate caries, or is the effect of some simply that of extending the des­ truction of the tooth structure once caries has been initi­ ated? Accordingly, the following series of experiments was set up: EXPERIMENT NO. 1. The effect of substituting various sugars for cereal. A group of 29 selected animals was placed at weaning (21-28 days) on the basal rice, or caries producing diet, containing: 60 parts coarsely ground rice, SO parts whole milk powder, 6 parts flax­ seed meal, 5 parts alfalfa meal, 3 parts dried yeast, and 1 part sodium chloride. At the end of five to six weeks, depending upon the development of lesions as de­ termined by examination, the diet was changed to the ex­ perimental ration and feeding was continued for a period of 7-8 weeks, the experiment being terminated in the 8th week. The experimental rations used had the following composition: Diet No. 1 Carbohydrate 67$ Casein (comTl.) Salt Mixture Dried Yeast Corn Oil Cod Liver Oil Total 18$ 4$ (Sucrose. Glucose, or Lactose) (Modified Steenbock) In this experiment, the carbohydrate level was slightly higher than the cereal content. The animals were divided into eight groups with the distribution and rations as fol­ lows: Group A - 4 animals (2 M, 2 F); Ration containing 67$ Sucrose Group B - 4 animals (2 M, 2 F); Ration containing 67$ Glucose Group C - 4 animals ( 2 M, 2 F); Ration was Carbohydrate Free Diet using 20$ 30$ 19$ 30$ 1% Flax-seed Meal Alfalfa Meal C om,l. Casein Crisco Sodium Chloride Group D - 4 animals (2 M, 2 F); Ration containing 07% Lactose Group E - 2 animals (2 F); Ration Coarse Rice Control Diet containing 66$ 30$ 3$ 1$ Coarsely Ground Rice Milk Powder Whole Alfalfa Meal Sodium Chloride Group F - 4 animals (l M, 3 F); Ration containing a combination of Sucrose and Oatmeal as follows: 30$ SU crose 4$ Salt Mix 27$ Ground Oatmeal 5$ Dried Yeast 18$ Com1!* Casein 5$ Corn Oil 1$ Cod Liver Oil Group G - 4 animals (2 M, .2 F); Ration Oatmeal Control Diet as follows: 66$ 30$ 3$ 1$ Group H - 1 animal (l F); Oatmeal Whole Milk Powder Alfalfa Meal Sodium Chloride Ration containing Milk Powder as follows: 90$ Whole Milk Powder 9$ Alfalfa Meal 1$ Sodium Chloride At the beginning of the experimental period, there were four animals in Group E, but two died during the process of examination under ether anesthesia. However, the results with the coarse rice diet had been fairly well standardized so that the two animals remaining served as sufficient ba­ sis for comparison. The caries scores of these animals are found in Table V— 1, and the comparison of the scores is shown on Chart V-l. Result e Before the results are cited, it would be well to men­ tion tha.t the diets were apparently not as satisfactory as had been hoped. weight, Although the animals were able to maintain or to gain slightly, seemed to decline. particular, their general physical condition The animals fed the high lactose diet, showed definite signs of a toxic effect. in This necessitated lowering the level of carbohydrate in subsequent studies. For qualitative purposes, be of some interest. however, the results may The score of the rice controls with a value of 9 showed an average similar to that of other animals fed the same diet. The high score of the entire experiment was 9.4- for the average of the group on glucose. Sucrose gave a value of 6, lactose B.5, carbohydrate-free 2, oatmeal and sugar combination 2, oatmeal basal 0, and high milk 0. Discussion Because the diets were not entirely satisfactory, it is a bit difficult to draw any definite conclusions from this experi ment. There are one or two points that may be mentioned, how­ ever. The 6 7 level of sugars in the experimental diets was obviously too high, particularly in the Case of lactose, which is now known to be toxic at high intakes. Moreover, there does 66. not appear in this experiment to be any greater degree of caries on the high carbohydrate diets than was found with the coarse rice diet. All the control diets showed scores of around 2 or less which is no more than would be expected on the basis of the preliminary 5 weeks feeding on the caries producing diet. Ono difficulty is that it is not possible to assess accurately the condition of the teeth in live animals in the early stages of decay. The initiation of the carious lesion may take place some time before the lesion can be re­ cognized by gross examination— indeed such must be the case. It is, therefore, easy to see that too much reliance cannot be placed on the results of this first experiment with the sugars. EXPERIMENT NO. 2: The effect of substituting carbohy­ drates at a lower level, and their influence on dental caries. A group of 1^ animals was placed on the rice basal diet as given in Experiment 1. At the end of five to six weeks, de­ pending on the development of lesions as determined by exami­ nations, the diet was changed to the experimental ration and feeding was continued for a period of 7-8 weeks, ment being terminated during the eighth week. the experi­ The experimen­ tal diets used were based on the following formula: Diet No. 2 Carbohydrat e 35$ Casein (com11. ) Flax-seed Meal Alfalfa Meal Dried Yeast Crisco Sodium Chloride Total 20$ 20$ 100$ (Sucrose, Glucose, or Lacto se) Note that the carbohydrate level has been reduced from 67$ to 35^, as compared to the diet of Experiment 1. The animals were divided into five groups with distribution a.nd diets as follows: Group A — 3 animals (2 M, I F ) ; Ration containing 35/* Sucrose Group B ~ 3 animals (2 M, I F ) ; Ration containing 35/* Glucose Group C - 3 animals (1 M, 2 F); Ration containing ! 35$' Lactose ' Group D - 3 animals (1 M, 2 F); Carbohydrate-free Diet as given in Experiment 1 Group E - 2 animals (2 M) ; Coarse Rice Control Diet as given in Experiment 1 The caries scores of these animals will be found in Table V-2, and the comparison of the scores is found on Chart V-2. Results The high average score of the group is shown with sucrose giving a value of 10, followed by the rice control at 9«5» lactose at 10.3, glucose at S.3 , and the carbohydrate-free diet at 6. In this experiment, the animals on glucose showed a slight drop over the first experiment. Both sucrose and lac­ tose showed results slightly higher than the control rice, but probably not significantly greater. The carbohydrate-free diet gave a score of six which was a little higher than expected. Discussion From the results obtained, there are several points of 68. interest. It is apparent that when the animals were placed on the experimental rations, they must have attained a fairly uniform initiation of caries in the teeth, for the scores show fairly similar values. The usual score for animals on the caries producing diet is about 10, and in both Experi­ ments 1 and 2, this value was maintained. The average scores of the animals on the sugars were 10.3» 10> and 8.3 which shows that even after the rice was taken away, the lesions continued to develop and at about the same rate. In the ab­ sence of both coarse cereal and sugars, the rate of dental deterioration was appreciably slower and was probably due to the small amount of rice left in the ca.vities at the time the rats were put on the several modified diets. The average scores in the second experiment were generally higher than those in the first. The principal purpose of the second ex­ periment was to develop a ration nutritionally adequate end yet satisfactory for studying the decay of teeth in rats. In all subsequent experiments, the general proportions of this ration were used. Summary and Conclusions A 35$ level of sugars proved satisfactory for compara­ tive studies of tooth decay in rats. The modified ration gave better growth, weight, and a generally healthier experimental animal. The results indicate in general that the lesions develop on the sugar containing diets at about the same rate as on the coarse rice or caries producing diet. 69. The ca.rbohydrate-fr e e diet gave a slightly higher score than was anticipated. This was probably due to the presence of rice in the cavities at the time the diets were changed. EXPERIMENT NO. 3: The next step in the work with car­ bohydrates was to compare the rela.tive effects of the cereal, if finely ground, with those of the various sugars. All the animals were fed the coarse rice ration (The simpler diet composed of 66’ jo coarsely ground rice, JOfo whole milk powder, 3$ alfalfa meal, and l lactose, 11.^1, sucrose, 1 0 . glucose, 10.3» fine rice, J, and the cs.rbohydrate-free diet, 5*&* The -animals on the coarse rice and fine rice rations in Experiment 3 were somewha.t higher than expected, bxrt fell to about their normally expected values in Experiment U-. Discussion It was interesting to note that lactose gave the highest avera.ge of all the sugars. It is possible that the scores in this series were slightly higher due to the fact that the le­ sions produced by the coarse rice in the preliminary feeding period may have been somewhat more severe than previously. It would be expected from the work done by such workers as Rosebury et al (32), Hanke (15), Florestano (10), and Bran­ son (^), we would expect to find sucrose most active with glucose following. However, little work has been done with lactose, per se, and a final conclusion should probably be held in reserve until the results of some of the subsequent experiments have been reviewed. It would appear that the development of the lesion does continue on the diets con­ taining the various sugars but not as rapidly as when the coarse rice is fed. Vyith the fine rice, the lesions appar­ ently slow down in their rate of development for the score is considerably.lower than for any of the sugars. This seemed a bit surprising in view of what happened with the coarse rice. Once having been initiated, it was thought that the fine rice would shoxv at least as rapid a development of the lesions as was found with the sugars. Of course, one must always consider the possibility that the rapid rate with the coarse rice might be due in part to frac­ ture inasmuch as the carious nature of the teeth rendered them more fragile, cation. and less able to withstand the forces of masti­ Further, the fine rice may have exerted a filling ac­ tion on the lesions which, though they had started to develop, might still have been small enough that when the fine rice be­ came impacted it behaved as a filling keeoing out oral fluids, thus retarding bacterial fermentation. It might be well to reserve final judgement on the effects of the fine rice until the data from additional animals may be scrutinized. Summary and Conclusions Carious lesions continued to develop or enlarge under the influence of the various sugars. The rate was, apparently, not as rapid as with the coarse rice in the caries producing diet. The effect of all three sugars was about the same, although lactose gave slightly higher scores than sucrose and glucose. Fine rice showed lower results than were expected. EXPERIMENT NO. 5: of Experiments 3 and 4. This experiment was a repetition This was done to check two ques­ tionable points that arose in the preceding work. It seemed desirable to determine whether or not lactose was consistent in producing a higher caries score, and whether or not the score produced on fine rice would be consistent­ ly lower than that of the sugars. In addition, a modifica­ tion was made in that groups of anima.ls were placed on the experimental rations without the preliminary 4 to 6 weeks period bn the coarse rice diet. A group of 81 animals was selected. These were taken at weaning (21-28 days), and were divided into three groups: Group A: 4 units of four animals each. These units were placed on the experimental rations at once, having no preliminary feeding period on the coarse rice diet. From time of birth until about 28 days old they had access to the stock diet which contained some ground corn. fed these four groups were: The rations fine rice ration, 35$ sucrose ration, 35$ glucose ration, 35$ lactose ration (rations described In Experiment 2). The experimental rations were fed for a period of 14 weeks. Group B: 6 units of ten animals each. These units were placed on the coarse rice diet for from 4 to 6 weeks for the initiation of carious lesions as determined by ex­ amination. When the lesions were found to have started, the animals were immediately transferred to the experimen­ tal rations for a period of 8 weeks. The rations used (given in Experiment 2) were as follows: coarse rice, fine rice, carbohydrate-free, 55$ sucrose, 55$ glucose, and 55$ lactose. Group C: 1 unit of five animals was placed on the coarse rice diet at weaning. This was fed for a period of six weeks, at the end of which time the animals were des­ troyed and the mandibles removed and scored as usual to give a standard of comparison for the animals on the experimental rations. The caries scores for Group A are found in Table V-5a, for Group B and Group C in Table V-5b. The comparison of the scores for all three groups is shown on Chart V-4. Results In the case of Group A the score for the fine rice was 7.2, for sucrose 3.5, for glucose 7.7, and for lactose 4.5. These scores were obtained with the animals having no pre­ liminary feeding period on the caries producing diet. The scores of the animals in Group B, which had a pre­ liminary feeding period, were much higher and as follows: fine rice IS, coarse rice IS.2, lactose 10.5, sucrose 10, glucose 7.7, and carbohydrate-free 8.9. It is interesting to note that fine rice gave the highest score, sucrose and lactose were about the same, while glucose gave a lower score than the carbohydrate-free diet. The reference group, Group C, which was terminated after 6 weeks feeding on the coarse rice diet showed a score of 5.4. This will give an idea as to the average at the time of transfer to the experimental rations. Discussion The results of Group A would lead one to believe either that the experimental rations would initiate carious lesions, or that some impaction had taken place on the stock diet, thus initiating caries which further developed on the experi­ mental rations. most valid. It would appear the latter explanation was It Is interesting to note that glucose gave the highest score in this group, while in the following group, it gave the lowest value. Fine rice also gave a high value. The results of Group B were quite consistent and about what might be expected. It was thought the fine rice should show as great or greater effect in the development of caries after the lesions were once started, since the finely ground cereal would furnish a source of some carbohydrate and also a continuous supply of the aciduric organisms. to be the case. Such proved With the sugars, sucrose and lactose, the average scores were lower than either the fine or coarse rice but were appreciably higher than the carbohydrate-free diet and also much higher than the score of the comparison Group C at 5.4. The score of the glucose ration was con­ sistent from individual to individual but for some reason was low. It appears that there is a possibility of a de­ crease of the number and strength of the acid producing bac­ teria where the cereal is completely removed and a more or less pure form of carbohydrate substituted. Further, the production of acid by the bacteria present might be great enough, in the areas where impaction and lesions were found, to kill the bacilli. To exceed the maximum pH for existence of the bacteria at certain focal points of infection is not inconceivable. Summary and Conclusions It may be concluded that a diet containing a high level of carbohydrate in the form of some sugar will cause the carious lesions to become enlarged. The sugars alone are not likely to be the cause of the initiation of caries. The cereal, whether fine or coarse, usually causes more severe lesions, since it not only furnishes a form of carbohydrate but also a source of the acid forming organisms. Further work with the sugars would be of interest, par­ ticularly in light of the results with glucose. EXPERIBIMT HO. 6 : This is a repetition of some of the previous experiments with slight modifications. group of 48 animals was selected, and at the beginning of their 6th week, the animals were placed on the coarse rice diet for the initiation of caries. This preliminary period was from 5-6 weeks as determined by the examination of the molars. When teeth showed the initial development of caries, the animals were placed on the experimental ration which was fed for a period of 6 rather than 8 weeks. The 8 week peri­ od seemed to allow the destruction of the tooth to advance too far for good scoring. '-L'he distribution of animals was as follows: (b) (c) (d) (e) 8 animals continued on coarse rice ration. 10 animals on the sucrose ration. 10 animals on the glucose ration. 10 animals on the lactose ration. 10 animals on the carbohydrate-free ration. The fine rice diet was eliminated from the series. At the end of the sixth week on the experimental ration, the ani­ mals were destroyed, the mandibles removed and scored as usual. Scores of these animals are found in Table V-6 , with the comparison of the scores shown on Chart V-5. Results The average scores obtained are as follows: coarse rice 11.6, sucrose 10, glucose 10.6, lactose 9.7, and carbo­ hydrate-free 8 . The scores are slightly lower than in pre­ vious work due to the shorter period on experimental rations but on the whole seem to be more consistent. Discussion The over-all averages of this group are more consistent and form a good pattern. normal response. The glucose group showed a more The cartoohydrate-free diet gave a score slightly higher than expected, tout even so, the other scores are still from 2 to 3 points higher, which shows that lacking available carbohydrate the development of the carious lesions is slowed down. In this work, the three sugars showed scores closely grouped which would indicate that they all function in the same way and to about the same degree. Summary and Conclusions The lesions develop more rapidly in the presence of carbohydrate in such form as sugars. The most rapid development requires the presence of the cereal which furnishes carbohydrate and acid forming organism as well. EXPERIMENT NO. 7: animals was selected. In this experiment, a group of 30 The group was divided into 4 units of 7 animals each, and 1 unit of 2 animals. They were 28-30 days old, and all were placed on the coarse rice (caries producing) ration until on individual examination caries could be detected. At this stage, the score toeing a minimum of 1 and i a maximum of 3, the animal was placed on the experimental ration of its respective unit for a period of 8 weeks. The animals were destroyed at the end of the eighth week, the mandibles removed and scored as usual. The feeding plan was as follows: Unit Is 7 animals on carbohydrate free diet. Units 2, 3 and 4: 7 animals each on the high car­ bohydrate diet with the following modifica­ tions 20% Flax-seed meal, 20% Casein (com1!.), 20% Alfalfa Meal, 5% Crisco, Dried Yeast, 1% Sodium Chloride, and 30^5 Carbohydrate (Su­ crose, Glucose, or Lactose). All ingredients were carefully blended before adding the car­ bohydrate, which was mixed in the last thing to avoid coating the sugars with the fat. Unit 5: 2 animals as controls on the coarse rice. The scores of the above animals may be found in Table V-7 with the comparison of the scores shown on Chart V-5. i Results The scores again conformed fairly well with those previously obtained. Glucose gave a value of 14*3, Sucrose 13.3, Lactose 12.3, Carbohydrate-free 9.6, and the 2 ani­ mals on the coarse rice, which served as controls, 15.5. Discussion It is again seen that the carious lesions continue to enlarge on diets containing sugar, but the advance is not as rapid as when the animal is continued on the coarse rice diet. The average scores of all the animals on the sugar diets was 13.3, an increase in the average score of 3.7 over the carbohydrate free diet. This 13.3 average is 2.2 less than that of the animals on the caries producing diet. From these results, and those of preceding experiments, it would seem to have been definitely established that though the sugars do cause an increase in the severity of lesions that have been initiated, the lesions are more severe if the animal is kept on the caries producing diet. The su­ gars may cause an increase in severity though they do not initiate the lesion. A point might be made of the fact that it is highly probable, in the case of the rat at least, that the sugars themselves are not so harmful unless fed in a diet con­ taining the caries producing factor, i.e. the coarsely ground cereal as rice or corn. If such a combination were fed the effect of the sugars superimposed on the coarse cereal would undoubtedly be a more drastic one. As has been mentioned before, the possibility of accurate and sig­ nificant scoring diminishes after the breakdown in tooth structure has advanced beyond a certain point. Thus, a feeding experiment involving both factors would have to be checked by careful individual examinations. Otherwise, the scores would approach a maximum in every case, and in less time than the eight week period used when the foods in question were fed separately. Summary and Conclusions It may be concluded that carbohydrate in the form of sugars causes the lesion to develop and become larger. The development is fairly rapid and severe. When sugars are included in the diet, the lesions are not as severe as when the caries producing diet is fed a similar length of time. If the sugars were fed with the caries producing diet, maximum severity of lesions and rapidity of breakdown in tooth structure might be expected. EXPERIMENTS NO. 8 and 9: These two experiments were designed to give proof to the point mentioned in the dis­ cussion and summary of the preceding experiment. What would be the effect of feeding a combination of sugar with the caries producing diet? To do this, a ration was devised which contained both coarse rice and sugars in the form of a c o m syrup. The syrup was first mixed thoroughly with the coarsely ground rice so that the rice was quite effectively coated with the syrup. The syrup-rice mixture was allowed to dry, then crumbled, and the other ingredients added and mixed by hand-, The formula of the ration is as follows: 66 parts by weight coarsely ground rice SO » n ” whole milk powder 5 * n w alfalfa meal 1 a n * sodium chloride 10 n " M corn syrup (Dark Karo) It was also decided that it would be of Interest to observe the effect of a high Vitamin D intake with such a ration. The procedure was as follows: Experiment 8 was carried out with 16 animals 28-30 days old, which were divided into one group of 10 animals (4 M, 6 F) fed the corn syrup diet for 8 weeks, and one group of 6 animals (2 M, 4 F) fed the coarse rice diet for 8 weeks. Experiment 9 was carried out with the offspring of 5 young females fed the regular stock diet containing Z% 81 irradiated yeast of high Vitamin D potency. The diet is reviewed here: 40% Ground yellow corn Z0% Oatmeal 20% Whole milk powder 6% Alfalfa meal Z% Irradiated yeast 1% Sodium chloride From the litters, 24 young were selected and at 28-50 days were divided and placed on the experimental rations as follows: Group 1 - 5 Group 2 - 8 animals (Z M, 2 animals (4 M, 4 F} Corn Syrup Ration F; Corn Syrup Ration plus Z% Irradiated Yeast Group 5 - 8 animals (4 M, 4 F) Coarse Rice Ration plus Zfa Irradiated Yeast Group 4 - 2 animals (S F) Coarse Rice Ration The experimental rations were fed for a period of 10 weeks. Experiments 8 and 9 were terminated during the 10th week, when the animals were destroyed, mandibles removed and scored as usual. The scores of Experiment 8 are found in Table V-8 , for Experiment 9 in Table V-9, with compari­ son of scores for both experiments being shown on Chart V-6 . Results The results were very much as anticipated and the scores for animals in Experiment 8 gave averages of 14.2 for the corn syrup ration against 12.5 for the coarse rice ration. The results in Experiment 9 gave averages of 16.8 for the corn syrup ration, 15.9 for the same ration containing the irradiated yeast, 15.2 for the coarse rice ration con­ taining the irradiated yeast, and 15 for the coarse rice ration alone. The results are quite consistent and do in­ dicate a protective action for the Vitamin D. Discussion A ten weeks period was used in Experiment 9 for feed­ ing the experimental rations inasmuch as all the previous experiments using carbohydrates had been preceded with a 4-6 week preliminary feeding on the caries producing ra­ tion. The differences were not as marked as was antici­ pated. It is entirely possible, however, that had the period of feeding been somewhat shorter, there would have been greater differences. The action of the cereal as initiator and of the su­ gars as accelerator is quite plainly established. The re­ sults show that corn syrup acted as did the sugars in pre­ vious experiments causing lesions to become more severe, and that its effect superimposed on the action of the caries producing diet gave a score that was greater than would be obtained with either one alone. The results confirm the theories and observations of such workers as Bunting and co-workers, Rosebury, Belding and Belding, Cox, and many others. The protective action of the Vitamin D observed in previous work was again apparent. The work done with the corn syrup ration indicates the desirability of a standardized caries producing diet. Such standardization would allow the application of statistical methods, and it would then be possible to evaluate the effect 83, of each factor. The availability of animals of uniform response would also be of assistance. This is largely a problem of selective breeding in which some progress has been made by Hunt and Hoppert who have developed strains of caries susceptible and caries resistant rats (19), The adoption of standardized procedures and improvement in accuracy, in selecting rats of approximately identical de­ gree of decay initiated by the use of coarse rice, would materially aid in a more quantitative comparison of the materials studied in this research. Summary and Conclusions It may be concluded that when a combination of sugar and cereal is fed, more severe lesions are produced than when these foods are used singly in suitable diets. Under the conditions of the experiments performed, the influence of Vitamin D is protective rather than preven­ tive. 84. TABLE V~1 CARIES SCORE RAT NO* Sex Upper Lower Lower Total Average Remarks Lft. Rt. a. 1 2 5 4 M M F F 5 6 7 8 M M F F 9 10 11 12 M M F F 15 14 15 16 M M F F 19 20 M M 21 22 25 24 M F F F — — — 25 26 27 28 M M F F ______ — — — — ------- ------ - — — — — — 29 F —-— — .— 2 1 . --— — — t— — ------— r— • 6 1 1 2 7 5 1 5 13 4 2 5 6 5 5 7 4 7 0 15 9 15 2 1 2 --- —— 4 4 7 11 1 5 3 ----------- — . -— ___ Glucose Carbohydrate-free 2.5 Lactose Coarse Rice 8 0 0 0 Oatmeal and Sucrose Oatmeal Basal —— 0 0 0 0 ___ 0 High Milk 5 ------ 9.4 2 1 4 1 2 2 6 0 2 1 5 ------- - ' .. 2 1 5 5 2 — — .. — Sucrose — -- —— —— CoAZS&g/C£ ‘SUCZOEJz CfLucose L A o to s e CAfZ0OtfYD. '/=&££ C >atm e A L+S u c# o s£ \ Oatmeal 0aSAJ- tf/GH /V/A.K Z£&o £/m £ , CHART V-l Prom Table V-l; Preliminary Results on High Sugar Diets. T AB L E V - 2 0ARI ES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 2 3 F M M 4 5 > O M M F 7 g 9 M F F 10 Xi 12 ^ F F U ■- — _ ——— 2 7 6 4 7 4 6 l4 10 10 Sucrose 3 4 4 7 S 10 s.3 Glucose —— 4 4 6 — " --- 7 5 5 6 .... 5 3 13 . 10 & 10,3 Lactose ------------- M III 4 2 4 1 3 4 6 \ 5 5 S 12 6 Carbohydrate-free 9.5 Rice Control CoARstZ fact S ucrose . Gi-ucose. LACTOSE CAE6oyr°- CHART V-2 Prom Table V-2; Feeding Various Sugars >t 35% Levels. 885. TAB LE V-3 r >V* no . CARIES SCORE Sex Upper Lower Lower Tot. Rt. Lft, 1 3 M M I 4 5 M M F 6 7 ■8 9 M M F F t 10 11 12 13 M M F F ___ l4 15 16 17 M M F F 18 19 F F 9 8 9 10 18 21 5 3 4 4 4 4 9 7 8 6 7 4 7 5 10 5 8 11 17 9 15 5 7 3 10 4 8 6 . 9 9 15 9 19 13 Glucose ------- . — —— 9 10 7 6 7 7 2 6 16 17 Q 12 13.5 Lactose --- . 6 4 6 3 12 7 9.5 3 ,„ ,~f —-. —T . -- ---_____ ___ 19.5 Rice Control Garboliydrat e— f ree Sucrose Fine Rice 39. TABLE V-4 CARIES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 f 2 f x f p -------________ 3 \ 3 y ________ ________ 2 2 5 5 F -------to M -------7 ,M — - ^*5 P 4 £^ 2.5 4 & 8 6,6 4 2 3 4 13 9 8 9 10 H M [-— M F F 2 2 7 5 3 2 6 ^ 12 M' M F 3 6 5 5 3 4 u 4 a 25 16 17 f M M --3 — 6 2.1 ------- 2 --—— 2 -2 - 3 2 10 12 9 - Rice Control Carbohydrate-free Glucose ^ 7.3 Sucrose 9.2 Lactose 4.5 Fine Rice to TA3C£ X-3 C&A£5£ 1&C£ £>OOEosd ■ {f/,UC£?S.e ' .: ....-...is Lrtc.ros£ CrtfieoHy&j-'tr&e£ f/A fE & /C E C b a e s e ~%/c£ E C/Lu Co^E L A c ro s s . CAE3oHY£>. F~b.s.i Z ^ /A /J S & /C E . L/,A / E CI-IAET V-o P r o m T a b 3.9s V - 3 , & 4 : Compara tiv e Effects of F e e di ng Various Sugars, and Pine Rice. 91 TAB LE V-5 A R A T NO* C A R IE S SCORE. Sex Upper Lower Lower Tot£ Rt. L ft. 1 2 3 4 F F M M 5 6 7 s F F M M 9 10 12 F F M l4 15 16 F F M M 3 3 4 4 ---* _ 2 3 — , 2 ______ --— —----, --- — . 4 3 3 5 7i 6 7 9 7*2 Fine Rice 3 2 ___ 2. 3 5 0 4 3.5 Sucrose 10 9 7*7 Glucose 4.B Lactose Rs 4 O* t . 3 5 2 6 2 2 2 2 2 2 S 2 4 1 4, 92* TABLE V - 5 B C A RI ES SCORE Rt, 17 IS 19 20 21 22 25 26 27 28 29 30 31 32 3 3 36 37 3S 39 42 4 4 45 46 47 4$ 49 50 H 52 54 55 56 M M M M M M F F F F M M M M M M F F F F M M M M ^ M M F F F F M M M M M “ M F F F F --—— _— , — — --- . --— —* _— --- ■ --_— 3 --— . _ ___ --t ^ „ 2 -— , —4— — —— --- “ — ___ Lf t . 6 7 6 5 6 4 10 10 7 6 5 6 4 7 5 7 7 9 9 5 6 11 13 10 12 11 11 19 19 12 12 13 Fine Rice 4 4 10 7 7 5 8 5 6 2 4 5 8 6 7 5 8 6 8 2 8 9 IS 16 l4 10 16 11 16 4 12*2 Coarse Rice 6 4 5 3 | 3 6 4 4 5 6 4 ^ b 6 3 I 2 I 3 10 11 11 7 I 8 12 7 10 s 5 2 9 4 7 6 5 6 3 S• 2 4 4 ? 4 ■9 2 4 7 5 4 3 7 R 17 6 11 10 ^ 10 l4 6 g s9 10 Carbohydrate-free Sucrose 7 l4 C o n t i n u e d on next page 93. T A B L E V-5B (con't.) C A R I ES SCORE Rt. 64 65 66 M M M M M M F F F F 67 62 69 70 71 72 7? 74 75 76 M M M M M M F F F F 57 52 59 60 61 62 §? — , . Died — —. —— --- —— Lft. 6 11 5 12 7 5 if 3 7 4 2 2 4 1 3 before completion 4 2 2 4 9 5 8 5 3 6 11 5 --— -—.--- ----- . — ---- --- 4 4 4 10 5 11 1 2 6 5 4 3 6 8 7 10 1 3 0 ,5 8 7 10 18 12 21 2 5 12 10 7.7 Glucose 10.5 'Lactose Control Group C: Terminate at 6 weeks for comparison 77 7& 79 SO 21 M ----M — ‘ 5> --F --F 3 3 2 1 3 ^ 7 ^ 1 3 3 3 7 4 6 5.4 Coarse Rice TX&L-e. V-SA <5iSGKO<5£ QtLc^gose 4*4C710S£ F4aS£ tt&£. 7Z8L£ J&S8 GoARS.gTP/c-E. 'Q o c e o s e CjrLo'COS B ^Across • &*/£. 7?/ce i C A eaortm &se41 CbAfiseffic-z & lA/ee/csG m /t^oL Z & X o /-/*£ CHART V-4 From Tables V-5a, & 5b; Comparative Effects . of Feeding Various Sugars, Fine Rice, and Coarse R i c e . TABLE V«6 CARIES SCORE RAT HO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. l a 3 4 5 6 7 8 M m — — M M F----- F----- F F - 6 6 5 4 6 3 6 7 9 M-----10 M 11 ' M — 12 M --13 M 14 F --15 F 16 F----- 17 F Xg F----- - 6 7 5 4 3 5 2 7 3 6 7 5 7 4 6 6 8 7 13 ii 12 g 12 11.6 Coarse Rice 10 Sucrose 10.6 Glucose l4 12 i4 11 4 - 8 5 10 4 - 9 2 4 8 15 3 6 5 11 6 7 6 - F 5 5 10 F — 3 4 7 7 11 18 F----- F-----7 8 15 -------448 23 M -------6 5 11 24 M 25M -— 9 6 15 26 M 1 3 4 27 M -------3 5 8 28 M Died before completion !9 20 21 29 F 30 F 31 32 F ---------F --- 6 4 6 7 4 - 3 3 4 5 4 5 5 5 5 5 6 10 13 7 7 9 9 10 11 35 M 36 m M --T.ir ----------- 37 M ----- 2 6 8 38 M ------ 7 6 13 9 -7 Lactose Continued on next page 96. TABLE V-6 (cont*d,) CARIES SCORE RAT NO. Sex Upper Lower Lower Total Average Remarks Rt.Lf t $ £ itl IS 4-1 4-2 & I F F F F M M M M M M 5 c; 10 3 6 9 2 2 6 9 k3 3 3 6 2 k 3 3 I 8 15 6 I 6 8 Carbohydrate-free 97 TABLE V-•7 CARIES SCORE RAT HO. Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 2 3 4 R v 6 7 F F F F F M M S 9 10 11 12 13 14 F F M U M M M _ _ — ----—— ---- - — ... — r . -----. --- R S 3 3 4 6 5 4 7 4 3 4 6 5 9 15 7 1 6 8 12 10 9,6 7 7 9 .. 8 7 5 6 2 R 8 9 6 8 6 9 12 17 17 13 13 12 13.3 Sucrose: , Carbohydr at e 15 M ----16 M 17 F ----IS .F 19 M ----20 M 21M — 9 7 5 7 6 9 4 10 2> s 7 5 10 7 19 15 11 14 11 19 11 14.3 Glucose 22-----F------23-----p------gl}. p 25 p _— . 26 M 27 M 2S M 7 6 5. g 6 6 2 S 13 4 - 1 0 7 12 S 16 5 11 7 13 7 9 12-3 Lactose fo 7 li 1 5 -5 0o“ S8 Rlcs M --- ’7 98 TABLt- 3f~ CsfA/zse Mose GtL u c o s b jLa c t o s b ® « Ca x s o w y ?.fke& 8 Z&tfo L/y£ CHART V-5 Prom Tables V-6, & 7; Feeding Various Sugars for 6 and 8 week Periods. 99. TABLE V-8 RA T NO, P $ •I Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 F ------- 6 g 14- 2 F ------ g 6 14- 3 M ------ 6 g 14- 4- M ----- . s 7 15 5 F — 6 10 6 F ------ 4- 7 11 7 M o S 7 16 g M ------ 9 7 16 9 F ------ g 7 15 10 F — — g 9 17 11 F _ — 9 g 17 12 F ------ 6 5"' 11 13 M — — 7 6 13 _ l4-. 2 --- 5 6 11 15 F --- 4- 3 7 16 F S 7 15 Corn Syrup Ration m 12.3 'M X'*4- I I R 9fev CARIES SCORE Coarse Rice Only 100 TABLE V-9 CARIES SCORE RAT NO* Sex Upper Lower Lower Total Average Remarks Rt. Lft. 9 S 7 9 10 10 s 7 7 9 19 16 14 16 19 l6.S Corn Syrup Ration r F F M M M 6 7 S 9 10 11 12 13 F F F F M M M M 6 5 R -<• 5 9 10 9 11 S 7 7 5 10 10 9 .11 14 12 12 10 19 20 IS 22 15.9 Corn Syrup Ration plus 1% Irradiated Yeast Ik 16 17 IS 19 20 21 F F F F M M M M 7 7i 6 6 6 6 6 10 R R 6 6 6 9 5 9 12 12 12 12 12 15 11 19 13.2 Coarse Rice Ration plus 1f0 Irradiated Yeast 22 Ai*0 7 c 2M F F F 10 9 5 7 S 6 17 17 11 15 Coarse Rice Ration 1 2 3 4- 77t&*.£ IT-8 Coarse TP/^-s T&t/o// 5 Cora/ 7A 0L£ Tf-f C o a r s e 7 ? /c e CoA/e^s 72/^e + Yeast C o r a / SyRv/^&tr/eR CoRA/CyRor far/otJ& + J1/ZR.AR. Ye a s t CZ/^ a z o *-//ve CHART V-6 Prom Tables V-S, & 9; Feeding Corn Syrup and Coarse Rice Simultaneously; Use of High Vitamin D Supplement. bibliography a h d l i t e r a t u r e c i t l d . 102 . BIBLIOGRAPHY AND LITERATURE CITED 1. . 2 Agnew, M.C., Agnew, G., and Tisdall, E.F. J. Amer. Dent. Assoc. 20; 195 (1955) Belding, P.H., and Belding, L.J. Dental Carles, 2nd Edition (1941) Findings and Conclusions on Its Causes and Control. Lancaster Press, Inc., Lancaster, Penn* Pages 47-50 (1940) S. Blayney, J.R.: Ibid.: pages 55-58 (1941) For himself and co-workers. 4. Branson, Charles B . : Ibid.: pages 67-68 (1940) 5. Breese, Frederick: Ibid.: page 68 (1940) 6* Brodsky, Ralph H . , Schick, Bela, and Volmer, Herman Ibid.: pages 74-75 (1941) 7. Bunting, Russel W . , and Jay, Philip Ibid.: pages 76-79 (1924-57) For themselves and co-workers in the Michigan Group Research on Dental Caries. a. Cox, Gerald J.: 9. Fleisch, Louis M. s Ibid.: pages 104-106 (1941) 10 . Ibid.: pages 86-87 (1941) For himself and co-workers. Florestano, H.J.: Ibid.: pages 106-107 (1941) Forshufvud, Sten: Ibid.: pages 107-108 (1959) n. . 12 Fosdick, Leonard S.: Ibid.: page 108 (1941) For himself and co-workers. IS. Gottlieb, Bernhard: Ibid.: pages 111-112 (1941) 14. Haber, G.G.: 15. Hanke, Milton T.: Ibid.: pages 116-118 (1940) In collaboration with the Chicago Dental Research Club. 16. Hanke, M.T., et al Dental Cosmos 75: 655 (1955) Ibid.: pages 115-117 (1941) For himself and co-workers, research in Germany and Switzerland. 103. 17. Idem.: Ibid. 75: 739 (1933) 18. Idem.: Ibid. 75: 933 (1933) 19. Hunt, H.R., and Hoppert, C.A. Inheritance in Hat Caries. Genetics: 24: 76 (1939) 20. Hoppert, C.A., Vtfeber, P.L., and Canniff, T.L* The Production of Dental Caries in Rats Fed an Adequate Diet. J. Dental Research 12: 161-173 (1932) 21. Klein, H . , and McCollum, E.V. J. Dental Research 12: 524 (1932) 22. Idem.: 23. Koehne, M . , and Bunting, R.W. A Review of Recent Studies of the Cause of Dental Caries. J. Am. Diet. Assoc. 9: 445-461 (1934) 24. Koehne, M . , and Bunting, R.W. Biochemical and Nutritional Studies in the Field of Dentistry. Ann. Rev. Biochem. 3: 441-458 (1934) 25. Koehne, M . , Bunting, R.W., et al Studies in the Control of Dental Caries, II. J. Nutrition 7: 657-678 (1934) 26. LeFevre, Marian L. Ibid. 13: 69 (1933) Dental Caries, 2nd Edition Pages 156-157 (1938) 27. Lilly, C.A. Failure to Produce Caries in White Rats. J. Nutrition 5: 175-181 (1932) 28. Lilly, C.A. Dental Caries, 2nd Edition Pages 164—165 (1938) 29. McClendon, J.F., and Foster, W.C. Ibid.: pages 171-172 (1941) 30. McCollum, E.V. and co-workers Ibid.: pages 172-174 (1940) 31. Mellanby,,May Ibid.: pages 175-176 (1939) 104. 32. Rosebury, Theodor, Ibid.: pages 200-202 (1939) For himself and co-workers. 33. Rosebury, Theodor and Karshan, Maxwell J. Dental Research 11: 121 34. Rosebury, Theodor and Foley, Genevieve Ibid. 12: 462-464 (1932) 35. Rosebury, T., Karshan, M . , and Foley, G. Ibid. 12: 247 (1933) 36. Idem.; 37. Wessinger, George D. Dental Caries, 2nd Edition Page 235 (1940) 38. McCollum, E.V. Physicians Vitamin Reference Book Second Edition Revised, 1938 E.R. Squibb & Sons, New York Page 114 Mellanby, May Ibid.: page 114 Ibid. 13: 379 (1933) 61. /9l^/Z» Cr£ k5 c OZ,£ OF C?fZOL/F> A < £■ & ■ A c£ ^ C i m R f IV ~ 2 F r o m T a b l e I V - 3; 13o e t E f f e c t i v e P e r i o d o f F e e d i n g V i t a m i n D. i P A R T III T H E EF F E C T OF F E E D I N G V A R I O U S CARBO H Y D R A T E S O N T H E D E V E L O P M E N T OF DENTAL CARIES P a r t III: The E f f e c t o f Feeding V a r i o u s C a r b o h y d r a t e s on the D e v e l o p m e n t o f D e n t a l Caries T h e l i t e r a t u r e is replete w i t h references to the sig­ n i f i c a n c e of c a r b o hydrates in the pic t u r e of d e n t a l caries* P r a c t i c a l l y every article w r i t t e n emphasizes the role o f carbohydrates. co—w o r k e r s kers (3), B u n t i n g and his group (32), Belding and Bel d i n g (7),.E o s e b u r y and (2 ), H a n k e and co—w o r ­ to m e n t i o n o n l y a few, h a v e all p o i n t e d out the s i g n i f i c a n c e o f carbohydrates. v/ork o f L i l l y In o n l y one instance, the (27), was it reported that there was no a p ­ p r e c i a b l e effect o f carbohydrates on the d e v e l opment of caries. The p o s s i b l e explanation in this case m a y be that a fine d i e t was u s e d w h i c h alone w o u l d fail to initi a t e carious lesions. M o d e r a t e use of sweets has long b e e n the a d v i c e o f the d e n t a l fraternity, and the w o r k of K o e h n e (25) w i t h a group o f children has a m p l y shown the effect of s u ­ gar 011 the i n c i d e n c e of caries. A g ainst this background, p l a n n e d to answer, a series of ex p e r i m e n t s was if possible, several q u e s t i o n s such as: W h a t types o f carbohydrates are m o s t active in their in­ f l u e n c e o n caries? Do all carbohydrates i n i t i a t e caries, or is the effect o f some s i m p l y that o f e x t e n d i n g the d e s ­ t r u c t i o n o f the t o o t h s t r u c t u r e once caries has been i n i t i ­ ated? A c c o r dingly, the f o l l o w i n g series o f experiments was set up: E X P E R I M E N T NO. 1. T h e effect of s u b s t i t u t i n g various sugars for cereal. p l a c e d at w e a n i n g A group of 29 selected animals was (21-28 days) caries p r o d u c i n g diet, ground rice, seed meal, containingr 60 p a r t s 30 parts w h o l e m i l k powder, coarsely 6 parts f l a x ­ 5 parts a l f a l f a meal, 3 parts d r i e d yeast, and 1 p a r t sod i u m chloride. weeks, on the basal rice, or At the end o f five to six d e p e n d i n g upon the d e v e l opment o f lesions as d e ­ t e r m i n e d b y examination, the diet was changed to the e x ­ p e r i m e n t a l ra t i o n and feeding was c o n t i n u e d for a p e r i o d o f 7 — 8 w eeks, week. the experi m e n t being t e r m i n a t e d in the 8 t h T he e x p e r i m e n t a l rations used had the following composition: Diet No. 1 Carbohydrate C a s e i n ( c o m Tl.) Salt M i x t u r e T-,-1 J -C7-_ „ „ 4Iccioo Corn Oil Cod L i v e r Oil Total 67$ (Sucrose. Lactose) 4$ (Modified Steenbock) CC^ Glucose, or 5$ 1$ 100$. In this experiment, the c a r b o hydrate level was s l i g h t l y hi g h e r t han the cereal content. The animals w e r e divi d e d into eight gr o u p s w i t h the d i s t r i b u t i o n and rations as f o l ­ lows : G roup A - 4 animals (2 M, 2 F ) ; R a t i o n containing 6 7 $ Suc r o s e G r o u p B - 4 animals (2 M, 2 F ) ; R a t i o n containing $ Glu c o s e G r o u p C - 4 ani m a l s ( 2 M, 2 F) ; R a t i o n was Car b o h y d r a t e Free Diet using 2 0 $ 30% 1.2% 30$ 1% G r o u p D - 4 animals F l a x - s e e d Meal A l f a l f a Meal C o m fl. Ca s e i n Crisco S o d i u m Chloride (2 M, 2 F ) ; R a t i o n containing 67$ L a c t o s e G r o u p E — 2 animals (2 F ) $ R a t i o n Coarse Rice Control Diet containing 66$ 30$ 3$ 1$ G r o u p F - 4 animals C o a r s e l y Ground R i c e M i l k Po w d e r W h o l e A l f a l f a Meal S o d i u m Chloride (l M, 3 F ) ; R a t i o n containing a c o m b i n a t i o n o f Sucrose and Oatmeal as follows: 30$ S u c r o s e 4$ Salt M i x 27$ Ground Oatmeal 5$ Dried Yeast 18$ Com*l, Ca s e i n 5$ Corn Oil 1$ Cod Liver Oil G roup G - 4 ani m a l s (2 M, 2 F ) ; R a t i o n Oa.tmeal Control Diet as follows: 66$ 30$ 3$ 1$ Group H - 1 animal (l F ) ; Oatmeal Whole Milk Powder Alfalfa Meal S o d i u m Chloride R a t i o n containing M i l k P owder as follows: 90$ W h o l e M i l k Pow d e r 9$ A l f a l f a M e a l 1$ S o d i u m Chloride At the b e g i n n i n g of the e x p e r i m e n t a l period, t h e r e were four a n imals in G r o u p E, but two d i e d du r i n g the proce s s of e x a m i n a t i o n u n d e r ether anesthesia. However, the results w i t h the coarse rice diet had been f a i r l y well stan d a r d i z e d so that the two animals remaining; served as sufficient ba­ sis for comparison. The caries scores of these anim a l s are found in Table V— 1, and. the comparison of the scores is shown on Chart V— 1. Result s B e f o r e the res u l t s are cited, it would be well to m e n ­ tion that the diets were apparently not as satisfactory as had been hoped. weight, Alth o u g h the animals or to gain slightly, seemed, to decline. were able to mai n t a i n their general physical The animals fed the high lactose diet, p a r t i c u l a r , showed definite signs of a toxic effect. n e c e s s i t a t e d lowering- the level of carbohydrate studies. be of For condition qualitative purposes, some interest. in This in subsequent h o w e v e r , the results may The score of the rice controls w i t h a. value of 9 showed an average similar to that of other animals f ed the same diet. The high score of" the entire experiment was 9.^ for the average of the group on glucose. a value of 6, lactose Sucrose gave carb o h y d r a t e — free 2, oatmeal and sugar c o m b i nation 2, oatmeal basal 0, a n d high milk 0» Discussion Because the diets were not bit d i f f i cult entirely satisfactory, it is a to draw any definite conclusions from this experi ment. There are one or two points that may be mentioned, ever. The 67c'Jo level of o b v i o u s l y too high, is n o w known how­ sugars in the experimental diets was particularly in the case of lactose, to be toxic at high intakes. Moreover, which there does not appear caries in this experiment on the h i g h to be a ny g r e a t e r carbohydrate the c o a r s e r i c e diet. All the diets on t h e b a s i s of th e p r e l i m i n a r y p r o d u c i n g diet. One d i f f i c u l t y assess accurately in t h e early lesion may the c o n d i t i o n s t a g e s o f decay. take showed 5 weeks f e e d i n g on the c a r i e s is that it is not the to animals of th e c a r i o u s l e s i o n c an be r e ­ c o g n i z e d by g r o s s examination— indeed such must It is, easy be pla c e d on th e r e s u l t s possible of t h e t e e t h in l i v e The i n i t i a t i o n to see sc ores t h a n w o u l d be e x p e c t e d plo.ee some time b e f o r e therefore, of than was f o u n d w i t h control diets of a r o u n d 2 or l e s s w h i c h is no m o r e degree be t h e case. that to o m u c h r e l i a n c e c a n n o t of this first experiment with the sug ars. EXP TRIM BINT NO. drates A at a lower 2: The e f f e c t of l e v e l , and their substi tut ing c a r b o h y ­ influence on d e n t a l caries, group of lU- a n i m a l s wa s p l a c e d on the r i c e b a s a l d i e t as given in E x p e r i m e n t p e n d i n g on t h e nations, feeding ment tal At the development the di et was 1. of was c h a n g e d c o n t i n u e d for end of f i v e t o six le si ons to as d e t e r m i n e d b y the 7-8 weeks, e i g h t h week. d i e t s u s e d w e r e b a s e d on t he f o l l o w i n g Diet No. Carb 0hydrats yyk C a s e i n ( c o m 11.) Flax-seed meal A l f a l f a tiea.l Dried Yeast Cri sco Sodium Chloride Total 20# 20 *jo 9f ic io 100 the and experi­ The e x p e r i m e n ­ formula: 2 (Sucrose, G l u cose, Lactose} de­ exami­ the e x p e r i m e n t a l r a t i o n a p e r i o d of being terminated during weeks, or 67. IS'Ote that the cs.rbohydrat e level has been r e d u c e d from 67# ‘bo 35^? animals diets as compared to the diet o f Experiment 1. were d i v i d e d into five groups The with di str itmtion a n d as follows: Gr oup a 3 animals 35 Group B (2 M, 1 F); R a t i o n containing Sucrose 3 animals (2 i/i, I F ) ; R a t i o n containing 35°/° Glucose Group C 3 animals (1 M, 2 F); Ration containing 3 5 % Lactose Group D 3 animals (l ii, 2 F); C a r b o h y d r a t e — free Diet as given in Experiment 1 Group E — 2 animals (2 ii) ; Coarse R i c e Control Diet as given in Experiment 1 The caries sc ores of these animals will be found in Table V — 2, a n d the c o m p a r i s o n of the scores is f o u n d on Chart V-2. Result s The high average score of the group is shown w i t h sucrose g iving a value of 10, f o l l o w e d by the rice control at 9.3? lactose at 6. at 10.3, In this glucose at 0.3? experiment, slight drop over the first and the car b o h y d r a t e — free diet the animals on glucose experiment. showed a Both sucrose and lac­ tose showed results slightly higher than the control rice, p r o b a b l y not s i g n ificantly greater. gave a score of six whic h 'was The carb o h y d r a t e - f r e e diet little higher than expected. Di s c u s s i o n From the res u l t s obtained, there but are several po i n t s of 68. interest. It is apparent that when the on the e xperimental rations, animals ware placed they must have a t t a i n e d a fairly u n i f o r m initiation of caries in the teeth, s h o w fairly similar values. the c a r i e s p r o d u c i n g diet The usual is about 10, shows that on and in b o t h Exp e r i ­ on the sugars were 10.3» The average scores 10, and 8.3 wh i c h even after the r i c e was taken away, the c o n t i n u e d to develop and at about the seme rate. sence scores score for animals m e n t s 1 and 2, this value was maintained. of the a n i m a l s for the of both coarse cereal and sugars, lesions In the a b ­ the r a t e of dental d e t e r i o r a t i o n ’w as appreciably slower a n d was p r o b a b l y due to the small amount of rice left in the cavities at t h e the rats were put on the several m o d i f i e d diets. scores those The average in the second experiment were gener a l l y higher than in the first. periment yet time The principal purpose was to develop of the second ex­ a ration nutri t i o n a l l y adeq u a t e and s a t i s factory for stud y i n g the decay of teeth in rats. all subsequent experiments, the general p r o p o rtions In of this r a t i o n were used. Summary and C o n c l usions A 35

and 4- are s c o r e s of E x p e r i m e n t s 3 a n d 4- h a v e show the The r e s u l t s The c a r i e s in T able V-3. 4- wa s a d u p l i c a t i o n T a b l e V — ^-i. c o m b i n e d to C har t were comparison of s c o r e s and discussion g i v e n on of these two experi­ ments will be considered together. R e suit s Th e find the average s c o r e s of th e a n i m a l s c o a r s e r i c e diet 1 1 .4-, su crose, nigh with a value 10. 4-, g l u c o s e , es.rbohydra.te-free diet, a n d fine r i c e r a t i o n s than expected., values but of l6*5> 10. 3> f i n e rice, The a n i m a l s in E x p e r i m e n t fell in E x p e r i m e n t from b o t h experiments to about 7> on th e 3 were a n d the coarse somewhat their n o r m a l l y lactose, rice higher expected 4. Discussion It w a s average this interesting of a l l series the we re to n o t e th at sugars. slightly It is p o s s i b l e higher period may have been somewhat w o u l d be expected from Rosebury et al son we w o u l d e x p e c t (4), glucose (32), following. in th e more (15)> to fi nd However, the the scores the in le ­ feeding s e v e r e tha n p r e v i o u s l y . Florestano such workers (10), as and Bran- sucrose most active lit t l e hig h e s t tha t preliminary the w o r k d o n e by Hanke tha t due to t h e fact s i o n s p r o d u c e d by the c o a r s e r i c e It lactose gave with w o r k has b e e n d o n e with lactose, per Be, and a final conclusion should, probably be held in reserve until the results of some of the subsequent experiments have been reviewed. It would appear that the development of the lesion does continue on the diets con­ taining the various sugars but not as rapidly as when the coarse rice is fed. bith the fine rice, the lesions appar­ ently slow down in their rate of development for the score is considerably lower than for any of the sugars. This seemed a bit surprising in view of what happened with the coarse rice. Once having been initiated, it was thought that the fine rice would show at least as rabid a, v-O • * development of the lesions as was found with the sugars. Of course, one must always consider the possibility that the rapid rate with the coarse rice might be due in part to frac­ ture inasmuch as the carious nature of the teeth rendered more fragile, and less able to withstand the forces of masti­ cation. Further, them the fine rice may have exerted a filling a.c— tion on the lesions which, though they had started to develop, might still have been small enough that when the fine rice be­ came impacted it behaved as a filling keeping out oral fluids, thus retarding bacterial fermentation. It might be well to reserve final judgement on the effects of the fine rice until the data from additional animals may be scrutinized* S u m m a r y and Conclusions Carious l e s i o n s continued to develop or enlarge under the i n f l uence of the various sugars. T h e rate was, apparently, not as rapid as w i t h the coarse r i c e in the caries p r o d u c i n g diet. T h e ef f e c t o f all th r e e sugars was about the same, a l t h o u g h l a c t o s e gave s l i g h t l y higher sc or es than sucrose and glucose. F i n e r i c e showed lower results than w e r e expected. E X P E R I M E N T NO. 5: o f E x p e r i m e n t s 3 and 4. This experiment was a repeti t i o n This was done to check two q u e s ­ t i o nable po i n t s that arose in the prece d i n g work. It seemed d e s i r a b l e to d e t e r m i n e whether or not lactose was consistent in produ c i n g a h i g h e r caries score, and w h e t h e r or n o t the score prod u c e d on fine rice w o u l d be c o n s i s t e n t ­ l y lower t h a n that of the sugars. In addition, a modifica­ ti o n was m a d e in that groups o f animals w e r e placed on the e x p e r i m e ntal rations w i t h o u t the p r e l i m i n a r y 4 to 6 weeks p eriod b n the coarse rice diet. A g r o u p o f SI animals was selected* at w e a n i n g These were taken (21-28 d a y s ) , and w e r e d i v i d e d into three groups: Group A: 4 units o f four animals each. These units w e r e p l a c e d o n the expe r i m e n t a l rations at once, having no p r e l i m i n a r y feeding p e r i o d o n the coarse rice diet. From time of b i r t h u n t i l about 28 days old t h e y had access to the stock diet w h i c h contained some ground corn. fed these four groups were: ration, 3 5 % glu c o s e ration, d e s c r i b e d in E x p e r i m e n t 2). The rations fine rice ration, 3 5 % sucrose 35% lactose rat i o n (rations The e x p e r i m e n t a l rations w e r e 73 fed for a pe r i o d of 14 weeks* G r o u p B: 6 units o f ten animals each. These units w e r e p l a c e d on t h e coarse rice diet for from 4 to 6 weeks for t h e i n i t i a t i o n o f carious lesions as d e t e r m i n e d by ex­ amination. W h e n the lesions w e r e found to have started, the a n i m a l s w e r e i m m e d i a t e l y transferred to the exper i m e n ­ tal r a t i o n s for a period of 8 weeks. The rations used (given in E x p e r i m e n t 2) w e r e as follows: rice, c a rbohydrate-free, coarse rice, 35% sucrose, 3 5 % glucose, fine and 35% lactose. G r o u p C: 1 unit o f five animals was pla c e d on the coarse rice d i e t at weaning. six weeks, This was fed for a period of at the end of w h i c h time the animals were d e s ­ t r o y e d and the m a n d ibles removed and scored as usual to give a standard of comparison for the animals on the experimental rations. T h e caries scores for Group A are found in Table V — 5a, for G roup B and Group C in T a b l e V-5b. T h e comparison of the scores for all three groups is shown on Chart V-4. R esults In the case of Group A the score for the fine rice was 7.3, for s u c r o s e 3.5, for glu c o s e 7.7, and for lactose 4.5. T hese scores w e r e obtained w i t h the animals having no p r e ­ l i m i n a r y f e e d i n g period on the caries p r o d u c i n g diet. T he scores o f the animals in Group B, w h i c h had a p r e ­ l i m i n a r y f e e d i n g period, w e r e m u c h higher and as follows: f i n e r i c e IS, coarse rice 12.2, l a c t o s e 10.5, g l u c o s e 7.7, and c a r b o h y d r a t e - f r e e 8.9. sucrose 10, It is interes t i n g to n o t e that fine r i c e gave the h i g h e s t score, sucrose and l a c t o s e w e r e about the same, w h i l e glucose gave a lower score than the carbohyd r a t e - f r e e diet. The reference group, Group C, w h i c h was terminate d after 6 weeks feeding on the coarse rice diet showed a score o f 5.4. This w i l l give an idea as to the average at the t i m e o f t r a n s f e r to the e x p e r i m e n t a l rations. Discussion T h e results of Group A vrould lead one to b e l i e v e either that the exp e r i m e n t a l rations would, initiate carious lesions or that some i m p a c t i o n had taken pl a c e on the s t o c k diet, thus i n i tiating caries w h i c h further developed o n the experi m e n t a l rations. m o s t valid. hi g h e s t It w o u l d appear the latter exp l a n a t i o n was It is interesting to n o t e that glu c o s e gave the score in this group, w h i l e in the following group, it gave the lo w e s t value. Fine rice also gave a h i g h v a l u e . T h e results of Group B w e r e quite consistent and about what might be expected. It was thought the fine rice should show as great or greater effect in the d e v e l o p m e n t o f carles after the lesions w e r e once started, since the fi n e l y g round cereal w o u l d f u r n i s h a source o f some car b o h y d r a t e and also a continuous su p p l y of the aciduric organisms. to be t h e case. W i t h the sugars, Such proved sucrose and lactose, the av e r a g e scores were lower than either the fine or coarse r i c e but w e r e a p p r e c i a b l y higher than the c a r b o h y d r a t e — free diet and also m u c h higher than the score of the compar i s o n G r o u p C at 5.4. T h e score of the glucose r a t i o n was con­ sistent f r o m i n d i v i d u a l to i n d i v i d u a l but for some reason was low. It appears that there is a p o s s i b i l i t y o f a d e ­ c r e a s e of the n u m b e r and stre n g t h of the acid produ c i n g b a c ­ t e r i a w h ere the cereal is co m p l e t e l y removed and a mor e or less p u r e form of carbohydrate substituted. Further, the p r o d u c t i o n o f acid by the bact e r i a present might be great enough, in the areas where i m p a c t i o n and lesi o n s were found, to k i l l the bacilli. To exceed the m a x i m u m p H for existence o f the b act e r i a at certain focal points of i n f e c t i o n is n o t inconceivable. S u m m a r y and Conclusions It m a y be concluded that a diet containing a h i g h level o f c a r b o h y d r a t e in the f o r m o f some sugar will cause the carious lesions to become enlarged. T h e sugars alone are not l i k e l y to be the cause o f the i n i t i a t i o n of caries. T h e cereal, whe t h e r fine or coarse, u s u a l l y causes m o r e s e v ere lesions, since it not o n l y furnishes a form of c a r b o h y d r a t e but also a source of the acid f o r m i n g organisms. F u r t h e r w o r k w i t h the sugars would be of interest, p a r ­ t i c u l a r l y in light o f the results w i t h glucose. E X P E R I M E N T HO. previous 6: This is a r e p e tition of some of the experiments w i t h slight modifications. group o f 4 8 a n i m a l s was week, selected, and at the beginning o f their 6 t h the animals w e r e placed on the coarse rice diet for the i n i t i a t i o n o f caries. This p r e l i m i n a r y p e r i o d was f r o m 5-6 w e e k s as d e t e r m i n e d by the e x a m i n a t i o n of the molars. W h e n t e e t h showed the initial d e v e l opment of caries, the an i m a l s w e r e p l a c e d o n the e x p e r i m e n t a l ra t i o n w h i c h was fed f o r a p e r i o d o f 6 rather t h a n 8 weeks. The 8 week peri­ od s e e m e d to a l l o w the d e s t r u c t i o n o f the t o o t h to advance too far for good scoring. The d i s t r i b u t i o n o f animals was as follows: fa} (b) fc) (d) (e) 8 anim a l s 10 animals 10 animals 10 anim a l s 10 animals continued on coarse rice ration. on the sucrose ration. o n the glucose ration. o n the lac t o s e ration. o n the carb o h y d r a t e - f r e e ration. The fine rice d i e t was e l i m i n a t e d f r o m the series. end o f the s i x t h w e e k on the expe r i m e n t a l ration, mals w e r e destroyed, usual. At the the a n i ­ the m a n d i b l e s removed and scored as Scores o f these animals are found in T a b l e V— 6, w i t h the c o m p a r i s o n of the scores shown o n Chart V-5. Results T h e average scores o b t a i n e d are as follows: rice 11.6, sucrose 10, glu c o s e 10.6, h y d r a t e - f r e e 8. lactose 9.7, coarse and carbo­ T h e scores are s l i g h t l y lower t h a n in p r e ­ v i o u s w o r k due to the shorter pe r i o d on e x p e r imental rations but o n the w h o l e seem to be m o r e consistent. Di s c u s s i o n T h e o v e r - a l l averages of this group are m o r e consistent and f o r m a good pattern. n o r m a l response. The glucose group showed a m o r e The c a r b o h y d r a t e — free d i e t gave a score s l i g h t l y h i g h e r than expected, but even so, the other scores are still from 2 to 3 points higher, w h i c h shows that l a c k i n g available carbohydrate the development of the carious lesi o n s is slowed down. In this work, the three sugars s h o w e d scores c l o s e l y grouped w h i c h would indic a t e that t h e y all func t i o n in the same w a y and to about the same degree. S u m m a r y and Conclusions The lesions develop m o r e rapidly in the presence o f c a r b o h y d r a t e in such form as sugars. The m o s t rapid d e v e lopment requires the presence o f the cereal w h i c h furnishes carbohydrate and acid forming o r g a n i s m as well. E X P E R I M E N T NO. animals was of 7 animals 7: selected. each, 28-30 days old, In this experiment, a group of SO T h e group was d i v i d e d into 4 units and 1 unit of 2 animals. T h e y were and all w e r e placed on the coarse rice (caries producing) ration u n t i l on I n d i v i d u a l examinat i o n caries could be detected. At this stage, the score being a m i n i m u m o f 1 and a m a x i m u m of 3, the animal was plac e d o n the e x p e r i m e n t a l ration of its r e s p e c t i v e unit for a p e r i o d o f 8 weeks. The animals were d e s t r o y e d at the end o f the e i g h t h week, the m a n d i b l e s removed and scored as usual. T h e feeding plan was as follows: U n i t Is 7 animals on carbohydrate free diet. U n i t s 2, 3 and 4: 7 ani m a l s each on the h i g h car­ b o h y d r a t e diet w i t h the following m o d i f i c a ­ tion: 2 0 $ Flax- s e e d meal, 2 0 $ C a s e i n ( c o m fl.), 2 0 $ A l f a l f a Meal, 5$ Crisco, 4 $ Dried Yeast, 1$ S o d i u m Chloride, and 30$ C a r b o h y d r a t e (Su­ crose, Glucose, or Lactose;. All ingredients w e r e carefully b l e n d e d before adding the car­ bohydrate, w h i c h was mixed in the last thing to avoid coating the sugars w i t h the fat. U n i t 5: 2 animals as controls on the coarse rice. T h e scores o f the above animals m a y be found in Table V — 7 w i t h the co m p a r i s o n of the scores shown on Chart V-5. Results T h e scores a g a i n conformed f a i r l y well w i t h those p r e v i o u s l y obtained. 13.3, L a c t o s e 12.3, Glucose gave a value of 14.3, Sucrose C a r b o h y d r a t e - f r e e 9.6, and the 2 a n i ­ ma l s o n the coarse rice, w h i c h served as controls, 15.5. Di s c u s s i o n It is a g a i n seen that the carious lesions e n large o n diets containing sugar, continue to but the advance is not as rapid as when the animal is continued on the coarse rice diet. T he ave r a g e scores o f all the animals on the sugar d iets was 13.3, an increase in the average score of 3.7 o v e r the c a r b o h y d r a t e free diet. This 13.3 average is 2 . 2 less than that o f t h e animals on the caries produ c i n g diet. F r o m these results, and those of preceding experiments, it w o u l d s e em to have been d e f i n i t e l y established that though the sugars do cause an increase in the severity of lesions that h a v e been initiated, the lesions are m o r e severe if the animal is kept on the carles producing diet. The su­ gars may cause an increase in severity though they do not initiate the lesion, A p oint m i g h t be made of the fact that it is h i g h l y p r o b able, in the case of the rat at least, that t h e sugars t h e m s e l v e s are n o t so harmful un l e s s fed in a diet con­ t a i n i n g the caries producing factor, ground c ereal as rice or corn. i.e. the coarsely If such a c o m b i nation were fed t h e effect of the sugars superimposed on the coarse cereal w o u l d u n d o u b t e d l y be a more d r a s t i c one. been m e n t i o n e d before, As has the p o s s i b i l i t y o f accurate and s i g ­ n i f i c a n t sco r i n g d i m i n i s h e s after the b r e a k d o w n in t o o t h s t r u c t u r e has advanced be y o n d a certain point. Thus, a feeding e x p e r i m e n t In volving both f a c t o r s w o u l d have to be checked b y careful Indivi d u a l examinations. Otherwise, the scores w o u l d a p p r o a c h a m a x i m u m in every case, and in less t i m e than the eight w e e k period u s e d w h e n the foods in q u e s t i o n w e r e fed separately. S u m m a r y and Conclusions It m a y be concluded that c a r b o hydrate in the f o r m of sugars causes the le s i o n to develop and become larger. T h e d e v e l o p m e n t is f a i r l y rapid and severe. When sugars are included in the diet, the lesions are not as severe as when the caries producing diet is fed a similar length of time. I f the sugars w e r e fed w i t h the caries p r o d u c i n g diet, 80 m a x i m u m s e v e r i t y of lesions and r a p i d i t y of "breakdown in t o o t h s t ructure m i g h t be expected. E X P E R I M E N T S NO. 8 and 9: T h e s e two experiments w e r e d e s i g n e d to give p r o o f to the point m e n t i o n e d in the d i s ­ c u s s i o n and summ a r y o f the prece d i n g experiment. What w o u l d be the effect o f feeding a com b i n a t i o n of sugar w i t h the caries produ c i n g diet? To do this, a r a t i o n was d e v i s e d w h i c h contained b o t h c oarse r i c e and sugars in the f o r m o f a corn syrup. ‘ ^ he syrup w a s first m i x e d t h o r o u g h l y w i t h the coarsely ground r i c e so that the rice w a s quite e f f e c t i v e l y coated w i t h the syrup, crumbled, ^ h e s y r u p - r i c e mix t u r e was allowed to dry, then and the o t h e r ingredients added and m i x e d by hand. T he f o r m u l a o f the r a t i o n is as follows: 66 parts b y we i g h t 50 * ” n 5 n ” n 1 » n * 10 ” ” ” c o a r s e l y ground rice w h o l e m i l k powder alf a l f a meal sod i u m chloride corn syrup (Dark Karo) It was also d e c i d e d that it w o u l d be of I n t e r e s t to o b s e r v e the effect of a h i g h V i t a m i n D intake w i t h suc h a ration. The p r o c e d u r e w a s as follows: E x p e r i m e n t 8 was carried out w i t h 16 animals 28-50 days old, w h i c h w e r e div i d e d into one group of 10 animals (4 M, 6 F) fed the corn syrup diet for 8 weeks, group o f 6 animals and one (2 M, 4 F) fed the coarse rice diet for 8 weeks. E x p e r i m e n t 9 w a s carried out w i t h the o f f s p r i n g o f 5 y o u n g f e ma l e s fed the reg u l a r s t o c k diet containing " 6% i r r a d i a t e d yeast o f h i g h V i t a m i n D potency. The diet is r e v i e w e d here: 4 0 $ Gro-und y e l l o w corn 30$ O a t m e a l 20$ Whole milk powder Fr o m the litters, 6$ A l f a l f a meal 3$ I r r a d i a t e d yeast 1$ S o d i u m chloride 24 young w e r e selected and at 28-30 days w e r e d i v i d e d and p l a c e d on the experimental rations as follows: Group 1 - 5 Group 2 - 8 animals animals Group 3 - 8 animals Group 4 - 3 animals (Z M, 2 Corn Syrup R a t i o n (4 M, 4 F) C o r n Syrup R a t i o n plus 3$ I r r a diated Y e a s t (4 M, 4 F) Coarse Rice Ra t i o n plus 3$ I r r a diated Yeast (3 F) Coarse Rice R a t i o n T h e e x p e r i m e n t a l rations were fed for a pe r i o d o f 10 weeks. E x p e r i m e n t s 8 and 9 were t e r m i n a t e d during the 10 t h week, w h e n the animals were destroyed, mandibles removed and s c ored as usual. in T a b l e V-8, The scores o f Experi m e n t 8 are found for Experi m e n t 9 in T a b l e V — 9, w i t h compari­ son o f scores for b o t h experiments being shown on Chart V-6. Results T he results w e r e v e r y m u c h as anticipated and the scores for animals in Experiment 8 gave averages of 14.2 for t h e corn syrup ration against 12.3 for the coarse rice ration. T h e results in Experiment 9 gave averages o f 16.8 for the corn syrup ration, 15.9 for the same ra t i o n containing the i r r a d i a t e d yeast, 13.2 for the coarse rice ration c o n ­ taining the irradi a t e d yeast, and 15 for the coarse rice r a t i o n alone. The resu l t s are q u i t e c o n s i s t e n t and do i n ­ d i c a t e a p r o t e c t i v e ac t i o n for the V i t a m i n D. Discussion A t e n w e e k s p e r i o d was u s e d in E x p e r i m e n t 9 for f e e d ­ ing t h e e x p e r i m e n t a l rat ions i n a s m u c h as all the previous experiments using c a r b o h y d r a t e s had b e e n p r e c e d e d w i t h a 4 - 6 w e e k p r e l i m i n a r y feeding on the caries p r o d u c i n g r a ­ tion. T h e d i f f e r e n c e s w e r e n o t as m a r k e d as was an t i c i ­ pated. It is e n t i r e l y p o s s i b l e , period of feeding been somewhat however, shorter, that had the there w o u l d h a v e b e e n g r e a t e r d i f f e rences. T h e a c t i o n o f the cereal as i n i t i a t o r and of the s u ­ gars as a c c e l e r a t o r is q u i t e p l a i n l y e s t a b lished. ^he r e ­ sults s h o w that corn syrup a c t e d as did the sugars in p r e ­ vious experiments causing l e s i o n s to b e c o m e m o r e severe, and t h a t its ef f e c t s u p e r i m p o s e d o n the a c t i o n o f the caries p r o d u c i n g d i e t gave a score that w a s g r e a t e r than w o u l d be o b t a i n e d w i t h e i t h e r one alone. The results c o n f i r m the t h e o r i e s a n d o b s e r v a t i o n s of s u c h w o r k e r s as B u n t i n g and co -workers, others. Roseb u r y , Belding and Belding, Cox, and m a n y T h e p r o t e c t i v e a c t i o n o f the V i t a m i n D o b s e r v e d in p r e v i o u s w o r k was a g a i n apparent. The work done with the corn syrup ration indicates the desirability of a standardized caries producing diet. Such standardization would allow the application of statistical methods, and it would then be possible to evaluate the effect 83. o f e a c h factor. T h e a v a i l a b i l i t y of ani m a l s of u n i f o r m r e s p o n s e w o u l d also be o f assistance. This is l a r g e l y a p r o b l e m o f s e l e c t i v e b r e e d i n g in w h i c h some p r o g r e s s has b e e n m a d e b y Hunt and Hop p e r t who h a v e d e v e l o p e d strains of caries s u s c e p t i b l e and caries r e s i s t a n t rats T h e a d o p t i o n of s t a n d a r d i z e d p r o c e d u r e s accuracy, (19). and i m p r o v e m e n t in in s e l e c t i n g rats o f a p p r o x i m a t e l y i d e n t i c a l d e ­ gree o f d e c a y i n i t i a t e d b y the use of coarse rice, w o u l d m a t e r i a l l y aid in a m o r e q u a n t i t a t i v e c o m p a r i s o n o f the materials s t u d i e d in this research. S u m m a r y and Con c l u s i o n s It m a y be c o n c l u d e d that w h e n a c o m b i n a t i o n o f sug a r and c e r e a l is fed, m o r e severe les i o n s are p r o d u c e d t h a n w h e n t h e s e foods are u s e d si n g l y in s u i t a b l e diets. U n d e r the c o n d i t i o n s of the e x p e r i m e n t s performed, i n f l u e n c e o f 'vitamin D is p r o t e c t i v e ra t h e r than p r e v e n ­ tive • the TABLE V-l R A T NO* CARIES SCORE Sex Upper Lower Lower Total Average Remarks Rt. Lft. 1 2 3 4 M M F F 5 6 7 8 M M F F 9 10 11 12 M M F F 13 14 15 16 M M F F 19 20 M M — w. 21 22 23 24 M F F F __ — — — 25 26 27 28 M M F F — 29 F __ — ——— —— 2 1 fa* — —. ——— *^ 6 1 1 2 7 3 1 3 13 4 2 5 6 5 5 7 4 7 0 15 9 13 ,, 2 1 2 2 1 —— -- — 2 Sucrose 9*4 2 1 4 1 Carbohydrate-free 2*5 — 2 2 6 0 4 4 7 11 9 2 1 3 1 3 ---- 3 5 Glucose Lactose Co a r s e R i c e _ — — — 5 3 --- --- - ——_ ---— 8 0 0 0 O a t m e a l and S u c r o s e _ — •»«*«.CMT — ---—— — _— — — *—-* 0 0 0 0 Oatmeal Basal —— — 0 — __ _ 0 High Milk Coa&s&&/0£ m . O atme AJ--h5oc,/Zose* C>ATS-tJ=al* &A$Al- p ///&# M/A-K P Z jEZ&O JL/AJ0 , CHART V-l F r o m Table V-l ; P reliminary Results on H i g h Sugar Diets. 86. T A B L E V- 2 RAT Sex U p p e r F Ivl M 4 5 O M M F 7 i 1:1 8 9 F F s' 10 11 12 F F Ivl 11 M M ' --- 2 ---- 7 6 ------ — ---- 4 4 6 7 R 5 Lower Total Average Remarks Lf t . 4 7 4 6 i4 10 10 Sucrose ? 4 4 7 8 10 8.3 Glucose 6 5 3 13 io g 10.3 L ac t o s B 4 2 4 i ------ 3 4 E 5 8 CTn -R 1 2 3 Lower Rt. SCORE I 1 1 I 1 1 NO. 0ARIES I 12 ------ 6 Carbohydrate' 9.5 R i c e 'Control 4 Coar se fZ/CE CiscRose CrLucos £ LA OTOSE C A E S O V E&- CR£ ^EJEO JL/*S_E CdjriHl' V-2 P r o m Table V —2; .Pee>11up’ Various S u p e r s e t 35/» L e v e l s . gg. T A B L E V-3 C A R I E S SCORE NO. Sex U p p e r 1 2 M M — -__ 3 9 g 9 10 IS 21 3 4 5 M M F — --, — 5 3 4 4 4 4 9 6 7 S 9 M M F F 6 7 4 7 5 10 3 g 11 17 10 11 12 13 M M F F 5 7 3 10 4 g 6 9 9 15 9 19 l4 15 16 17 M M F F 9 16 6 7 7 2 6 IS 19 F F 6 4 6 3 — — _t i — — — ■— ____ — _ . — --------— — -------- . Lower Rt. 10 7 Lower Lft, i Total 7 s 19«5 g R i c e Control C ar b o layd r a t e - f r e e 13 Sucrose 13 Glucose 17 9 12 13.3 Lactose 12 7 9.g 9 15 M Fine R i c e I 2)9• TABLE V-4 R A T NO. CARIES.SCORE Sex U p p e r Lower L o w e r Total A v e r a g e R e m a r k s Rt. Lft. 1 g F F 3 f £j_ p -----2 _________ M -----~. ------ 3 6 7 8 q 10 H F 12 M 13 17 H 3 5 7 ll P £^ 4 2> 2 3 4- 1--------------------- 2 2 7 5 3 2 6 ------ 5 5 10 li 3.4 F 18 F 3 4 2 5 4 M M j/i F F ___ 3 Rice C o n t r o l 4 2.5 Carbohydrate-free 6.6 Glucose 8 5 4 13 4 - 9 7-3 Sucrose 6 3 12 5 4 9 9.2 Lactose ----- 2 4 - 6 ___ 2 1 E **5 FinH R l c e 90 . TA&t-£ 3C-3 Cc>A£S-£ fc/CE (5o>c^.as^ GLucosB LSHZ.roSB CAB&orty&J B&eE f7A/*= /2 /CE 7%&l-e3Z'/1Co/tese ^?/c£<5i/C,fSo^>£ GtLUCO&E Ljic-t o s e B C4/e0oHY&. /Oe^ /=/mj£ rZ/C-E E/ASE CHART V-3 P r o m Tao3.es V-3, ^ 4: Comparative E f f e c t s of P e e d i n g V a r i o u s S u g a r s , and Fine Rice. TABLE V-5A R A T NO* C A R I E S SCORE Sex U p p e r L o w e r Lower Rt. Lft. 1 p 2 F ------- M M 5 6 y M S M q 10 12 f F F ----- 43 3 5 2 3 2 ______ 3 ___ — , ------ 2 ------ R 4 2 F M 5 3 44- ------ 2 5 5 2 Total A v e r a g e Rem a r k s 6 7 9 5 5 q 0 4- 10 9 7.2 Fine R i c e 3.5 Sucrose 7* 7 Glucose TABLE V-5B RAT NO. 17 is 19 20 21 2? 22 24 25 26 27 28 29 30 31 32 j>3 34 36 37 38 8 41 42 43 44 4r 4§ 47 48 49 50 R1 §2 U U C A R I E S S CO R E Sex U p p e r M M M M M M F F F F ---— _— * --- * — _ -------—— , ---- M M M r ,^ — -- ——— K 3 M M F F F F -----— —M— —•—.— ---.^ M M M M M F F F F M M M i: M M F F F F 2 MM_ — —— -- L —.- — --- MM— — --- - t|nT_ — —— M-*— _ --r tt _ «*— — ——— .-- - Lower Rt. Lower Lft. 6 7 6 5 6 4 10 10 7 6 B § 4 7 5 7 9 9 5 6 li 13 10 12 11 11 19 19 12 12 4 4 10 7 7 5 8 4 3 8 6 7 R 8 6 8 2 8 9 18 16 l4 10 16 11 16 4 6 4 R 3 X * 6 4 5 4 2 4 5 6 4 h 6 3 R 4 10 11 11 7 8 12 7 10 3 P 2 9 4 7 6 6 5 4 3 7 3 8 • 2 4 4 4 9 2 4 7 4 8 (Z Total Average Remarks 13 Fine R i c e Coarse Rice 8.9 Cs.rbolivd.rate— f r e e c* 0 0 r~ R 17 6 11 10 10 14 6 7 l4 10 Sucrose C o n t i n u e d on next pa g e 93 TABLE V-5B R A T NO. M 2vi M M 11 M F F F F 67 66 69 70 71 72 M M M M Ivi M F F F F R Control 77 76 79 SO Si C A R I E S SCORE Sex U p p e r Lower L o w e r Total A v e r a g e R e m a r k s Rt . Lft. 57 5S 59 60 61 Oi *9 6s 64 65 66 75 76 (con't.) M M F F F 6 --5 „— . 3 —.— 2 ___ 3 Died before — — —. 2 --146 --§ ----_.— ------- . — --- --- . Gro\xp C: ■ --------- 4 4 4 10 5 11 1 2 6 5 11 5 12 7 71 4 4 2 4 1 c o m p l e tion 0. 4 L 9 5 S 3 11 5 4 3 b S 7 10 1 3 6 ,5 6 7 10 IS 12 21 2 5 12 10 4 4 1 3 3 Glucose 10.5 'Lactose 6 weeks T e r m i n a t e at 3 3 2 1 3 7.7 7 7 3 4 6 ,.4 for c o m o a r i s o n C oarse R i c e 7X&JUB. 1f~^A Cr/.C^BO-S B /^icr&ee ^?/o£ 7A &BB V-^3 do/J/SS£ A?/C-£ 'SlJC/Sc’SB C^JLly'C.CfSB ^Aor&sB CrtfZ0os/>rt?. /^eeA /dfa/B 7 IA/0JS/CS Go*JTg6‘ X£=*?o /-r//£ C H A R T V -4 Prorn T a b l e s V - 5a, & 5 b; CGuipy.rH.tive Cff'ec ts of F e e d i n g V a r i o u s S u g a r s , F i n e R i c e , a n d Coarse Rice 95 T A B L E V~6 HO. 1 2 \ 5 6 7 s q 10 n 12 13 14 16 17 IS 19 20 21 22 24 25 26 27 28 29 30 31 32 p 36 37 36 C A R I E S SCORE Sex U p p e r Lower Rt. ~ —«• --- £ •^s 6 M Ivi M M F F F F --—— —— --- 3 6 7 M M M M M F F F F F ____ --, ----—— —— --------- 6 7 5 4 6 5 2 7 3 6 F F F F M M M M M M F F F F M 11 M M M M I6 —.— 5 --3 --7 _— 7 —— 4 r --o •fin*— —• 9 --1 —— 3 Di ed before —— _— --—— — —— --- Hs', 6 4 3 5 5 5 5 2 7 Remarks Lf t . 7 5 7 4 6 6 8 7 13 11 12 8 12 6 7 6 4 5 4 12 14 11 8 10 q 4 18 6 11 2 8 3 5 5 4 11 8 4 r~ ■f- 1 1 .6 Coarse R i c e 10 Sucrose 10.6 Glucose .7 Lactose ? 14 l4 10 7 18 15 8 -1.-b b 4 3 8 5 iomple t io] 4 7 3 4 4 4 p; s 6 6 6 10 13 7 7 9 9 10 li 8 13 C o n t i n u e d on next page 96 TABLE V-6 R A T NO. $ O 41 42 Ify ^5 H-6 H-S (cont'd, ) C A R I E S SCORE Sex U p p e r Lower Rt. F F F F M 5 M M M M M — _ _ — —— --- . ____----___ — -_ — -------------- 5 4 2 3 2 9 9 4 3 3 3 L o w e r Total A v e r a g e R e m a r k s L f t .. p; 3 4 6 6 6 a. 2 4 3 3 10 71 6 9 8 15 15 6 7 6 6 g Carbohydrate-free 97 T A B L E V~7 NO. C A R I E S SCORE Sex U p p e r Lower T -« xDt4u L o w e r T o t al Lft. 1 2 3 4 R § 7 F F F F F M M ___ ---------------_ _ —. R s 3 3 4 6 5 4 7 4 3 4 6 5 9 19 7 6 8 12 10 9,6 S 9 10 11 12 1? 14 F F M M M M M ____ --- . ---—— ------ . —>— — 7 7 9 S 7 R 6 2 R •y 8 9 6 8 6 9 12 17 17 13 13 12 13,3 Sucrose 19 16 17 IS . 19 20 21 M Ivl F F M LI M _— —— ---_ — 9 7 5 7 6 10 8 6 7 9 10 7 t 19 19 11 l4 11 19 11 14.3 Glucose 22 23 24 25 26 27 28 F F F F LI M LI -----_— — ---—“ 7 6 R 8 6 S£ /^AC-TC?se Csi/es&MYO. /?&££» 7'AB/-& Y - 7 gw££AcS T-h&oO CcPAMS.se ~%?/COAC>SE GrLu c&s £ assas; jLAC-TOS£ Camz& ommvp.foe- £ 0 /to jL/ASif CHART V-5 P r o m T a b l e s V - 6 , & *7 5 Peocting V a r i o u s 3upairs f o r 6 arid 8 w e e k P e r i o d s . 99. TABLE V-g RAT NO. C A R I E S SCORE Sex U p p e r Lower Rt. Lower Total Av e r a g e Re m a r k s Lft . 1 F --- 6 g i4 2 F --- g 6 14 3 M --- 6 g l4 4 M --- S 7 13 5 F — _ 6 4 10 6 F ------- 4 7 11 7 Li --- o s' 7 16 g i\n --- . O -> 7 lb Q > F -------- g 7 15 10 F g 9 17 H F — — 9 g 17 12 F ---- 6 5"' 11 13 M ---- 7 6 13 14 M ---- 5 6 li 15 F _ — 4 3 7 16 F — — — S 7 15 l4. 2 Corn Syrup Ration 12.3 Coarse Rice Only 100. TABLE V-9 R A T NO. 1 p C A R I E S SCORE Sex U p p e r F Lower Rt. 10 10 8 7 7 Q V 19 16 i4 1-5 19 6 5 R 5 9 10 9 11 8 7 7 5 10 10 9 . 11 14 12 12 10 19 20 IS 22 7 7 6 . 6 6 6 6 10 R R 6 k 6 9 5 9 12 12 12 12 12 15 11 19 10 7 8 17 17 11 9 S 9 IS 6 71 8 9 10 11 12 15 F T p £' F F M M li M l4 15 16 17 is 19 20 21 F F F F M M M M 2? 24 F F F --__— . --------— —____ --—— — — ---------- _ _ _ 9 5 i Lower Total A v e r a g e R e m a r k s Lf t * 6 Corn Syrup R a t i o n Corn Syrup R a t i o n plus 1f0 Irradiated Yeast Coarse R i c e R a t i o n plus Xfo I r r a d i a t e d Y east 15 Coarse R i c e R a t i o n 101 . 1T' 8 R$£ 7x?/a && e 7E!/y£AST &>/z*s ■Sy&c's*^at/o m CjoksJ v5V m u e ffilr/ofAKt y--Z/Z/Z.A r. Ye a * r d * C H A R T V- 6 Prom. Tables V-8, *c 9; F e e d i n g Corn S y r u p a n d C o a r s e R i c e Simultaneously; U s e o f H i gii Vitamiri D Su pp 1 s m e n t . BIBLIOGRAPHY AND LITERATURE CITED. 102 . BIBLIOGRAPHY AHD L I T E R A T U R E CITED 1. A g n e w, M.C., Agnew, G., and Tisdall, E.F. J. Amer. Dent. Assoc. .20; 193 2. Belding, 3. B l a y n e y , J . R . : Ibid.: pages 5 5 - 5 8 (1941) F o r h i m s e l f and c o - w o r k e r s . 4. B r a n son, 5. B r e e se, 6. B r o d s k y , R a l p h H . , Schick, Bela, and Volmer, Ibid.: p a g e s 7 4 - 7 5 (1941) 7. Bunting, 8. 9. Cox, P.H., and Belding, L.J. D e n t a l Caries, 2 n d E d i t i o n (1941) F i n d i n g s and C o n c l u s i o n s on Its Causes and Control. L a n c a s t e r Press, Inc., Lancaster , Penn. P a g e s 4 7 - 5 0 (1940) C h a r l e s B . : Ibid.: Frederick: pa g e s 6 7 - 6 8 Ibid.: p a g e 68 (1940) (1940) Gerald J . : Ibid.: pages 8 6 - 8 7 (1941) F o r h i m s e l f and co-v.rorkers. F l e i sch, L o u i s M.s Ibid.: p a g e s 104-106 (1941) Florestano, H.J.: Ibid.: pages 106 - 1 0 7 (1941) 11. Forshufvud, Sten: Ibid.: p a g e s 1 0 7 —108 (1939) 12. F o s d i c k , L e o n a r d S.: Ibid.: p a g e 108 (1941) F o r h i m s e l f and co-workers. 14. G o t t lieb, Haber, Herm a n R u s s e l W. , and Jay, P h i l i p Ibid.: p a g e s 7 6 - 7 9 (1924-37) F o r t h e m s e l v e s and co—w o r k e r s In the Michigan Group Research on Dental Carles, 10. 13. (1933) Bernhard: G.G.: Ibid.: p a g e s 1 1 1 —112 (1941) Ibid.: pa g e s 1 1 5 - 1 1 7 (1941) For h i m s e l f and co-workers, in G e r m a n y and Switzerland. 15. Hanke, M i l t o n T.: 3.6. Hanke, M.T., research Ibid.: pa g e s 1 1 6 — 118 (1940) I n c o l l a b o r a t i o n w i t h the Chicago D e n t a l R e s e a r c h Club. et al D e n t a l Cosmos 75: 635 (1933) 103. Idem.: Ibid. 75: 739 (1933) Ibid. 75: 933 (1933) • 00 H 17. Idem.: 19. Hunt, SO. H o p p ert, SI. Klein, H.R., and Hoppert, C.A. I n h e r i t a n c e in R a t Caries. G e n e t i c s : 24: 76 (1939) C. A., Weber, P.L., and Canniff, T.L. T h e P r o d u c t i o n of D e n t a l Caries in R a t s F e d an A d e q u a t e Diet. J. D e n t a l Research. 12: 1 6 1 — 173 (1932) M cCollum, E.V. H . , and ! J. D e n t a l R e s e a r c h 12: 5 2 4 Ibid. 13: 6 9 (1932) 22. Idem.: (1933) 23. Koehne, M . , and Bunting, R.W. A R e v i e w o f R e c e n t S t u d i e s of the C a u s e o f D e n t a l Caries. J. Am. Diet. Assoc. 9: 4 4 5 - 4 6 1 (1934) 24. Koehne, M . , and Bunting, R.W. B i o c h e m i c a l and N u t r i t i o n a l Studies in the F i e l d o f Dentistry. Ann. Rev. Biochem. 3: 4 4 1 —4 5 8 (1934) 25. K o e h n e , M . , Bunting, R . W . , et al S t u d i e s In the Con t r o l o f Denta l Caries, II. J. N u t r i t i o n 7: 6 5 7 - 6 7 S (1934) 26. L e F e vre, M a r i a n L. D e n t a l Caries, 2 n d E d i t i o n P a g e s 1 5 6 - 1 5 7 (1938) 27. Lilly, C.A. F a i l u r e to P r o d u c e Caries in W h i t e Rats. J. N u t r i t i o n 5: 1 7 5 - 1 8 1 (1932) • 00 N L illy, C.A. D e n t a l Carles, 2 n d E d i t i o n P a g e s 1 6 4 —165 (1938) 29. 30. 31. McClendon, McCollum, J.F. , and Foster, W.C. I b i d . : p a g e s 1 7 1 —1 7 2 (1941) and co —w o r k e r s Ibid.: p a g e s 1 7 2 —1 7 4 (1940) E.V. M e l l a n b y , .M a y Ibid.: p a g e s 175-176 (1939) 104 . 32. Rosebury, Theo d o r , 33. Rosebury, T h e o d o r and K a r s h a n , M a x w e l l J. D e n t a l R e s e a r c h 11: Ibid.: p a g e s 2 0 0 - 2 0 2 (1939) For h i m s e l f and co— workers. 121 (1931) 34. Rosebury, T h e o d o r and Foley, G e n e v i e v e Ibid. 12: 4 6 2 - 4 6 4 (1932) 35. R o s e bury, T., K a r s h a n , M . , and Foley, G. Ibid. 12: 247 (1933) 56* Idem.: 37. W e s s i n g e r , G e o r g e D. D e n t a l Caries, 2 n d E d i t i o n P a g e 235 (1940) 38. McCollum, E.V. P h y s i c i a n s V i t a m i n R e f e r e n c e Book S e c o n d E d i t i o n Revised, 1 9 3 8 E.R. S q u i b b & Sons, N e w Y o r k Page 114 Mellanby, May Ibid.: p a g e 114 Ibid. 13: 379 (1933)