Studies on Inorganic Salt Metabolism: V. The Effect of Faulty Diet on the Dentition of Brood Bitches, and the Dental and Skeletal Development of Their Offspring

I. Introduction

A review of the literature has revealed surprisingly few studies on the new-born, and none in which dental and skeletal findings in mother and offspring have been correlated with each other and with the maternal diet.

In a long series of detailed experiments, it was observed that brood bitches living in an environment that afforded a negligible amount of ultraviolet radiation, and fed a diet low in calcium, vitamins, and protein, and potentially basic in reaction (bread, meat, and potato), gave birth to young which showed marked differences in body weight, general well-being, body ash, and mineral content and structure of the bones. Surviving members of successive litters of puppies born to bitches fed continuously on this type of diet showed an increasing proneness to skeletal disorders. Ultimately, 100 per cent of the survivors of four different litters (twenty-two puppies) born to four different mothers developed clinical rickets before they had been weaned.

In this paper, the dental findings on certain mothers and their offspring will be described and correlated with the maternal diet. Puppies in Group I were, with one exception, representatives of the last litters of Bitches 5 and 7. The father of both litters was Dog l. All three of the dogs were large mongrels, collie predominating, and had lived in the kennels and been fed a stock diet consisting essentially of bread, meat, and potato for more than eighteen months prior to mating. Before and during the last pregnancies, the bitches were fed quantitatively. The diet was similar in composition to the stock diet previously used, but was prepared in such a manner that it was impossible for the animal to exercise choice in the selection of food.

Puppies in Group II were offspring of mothers whose histories were either unknown, or had been in the laboratory kennels and fed a stock diet of bread and meat for varying lengths of time.

II. Experimental Procedure and Results

A. Diets

Diet I consisted of the following combination:

Patent wheat-flour bread (10 per cent moisture)	225.0 grams
Lean ground beef	75.0 grams
While potato	600.0 grams
NaCl	5.0 grams
Distilled water	1200.00 cc.

 

The bread was dried and ground to a fine crumb. The lean beef was finely ground, and the potatoes peeled and boiled. The meat was placed in about 500 cc. of distilled water, the mixture brought to a boil, and the potato, which had already been cooked, was added and mashed while the mixture was hot. The bread was then added and the whole thoroughly mixed into a uniform mash, which was thinned to the consistency of a thick gruel with the remainder of the water. The composition of the diet was estimated to be as follows: total calories, 1560; protein, 59 grams; calcium, 0.15 gram; phosphorus, 0.72 gram; potassium, 3.0 grams. The excess of base was equivalent to approximately 13 cc. of normal solution.

Diet 2 consisted of bread and meat exclusively. Scrap meat, usually beef ribs, was purchased from a butcher. It was boiled with salt, and when cold was stripped from the bone and cut into small pieces. Wheat-flour bakery bread was used. It was cut into uniform slices on a machine and air dried. When fed to the dogs, it was softened in hot water and beef stock. The bread and meat were roughly measured–about 225 grams of each were fed daily. While it is impossible to state the exact composition of a diet prepared in this manner, it can be approximated with a fair degree of accuracy. It is certain that the diet contained more protein and fat and less carbohydrate than Diet 1. The calcium content was estimated to be 0.09 gram; phosphorus, 0.69 gram; and potassium, 1.0 gram. The excess of acid was equivalent to approximately 46 cc. of normal solution.

B. Cages

The dogs occupied adjoining cages in a well lighted and ventilated room. Each animal was fed separately in his own cage. During a part of the day they were allowed the freedom of an outside enclosure paved with concrete. The run was built on the north side of a hill and received little direct sunshine. The heavy fog which envelops the district during a large part of the year, and especially during the summer months, greatly reduces skyshine, so that it is believed that the amount of ultraviolet radiation received at any time was negligible. Brief histories and findings on six bitches and thirty-eight new-born puppies, representatives of seven litters, will be given.

C. Group I: general data

The bread, meat, and potato diet described above (Diet 1) was fed quantitatively to four bitches, Nos. 5, 7, 8 and 9, all of which had previously given birth to litters. Bitches 8 and 9 were given sodium carbonate in addition, in order to determine the effect of excessive amounts of alkali. All of the animals appeared to be in excellent physical condition. Their weights ranged from 20 to 26 kilos. Bitches 5 and 7 were successfully mated, 8 and 9 were not. After the latter two animals had been on the alkaline diet for one hundred and thirty days, dental tissue was excised for histologic study, and the soda was discontinued. Subsequently, an equivalent amount of rice was substituted for the potato. Within thirty days after this change in diet, both animals died in convulsions. The biopsy tissue (upper second and third incisors with investing tissue) showed marked retrograde changes in the alveolar process and resorption of the roots of the teeth. The findings were quite comparable for the two animals. The corresponding tissue excised post-mortem indicated a reversal of the calcifying mechanism. Marked reconstructive changes were evident. Old dead bone was overlaid by new. Marrow spaces which had previously coalesced were separated again by bone growths between them. Along the crests of the alveolar process, and in excavated regions in the dentin of the roots, new bone formation was taking place (fig. 1). Histories of Bitches 5 and 7 are given below.

D. Bitch 5

Bitch 5 was received at the kennels when she was about two years of age with a litter of nine vigorous puppies three weeks old. A superficial dental examination was made on the mother. Her mouth was found to be extraordinarily clean, and her teeth white and apparently sound. After the second pregnancy, which occurred eight months later, both upper and lower incisors were found to be badly worn, the latter almost to the gingivae. The diet during this time consisted almost entirely of scrap beef. Subsequently, her diet was changed to bread, meat, and potato in approximately the amounts given above. During the next eighteen months two more litters were born. Representatives of both litters were killed at birth, and certain observations made on the bodies and bones. The findings were correlated with the subsequent behavior of their surviving litter-mates. The mineral content of the bodies and bones of the puppies killed at birth was found to be low, and the bone structure abnormal. The surviving members of the litters showed an increasing susceptibility to skeletal disorders, all of those in the last litter developing clinical rickets before weaning.

The dietary regimen was continued, but four and one-half months prior to the next mating, quantitative feeding was instituted, and certain metabolic and blood studies made before and during pregnancy. Detailed dental examinations were also made at intervals. The metabolic process, which earlier resulted in disintegration of the tooth substance, had already become static and remained so during this period. There was some gingivitis. Her mouth was less clean than formerly, but no striking abnormalities were observed. During pregnancy, the bitch was occasionally seen in convulsions. She had not been known to have them before. They were not severe, and she apparently suffered no serious ill effects. Her appetite was good, and she consumed practically all of the food offered during the entire gestation period. At the time of parturition she had a convulsion and died before the birth of her young. On postmortem examination her dental condition was found to be essentially the same as that previously observed. Of the tissue prepared for microscopic study, the upper right first, second, and third incisors revealed a most extraordinary condition of the alveolar bone. Scattered throughout the compact bone, but more on the margins of the septal process, were regions that appeared as though the inorganic constituents had been leached out. Fibers and bone corpuscles in various stages of degeneration stood out in bold relief (fig. 2). Numerous small lacunae, some containing giant cells, bordered the margins of the septal process, but the peridental membrane was narrow. The pulp of the teeth was fibrous, and the layer of odontoblasts entirely absent in regions.

Fig.1, Fig. 2, Fig. 3

Fig.1. Bitch 8. Post-mortem tissue. Note excavated regions in dentin and cementum, which have been filled in with haversian bone.

Fig. 2. Bitch 5. Post-mortem tissue. Alveolar bone from which inorganic constituents appear to have been leached. Note apparently intact haversian system surrounded by corpuscles in a matrix of fibers.

Fig. 3. Bitch 7. Pulp and dentin of tooth excised post-mortem. Note fibrous pulp, P; large blood vessels, BV; and absence of odontoblasts.

 

Dental findings on new-born Puppies, 22 and 38, representatives of the last two litters born to Bitch 5 and Dog 1, are described below.

Puppy No. 22 was the smallest of a litter of ten; weight of the litter, 2550 grams. The weights of the individuals varied from 160 to 330 grams. Five of the ten puppies were sacrificed immediately after birth. The bones of three were prepared for ash analyses and histologic sections, and the whole bodies of the other two for ash determinations. The mineral content of the bones was found to vary from 8.7 to 13.2 per cent. The body ash in the largest and in one of the smallest of the litter was found to be 1.87 and 1.9 per cent, respectively. The bone structure was abnormal. A photomicrograph of a lower canine of this puppy is shown in fig. 10. The section appears to be exactly median. The most striking abnormality is the large mass in the pulp. It stains and looks like dentin. Following this through serial sections, it appears first as a small island in the interior of the pulp chamber, and gradually increases in size until it becomes attached to the inner wall of the dentin layer. It appears to have been laid down irregularly and is surrounded by cells. In the section shown, a large clump of cells is found in the interior of the mass. The space on the margin of the tooth, which appears as a white spot, was an irregular deposit of the same substance lost in sectioning. The odontoblasts are clumped together leaving large intercellular spaces, and the dentin layer is much thinner than that in the teeth of the control puppies. The dental papilla is also less vascular. Compare figs. 10 and 13.

Puppy 38 was the smallest of a litter of ten full-term but unborn offspring of Bitch 5 and Dog 1. Three litters had previously been born to these parents. For four and one-half months prior to the last mating both had been fed quantitatively on Diet 1 (bread, meat, and potato), and appeared to be in excellent physical condition. Quantitative feeding was continued during pregnancy. On the completion of the gestation period, the bitch had convulsions and died before parturition. The total weight of the foetuses was 1690 grams, which was markedly less than the birth-weight of the preceding litter. The mineral content of the body substance of the unborn puppies was found to be extraordinarily low–an average of 1.73 per cent, compared with 1.9 and 2.35 per cent in the representatives of the third and second litters, respectively. The total calcium content of the last litter was estimated to be 5.9 grams. The histologic findings in the bones of these puppies were in complete agreement with the chemical. A photomicrograph of a costochondral junction of a rib of Puppy 38 is shown in fig. 7. There was marked retardation in calcification. The proliferating-cartilage zone is extraordinarily wide, and the trabeculae thin and poorly calcified. A photomicrograph of a cusp of a lower premolar of this puppy is shown in fig. 8; in fig. 9, a high magnification of the coronal region of pulp and dentin in a lower canine. Here again, the most striking structural abnormality appears to be in the odontoblasts and dentin formation. In spite of the fact that the section shown in fig. 8 may have been cut obliquely, the dentin layer is relatively thin; and the odontoblasts, which are cut transversely, appear to be widely separated. In fig. 9 the predentin layer is poorly defined, and is separated from the layer of odontoblasts by a space that is traversed by a network of processes from the odontoblasts. The scarcity of blood vessels in this region is conspicuous. Compare fig. 9 with fig. 18, which is a photomicrograph of comparable tissue from Puppy 49 in Group II.

Fig. 7, Fig.8, Fig. 9

Fig. 7. Costochondral junction. note wide proliferating cartilage zone. Trabeculae in shaft are thin and poorly defined. An osseous cylinder surrounds the enlarged cartilage cells. Compare fig. 7 with fig. 16 showing analogous tissue from Puppy 49, Group II.

Fig. 8. Cusp of premolar. Note the thin layer of dentin and large intercellular spaces between the odontoblasts. Compare fig. 8 with fig. 17 showing analogous tissue from puppy 49.

Fig. 9. High magnification of coronal region of pulp and dentin. The odontoblasts are scattered, and the intercellular spaces large. The predentin layer is poorly defined. Scarcity of vessels is conspicuous.

 

E. Bitch 7

Bitch 7 was a large, robust, mongrel collie. Her weight averaged about 23 kilos. She had been in the kennels for more than two years, and during that time had given birth to three litters of puppies. The father of all her offspring was Dog 1. The two dogs were received at the kennels at about the same time, and had lived under identical conditions of diet and hygiene. Successive litters born to these parents showed an increasing proneness to skeletal disorders, and all of the survivors of the last litter developed clinical rickets before they were weaned. Representatives of the last two litters were killed at birth, and certain observations were made on the new-born puppies. The mineral content of the bodies and bones was low, and the bone structure abnormal.

Prior to the next mating, quantitative feeding was instituted, and a detailed dental examination made. Before and during pregnancy certain observations were also made on blood and urine. Dental examination revealed pyorrhea-like lesions. There was gingivitis, recession of the gum with detachment, and heavy deposits of tartar. The mouth was dirty, but the teeth were sound. Tissue was excised for microscopic study. The tartar at the gingival margins was very hard and flint-like, and was stained black. Histologic examination of the excised tissue showed osteoporotic changes in the alveolar bone, with evidence of an inflammatory reaction. During pregnancy, and on the completion of the gestation period, the bitch had convulsions. Immediately after the birth of her young, she went into coma, and the following day was chloroformed.

There were nine puppies in the litter, the total birth-weight being 1725 grams. The weights of the two preceding litters were 2850 and 2440 grams. The weights of the individuals of the last litter varied from 99 to 240 grams. The percentages of bone ash in the three puppies varied from 7.8 to 10.4 per cent. The ash content of the bodies of six averaged 2.09 per cent. As was found in successive litters born to Bitch 5 and Dog 1, there was a progressive decrease in the mineral content of the offspring of Bitch 7, if it may be assumed that values found in certain individuals were representative of the litter as a whole. The total calcium content of the litter was estimated to be 7.9 grams, and the amount of this element in the body substance of the two preceding litters, 15.4 and 12.7 grams.

Post-mortem dental examination of Bitch 7 showed remarkable healing of the region from which dental tissue had been excised. All of the oral conditions previously observed were slightly exaggerated, but in general there were no marked changes. Histologic examination of the site of the canine, in which there remained a considerable portion of the root, revealed an astonishing amount of inflammation and resorption of cementum, dentin, and alveolar bone. The histologic picture in other regions showed profound resorption of bone and tooth substance, but less evidence of an inflammatory reaction. In certain regions in the alveolar bone, but more marked in the cementum, the histologic picture was strikingly like that observed in Bitch 5. Here again it appeared that the inorganic constituents had been leached out leaving the corpuscles in various stages of degeneration in a matrix of fibers. In the cementum, both on the margins and in the interior, there were numerous light regions that stained like osteoid. In them were many faintly-visible round-bodies or spaces, the structure of which could not be determined. In the interior, bordering the band of “osteoid,” were several rows of mononuclear cells. The pulp of the teeth was fibrous. The layer of odontoblasts was entirely missing in regions, and normal pulp elements could not be recognized. Fig. 3 is a photomicrograph (high magnification) of a region of pulp and dentin in an upper third incisor.

Puppy 23, largest representative of the last litter born to Bitch 7 and Dog 1, weighed 240 grams; average weight of litter (nine), 191 grams. The percentage of ash in his bones (10.4 per cent) was found to be the highest for the three puppies examined. The structure of the ribs was abnormal–he had suffered an intrauterine fracture, which had partially been repaired with fibrous tissue. A photomicrograph of a cusp of a lower premolar of this puppy is shown in fig. 11, and a high magnification of a lateral region of this tooth in fig.12. The same general findings described above are characteristic of this litter also. The dental papilla is less vascular than that in the control group of puppies, the odontoblasts are clumped together irregularly, and the layer of dentin is relatively thin. Under high magnification, the zone separating the odontoblasts from the predentin layer is clearly visible. The ameloblasts are also separated from the enamel layer by a network of cells and fibrils, in which enamel globules are irregularly scattered. Compare fig. 12 with fig. 15, which shows comparable tissue, under the same magnification, from Puppy 43 in the control group.

Fig. 10, Fig. 11, Fig. 12

Fig.10. Lower canine of Puppy 22. Note large mass in pulp. It has been irregularly laid down, and in the interior is a clump of odontoblasts. It is also surrounded by cells. The odontoblasts bordering the pulp are bunched together, leaving large intercellular spaces. Compare with fig.13.

Fig. 11. Cusp of premolar of Puppy 23. The dentin layer is relatively thin, and odontoblasts are irregularly arranged. There is also a disturbance in the enamel-forming mechanism.

Fig. 12. High magnification of a lateral region of canine of Puppy 23. The odontoblasts are scattered and irregularly arranged, and separated from the predentin layer by a space transversed by fibrils. The ameloblasts are also separated from the enamel matrix by a network of fibers in which enamel globules are scattered.

 

F. Group II: general data

Dental and skeletal tissues from fourteen puppies in the control group were examined. They were representatives of four litters from four different mothers. The histories of two of the mothers were unknown. They were “street” dogs, and gave birth to litters soon after their arrival at the kennels. The third bitch, No. 28, arrived at the kennels during the third week of pregnancy, and the fourth, No. 31, had been in the kennels for a year before the birth of her last litter. The histories of the latter two animals will be given in detail.

G. Bitch 28

Bitch 28 was a large mongrel terrier. She weighed 18 kilos, and her age was estimated to be about three years. Her mouth was clean; her teeth were white and sound. When she arrived at the kennels she was in the third week of pregnancy. During the remainder of the gestation period her diet consisted of bread and meat. A litter of eight vigorous puppies was born. The total birth-weight was 1716 grams, the weights of the individuals ranging from 157 to 323 grams. Four of the litter were killed.

Roentgenograms of the new-born puppies showed differences in the density of the bones, which were exactly parallel with the percentages of bone ash. The latter varied from 9.7 to 14.7 per cent. The puppies left with the mother grew rapidly. At 21 days of age, roentgenograms showed differences in skeletal development, which, at 40 days, could no longer be detected. Subsequently, however, when two of the puppies were placed on the basal diet of bread, milk, meat, butter fat, and orange juice, the earlier differences rapidly became apparent. A third puppy of this litter was kept in the open and fed on a mixed diet. Even though ossification appeared to be least advanced in him at the age of 21 days, his development, as far as could be determined by clinical examination and roentgenograms, was normal. The behavior of the surviving individuals of this litter was altogether comparable to that observed in the offspring of the average “street” dog.

The chemical and histologic findings on two of the new-born of this litter have arbitrarily been chosen as standard, and are described below.

Puppies 42 and 43. The birth-weights of these puppies were 190 and 203 grams respectively, both of which were less than the average (215 grams) for the litter. The mineral contents of their bones were 14.2 and 14.7 per cent. Histologic findings on bone and dental tissues were in close agreement. A photomicrograph of a section of rib of Puppy 42 is shown in fig. 13. The junction line is fairly straight, and the proliferating-cartilage zone relatively narrow. The provisional zone of calcification is well defined, and the arrangement of cells and trabeculae orderly. The marrow is cellular on the whole, but there is also a considerable amount of fibrous tissue in regions. Mononuclear cells are much less numerous than in some of the ribs of puppies in Group I, and polynuclear cells are relatively few in number. A photomicrograph of the coronal portion of a lower canine is shown in fig. 14. The closely packed, orderly arranged odontoblasts, and the relatively thick layer of dentin, are in striking contrast to those observed in the puppies in Group I. Compare figs. 11 and 14. As in the other group, there appears to have been a disturbance in the ameloblasts. In fig. 15 is shown a high magnification of the lateral region of a lower canine of Puppy 43. Compare fig. 15 with fig. 12, which shows comparable tissue from Puppy 23 in Group I.

Fig. 13, Fig. 14, Fig. 15

Fig.13. Canine of Puppy 43. A–ameloblasts; E–enamel; D–dentin; O–odontoblasts; P–pulp. Note large blood vessels, densely packed; orderly arranged odontoblasts; relatively thick layer of dentin. Compare with fig. 10.

Fig. 14. Coronal portion of canine of Puppy 42. The ameloblasts in places are separated from the enamel matrix.

Fig. 15. High magnification of a lateral region of canine of Puppy 43. Compare with fig. 12.

 

H. Bitch 31

Bitch 31 was a mongrel–police-collie. She weighed 25 kilos, had been reared as a pet on a near-by ranch, and was about four years old. Her diet had consisted of table scraps, with very little meat but an abundance of skimmed milk. She had lived in the open all her life, and was received at the kennels with a litter of six puppies five weeks old. The mother and puppies appeared to be in excellent condition. The puppies were weaned, the mother was put on a diet of bread and meat, and all were given the ordinary care. A dental examination revealed a most extraordinary oral condition. Her mouth was dirty. The two upper canines were missing. There was gingivitis, tartar, and recession of the gums. The upper left second and third incisors, with investing tissue, were excised for histologic study. The process of resorption was active. The peridental membrane was wide, and the margins of the septum fringed with lacunae, which contained numerous multinuclear cells. The structure of the pulp tissue was not clearly defined, and the odontoblasts were arranged in festoons.

During the following year, the bitch was kept on the diet of bread and meat described above. In July, 1927, she gave birth to a litter of six puppies whose birth-weight was only 1250 grams. The mother had gained in weight and appeared to be in excellent condition. The entire litter of puppies and the mother were sacrificed. Postmortem examination showed that a profound change had taken place in the dentition of the mother. Many of the teeth were almost denuded of enamel, and even the dentin had suffered disintegration in regions. There were heavy deposits of tartar on the buccal surfaces of the molars. The histologic pictures of the post-mortem material were in striking contrast to that observed in the tissue excised twelve months previously. There was less lacunar resorption, and the peridental membrane was narrower than in the biopsy tissue. The pulp was less fibrous, but the structure of the alveolar bone presented the leached-out appearance observed in the post-mortem tissues of Bitches 5 and 7 (figs. 5 and 6). The localization of dental lesions in animals as well as man, and the large part played by chance in the selection of tissue for examination, make the interpretation of such findings as these extremely difficult. The disintegration of the crowns of the teeth of this animal–a phenomenon that has repeatedly occurred concomitantly with accelerated calcification in young puppies–indicates that the differences observed in the histologic pictures of the biopsy and post-mortem tissues were associated with metabolic changes, rather than the result of a chance selection of material for examination.

Fig. 4, Fig. 5, Fig.6

Fig. 4. Photograph of jaws. Note disintegration of tooth substance.

Fig. 5. Alveolar bone excised post-mortem. Note haversian systems surrounded by tissue from which the inorganic constituents appear to have been leached.

Fig. 6. Dentin and pulp. Tissue excised post-mortem. Compare with analogous tissue from Bittch 7 shown in fig. 3.

The physical condition of the offspring of Bitch 31 was so very poor that it was thought the dental and skeletal findings would be correspondingly abnormal. The reverse, however, proved to be the case. The percentage of ash in the bones was found to average the highest yet observed, the values ranging from 11.1 to 16.1 per cent, with an average of 14.06. The bone ash of ten puppies in Group I averaged 10.0 per cent, and of the offspring of Bitch 28, 12.8 per cent. There was considerable individual variation in bone structure. The outstanding characteristic of the histologic pictures was the large number of osteoclasts. In other respects, the findings were comparable to those observed in the offspring of Bitch 28, and in the new-born of other mothers whose histories were unknown.

The histologic findings in the dental tissues of these puppies were comparable to each other and to those observed in Puppies 42 and 43. Puppy 49 will serve to illustrate.

Puppy 49. The birth-weight of Puppy 49 was 125 grams, the smallest of a litter of six whose average weight was 208 grams. He was born July 5, 1927. The mineral content of his bones was 16.1 per cent, the highest value for any of the puppies examined. The histologic picture of his ribs was dominated by multinuclear cells. Mononuclear cells were also numerous. The junction line was straight and the proliferating-cartilage zone narrow, and the cells were orderly in arrangement. Compare figs. 16 and 7, which are photomicrographs of costochondral junctions of the ribs of puppies 49 and 38. A photomicrograph of a section of a cusp of a lower premolar of Puppy 49 is shown in fig. 17. The dental papilla is vascular, odontoblasts are closely packed and orderly in arrangement, and the layer of dentin is relatively thick. The ameloblasts are also separated from the enamel matrix. Compare figs. 17 and 8. Both of these sections appear to have been cut obliquely. The latter was taken at a slightly lower magnification, which would, of course, minimize the difference in thickness of the dentin layers. Fig. 18 is a high magnification of the coronal region of pulp and dentin in the tooth shown in fig. 17. The predentin layer is clearly defined, the odontoblasts are closely packed and orderly in arrangement, and the entire pulp is studded in blood vessels. Compare fig. 18 with fig. 9, which is a high magnification of the coronal region of pulp and dentin in a lower canine of Puppy 38 in Group II.

Fig. 16, Fig. 17, Fig. 18

Fig. 16. Costochondral junction. Compare with fig. 9, which shows comparable tissue from Puppy 38, Group I.

Fig. 17. Cusp of premolar of Puppy 49. Compare with fig. 8. Note the densely packed, orderly arranged odontoblasts; and relatively thick layer of dentin. The ameloblasts are separated from the enamel matrix in places.

Fig. 18. High magnification of a coronal portion of pulp and dentin of tooth shown in fig. 17. Compare with fig. 9.

 

III. Discussion

A comparison of the findings for Bitches 5 and 7 reveals interesting differences. Both were large mongrel collies of about the same size and age. They lived under identical conditions of diet and hygiene, were mated with the same male, and bore four litters of young, the total number of offspring being forty and thirty-nine respectively, during a period of two and one-half years. Their pregnancies, with the exception of the last, occurred at about the same times. Before and during the first gestation periods their diet consisted almost entirely of meat. It was usually scrap beef, and was cooked and fed with the bone. During this time Bitch 5 suffered marked disintegration of the tooth substance, both upper and lower incisors having been worn down almost to the gingivae. The exposed surfaces of the teeth of Bitch 7 remained intact. Subsequently, the diet was changed to bread, meat, and potato. During a period of eighteen months, Bitch 7 developed gross pyorrhea-like lesions, and Bitch 5 had only a slight gingivitis. There were no obvious changes in the enamel of the teeth of Bitch 7, and no further destruction of tooth substance in Bitch 5. Successive litters born to these mothers showed an increasing proneness to skeletal disorders, and representatives of these litters killed at birth had faulty bone structure, and a low percentage of ash in their bodies. Prior to the last mating, quantitative feeding was instituted. Both bitches had convulsions during pregnancy. Bitch 5 died at the time of parturition, and Bitch 7 was chloroformed when at the point of death a few hours after the birth of her young.

Differences in the metabolism of the two mothers is clearly shown in the mineral content of their offspring. During the last pregnancies, the two bitches received exactly the same diet, both in kind and amount. Their appetites were good, and both ate practically all of the food offered. The total birth-weights of the two litters differed by only 35 grams, but the offspring of Bitch 7 had 34 per cent more calcium in their bodies than those of Bitch 5. Representatives of successive litters born to both mothers showed a progressive decrease in mineral content, the offspring of Bitch 7 invariably having a higher average value than those of Bitch 5. Blood findings in the bitches, before and during pregnancy, were altogether comparable and well within the normal range.

Histologic examination of the dental tissues of the bitches showed marked degenerative changes. The peridental membrane was wider, and there was more resorption of the alveolar bone and roots of the teeth in Bitch 7 than in Bitch 5. In the former animal, all of the regions examined showed more or less evidence of an inflammatory reaction. This was not observed in Bitch 5, in which the histologic picture was dominated by regions in the alveolar bone that appeared to be undergoing disintegration by a process of solution of the inorganic constituents. In the interior of such regions haversian systems remained apparently intact. The same process was active in the tissues of Bitch 7, but the cementum was more involved than the alveolar bone. The pulps of the teeth of both animals were fibrous. In some regions the layer of odontoblasts had been entirely obliterated by fibrous tissue, and normal pulp cells could not be recognized (fig. 3). Histologic findings in the offspring of both mothers showed retarded calcification and faulty bone structure. There was considerable variation in the individuals of the same litter, both in respect to structure and composition of the bones. Dental findings showed less variation, and were comparable in the two litters.

Biopsy dental tissues from the two bitches fed Diet 1, supplemented with gradually increasing amounts of sodium carbonate (0.75-4.0 grams) for a period of one hundred and thirty days, were altogether comparable, and showed profound degenerative changes in the alveolar bone and roots of the teeth. These findings, with the subsequent history of the bitches, have already been described.¹

The substitution of bread and meat for a mixed diet, which consisted essentially of potato and vegetables with raw skimmed milk ad libitum, apparently exerted a profound influence upon the dentition of Bitch 31. In spite of the fact that she lived under most favorable hygienic conditions, and her diet had been rich in minerals and vitamins, pyorrhea-like lesions developed to a marked degree. Both upper canines were missing. Roentgenograms of the sites of the missing teeth revealed small bodies near the surface of the gum that looked like fragments of roots. The overlying tissue was smooth and showed no evidence of trauma. The owners of the bitch did not know of any injury she had received, nor that the canines were gone. It is almost inconceivable that the roots of canine teeth in an adult dog could be resorbed to such an extent that the teeth would be entirely lost. Yet, the profound degenerative changes observed in the biopsy tissue from this animal indicate that such was the case. Root resorption is a common occurrence in animals on diets that may be well proportioned in respect to calcium and phosphorus, but which contain a certain preponderance of basic ions, and has been so extensive in some of the puppies that permanent teeth have been spontaneously shed. The biopsy tissue from Bitch 31 presented a histologic picture that was altogether comparable to that of Bitches 8 and 9, which received Diet 1 supplemented with sodium carbonate, and closely resembled the diffuse atrophy in human tissues described by Gottlieb.² Interestingly enough, the owners of Bitch 31 had pyorrhea. Concomitant with a change in diet and hygiene, the “pyorrhetic” process appears to have been arrested, and during the following year, the crowns of the teeth suffered rapid and profound disintegration. Unfortunately, a dental examination was not made on this animal at the beginning of pregnancy, and it cannot be said with certainty, but it is believed that the destruction of the tooth substance occurred during this period. A similar condition has been observed in the teeth of a large number of puppies on experimental diets, and has been associated with accelerated calcification.³

A comparison of post-mortem dental findings for Bitches 5, 7, and 31 shows interesting similarities as well as differences. The extraordinary appearance of the alveolar bone–corpuscles in various stages of degeneration in a matrix of fibers–was a striking feature of the histologic pictures of tissues from all three animals. This suggests that the condition may be associated with pregnancy. The fact that it was not observed in the biopsy tissue from Bitch 31, and in neither the biopsy nor post-mortem tissues from Bitches 8 and 9, strengthened this view. It was observed, however, in biopsy tissue from a bitch whose history was unknown. Subsequently she was fed a diet of bread and meat, but the experiment was prematurely terminated, and the effect of the diet on the alveolar bone was not determined.

Differences in the chemical findings for the two groups of puppies are striking. The mineral content of the bones of representatives of the litter born to Bitch 7 averaged 9.4 per cent, and of that of the offspring of Bitch 31, 14.0 per cent. There were nine puppies in the former litter, and their average weight was 191 grams; there were only six in the latter, with an average weight of 208 grams. It could be argued that the size of the litter determines the absolute amount of minerals in the individual. That this is not necessarily true is shown by the fact that there were ten puppies, in the second litter born to Bitch 7 and Dog 1, whose average weight was 285 grams. The calcium content of the fresh body-substance was 0.54 per cent. The fourth litter born to these parents contained nine puppies, with an average weight of 191 grams and a calcium content of 0.46 per cent. Then again, the calcium content of two successive litters born to Bitch 5 and Dog 1, each litter containing ten puppies, was 10.45 and 5.9 grams, respectively, these values being calculated from percentages in representatives of the litters. Thus it seems that certain combinations of dietary and hygienic factors exert markedly different influences upon calcification in the dog.

A comparison of the dental findings in the two groups of puppies shows interesting differences. The most striking characteristics of the histologic pictures for the puppies in Group I are the degenerate condition of the odontoblasts, the relative thinness of the dentin layer, and the small blood-supply of the pulp. Compare figs. 8 and 17, 9 and 18, 11 and 14, 12 and 15, which are photomicrographs of dental tissues of puppies in Groups I and II, respectively. Examination of fig. 9, which is a high magnification of the coronal region of pulp and dentin in a canine of Puppy 38, shows an almost total absence of blood vessels bordering the predentin layer. The odontoblasts are scattered and irregularly arranged. These findings are in striking contrast to the histologic pictures of pulps in the teeth of the puppies in Group II (fig. 18). In keeping with the apparent disturbance in the odontoblasts, the dentin appears to be thinner in the teeth of the puppies in Group I than in those in Group II. This, however, is difficult to determine on account of differences in the angles at which the sections were cut. Since the teeth are unerupted and are irregularly set in the jaws, it is impossible to secure sections of the various regions that are altogether comparable. Regardless of angle, however, the odontoblasts in the teeth of the puppies in Group I are widely separated, compared with those in Group II, and appear as fibers clumped together in bunches, with large intercellular spaces.

A comparison of figs 9 and 18 shows also a striking difference in the formation of predentin. In fig. 9 the marginal lines are indistinct, there being no definite line of demarcation between the calcified and non-calcified areas. In other sections, the predentin layer is better defined (fig. 12), but even in these the odontoblasts are separated from the organic matrix by a space irregularly traversed by fibrils entering the matrix. Here again, technical differences, thickness and angles of sections, make interpretation difficult. The findings are so consistent, however, regardless of technique, that the conclusion that the differences are real is inevitable.

An interesting finding, which occurred frequently in the puppies of both groups, is the disturbance in the ameloblasts (figs. 11, 12, 14 and 17). The ameloblasts were separated from the enamel matrix by a network of fibers in which enamel globules were scattered. Obviously, if the failure in function of the enamel and dentin-forming cells was dietary in origin, the mechanisms were controlled by different factors. The fault that resulted in the failure of the ameloblasts was common to both diets, while that affecting the odontoblasts was present only in Diet 1. Wolbach and Howe⁴ reported that vitamin-A deficiency in the diet of rats exerted a profound influence upon epithelial tissues. Microscopic changes, usually of minor degree, were also regularly found in the odontoblasts and in the pulp. Atrophy of the dentin-forming cells from slight shrinkage to complete disappearance was noted; also osteoid tissue, which was incorporating as well as surrounded by osteoblasts. Such findings were not observed in the offspring of Bitch 31, which was fed a diet of bread and meat exclusively for several months both before and during pregnancy. M. Mellanby⁵ has shown that diets deficient in fat-soluble vitamins and high in cereals have a marked effect upon the form and structure of puppies’ teeth. Since both diets used in the experiments reported above were low in vitamins, the disturbance in the ameloblasts that occurred in the offspring of all the bitches could easily be explained on the ground of fat-soluble-vitamin deficiency. This, however, does not offer a satisfactory explanation of the observed differences in dentin formation and pulp structure in the two groups of puppies.

Numerous investigators have studied the effect of the antiscorbutic vitamin on dental tissues. Histological changes in the teeth resulting from vitamin-C deficiency have been shown to occur primarily in dentin and pulp. In extreme cases, in guinea pigs, the normal orthodentin is largely substituted by osteodentin. The pulp tissue is so degenerated that the normal elements can scarcely be recognized. The process begins with hemorrhages in the upper part of the pulp and extends toward the apex. Such a condition has not been observed in any of the puppies’ teeth. Furthermore, potato is relatively rich in antiscorbutic vitamin. Since the disturbance in the odontoblasts occurred in puppies whose mothers were fed the potato diet, the fault, if of dietary origin, could not have been due to vitamin-C deficiency. Findings reported in the literature, on the teeth of various laboratory animals suffering from vitamin deficiencies, do not closely agree with the observations on either the adult dogs or new-born puppies in these experiments, and it appears that some other factor or combination of factors regulates calcification in the canine foetus under this particular set of conditions. In a series of experiments on older puppies whose diet consisted of bread, milk, meat, butter fat, and orange juice, supplemented with a salt mixture, the composition of which was based on ash analyses of milk but contained an excess of sodium and potassium, gross skeletal and dental abnormalities were observed. The dentin and pulp of the permanent teeth were extensively involved. In one animal it was found that the dentin had been so altered that its identification was impossible. The roots had become so completely resorbed that the enamel shell of a canine was lifted from the gum by a slight pull with the fingers. The pulp had degenerated into a fibrous mass.²¹

The bread, meat, and potato diet described above was low in calcium, vitamins, and protein, and potentially basic in reaction. Compared with the bread and meat diet, on which Bitch 31 was fed for ten months before and during pregnancy, it was lower in protein and appreciably richer in minerals and vitamins, and from the latter standpoints would seem to be superior. Yet the skeletal conditions of the offspring of the mothers on the two diets were just the reverse of those expected. That the substitution of meat for potato in the diet of the mother should exert so profound an influence upon foetal calcification seems extraordinary. The most striking difference between the two diets was in their potassium contents and acid-base relations. The bread, meat, and potato diet had approximately three times as much potassium as the bread and meat diet, and an excess of base equivalent to 13 cc. of normal solution, while the latter had an excess of acid equivalent to approximately 46 cc. In metabolism studies on children, Sherman and Hawley⁶ have shown that the calcium of vegetables is poorly utilized compared with that of milk. The same has been shown, by McCluggage and Mendel,⁷ to be true in the dog. The low coefficient of digestibility of vegetables may account for this in part, but it is also probable that the high basicity of the vegetable ash is a contributing factor to the poor utilization of the calcium salts.

In this connection the data reported by McCluggage and Mendel are interesting. In a series of experiments on two dogs, a rise in the potential alkalinity of the diet was accompanied by an increase in the excretion of calcium. A fixed amount of this element was added to a basal diet first in the form of milk, then carrots, then spinach. The milk diet of one animal had a potential acidity equivalent to approximately 11 cc. of normal solution; that containing carrots, an excess of base equivalent to 33 cc.; that containing spinach, 77 cc. of base. The calcium balances (averages of the different metabolism periods) were–0.108, –0.215, and –0.270 gram, respectively. Givens and Mendel⁸ reported that “the administration of base or acid produced no significant effect upon the balance of nitrogen, calcium, magnesium and phosphorus in the dog.” Simultaneously with the appearance of their paper, Stehle⁹ reported that the oral administration of hydrochloric acid, in dogs, causes an increased excretion of calcium. Kochmann and Petzsch,¹⁰ from their studies on the effect of protein, fat, and carbohydrate upon the excretion of calcium in dogs, concluded that the character of the diet influences in a large measure the retention of this element.

As early as 1873 Bunge showed that the animal organism suffers an impoverishment in chlorin when there is a surplus intake of potassium. Vegetables are extraordinarily rich in this element, and the necessity of adding sodium chloride to the diet of herbivorous animals is attributed to this fact. In view of these findings, Seeman¹¹ arrived at the conclusion that the high incidence of rickets among the poorer classes was due to the excessive use of vegetables, which resulted in an insufficient formation of hydrochloric acid in the stomach, and thus prevented the transformation of the insoluble calcium salts in the food into the soluble forms. In Seeman’s experience, the addition of sodium chloride or hydrochloric acid to the diet of rickety children was beneficial. Subsequently, Zander¹² reported that the milk of mothers of rickety babies contained more potassium than that of mothers of normal infants. Some evidence tending to corroborate, and some to contradict, has been reported in the last half century, but no irrefutable data for or against Seeman’s theory have been presented. Scheer¹³ found better absorption and greater retention of calcium and phosphorus in children on an acid than on an alkaline diet. Recently Flood¹⁴ and the writer¹⁵ reported that certain additions of hydrochloric acid to cow-milk formulas promoted calcification in rickety children. Conversely, the addition of sodium bicarbonate to cow-milk formulas has been shown by Shohl and Sato¹⁶ to reduce the retention of calcium and phosphorus in infants. According to these investigators, hydrochloric acid in the amounts they used had the same effect. Aub and associates,¹⁷ in their studies on lead poisoning, have also shown that the excretion of calcium as well as lead is increased by certain amounts of both acid and alkali. Zucker and Barnett,¹⁸ in experiments on rats, converted a non-rickets-producing diet into a rickets-producing diet by the addition of sodium carbonate.

In the writer’s experience,¹⁹ alterations in the acid-base balance of certain experimental diets appeared to exert a profound influence upon calcification in dogs. The absolute amount of a food constituent, organic or inorganic, within certain limits, does not seem to be a prime factor in regulating the calcifying mechanism. Certainly, the excess of base in the bread, meat, and potato diet described above was not sufficiently great, nor the mineral, protein, and vitamin content, per se, sufficiently low, to account for the failure in calcification in the offspring of Bitches 5 and 7. Yet the combination of factors contained in such a food mixture, in the absence of ultraviolet radiation, appears to exert an unfavorable influence upon the calcifying mechanism in both man and dog. Bread, meat, and potato constitute the basis of the average American dietary, and in large cities where hygienic conditions are poor and ultraviolet radiation is negligible in quantity, rickets and dental disease are almost universal. Nature appears to have combined certain factors–fruits and vegetables, which are relatively low in protein, high in minerals and vitamins, and potentially basic in reaction–with an abundance of sunshine. As the intensity of the sun’s rays decreases, the diet of man tends to become higher in protein, lower in calcium, and potentially acid in reaction.

With certain combinations of food, ultra violet radiation and hygiene appear to assume minor roles in calcification. In this connection the Carnegie report on the Physical Welfare of Mothers and Children of Scotland is of interest. The hygienic condition of certain of the inhabitants of the Island of Lewis in the Hebrides could scarcely be worse. They live in “black houses,” the walls of which are often as much as five feet thick. There is no chimney and only one opening in the house. Peat fires are kept constantly burning. Domestic animals occupy the building with the family. Chickens roost on the beds, tables, and chairs. The children are rarely ever taken out of the house until they can walk, yet rickets is almost unknown on the island; and, according to the report, “the most striking fact in the adult population is their beautiful teeth–a testimony to the absence of rickets in infancy.” Furthermore, the infant death-rate is among the lowest in the British Isles, and has occasionally fallen as low as 40 per 1000. The staple articles of food are fish, oatmeal, and eggs. Milk is scarce and vegetables almost unknown. Fish liver is a favorite dish. Such a diet is high in protein and phosphorus, and low in calcium and potassium. It has a relatively high potential acidity, and is probably also rich in vitamins. Such a combination of dietary factors apparently is able to overcome the deleterious effects of poor hygiene, filth, and lack of ultraviolet radiation.

In addition to the factors already discussed, the influence of the father’s diet before mating, on the composition and structure of the bones of the offspring, should be considered. The observations reported in this paper throw no light on this point, since both parents received the same diet, and lived under identical hygienic conditions in each of the experiments. Korenchevsky,²⁰ in experiments on rats, has shown that feeding the father before mating on a diet deficient in “fat-soluble factor” had no influence whatever on the number, weight, water, calcium, phosphorus, or nitrogen content of the offspring at birth. It is conceivable that faulty diet and hygiene on the part of the father extending over a long period of time might exert a deleterious effect upon the offspring. Such experiments as these in dogs require years of time and large facilities. This is one of the many phases of the problem which remain to be studied.

Clinical and laboratory experience shows that ossification is not determined by the absolute amount (within certain limits) of any one known dietary factor, or of ultraviolet radiation. There are many interdependencies and interrelations. The ability of a cell to function seems to be equally dependent upon its integrity and its environment. It has been demonstrated that the composition of the body substance, and the structure of the bones and teeth of new-born puppies of the same litter, as well as of different litters, may vary greatly. This fact offers a logical explanation of the differences in the behavior of animals toward the same experimental procedures. Certain combinations of diet and hygiene appear to exert a favorable influence, others an unfavorable effect, upon the calcifying mechanism. The experiments reported in this paper are preliminary, and are not sufficiently numerous or varied to warrant deductions in regard to the mechanism of foetal ossification. They show, however, certain well defined differences in the composition of the body substance, and in the structure of the bones and teeth, of new-born offspring of parents fed different combinations of common foodstuffs, and suggest new lines of attack on human problems.

IV. Summary

1. Observations were made on six brood bitches and thirty-eight new-born puppies. Mothers and offspring were divided into two groups. In Group I were four large mongrel bitches, each of which had previously given birth to two or more litters, and twenty newborn puppies. The bitches and the male with which they were mated were subsequently fed quantitatively a diet of bread, lean beef, and potato. Two of the bitches were given in addition increasing amounts of sodium carbonate. All of the animals lived under identical hygienic conditions, and received a negligible amount of ultraviolet radiation.

In Group II were two bitches and eighteen new-born puppies. Eight of the latter were representatives of litters born to two bitches whose histories and dental conditions were unknown. The remainder were offspring of two bitches, one of which had been in the kennels for six weeks, and the other one year, before the birth of their young. Their hygiene was the same as that of the animals in Group I. The diet consisted of bread and meat exclusively.

2. New-born puppies of the same litters in both groups (I and II, above) showed marked differences in weight, general well-being, body ash, and structure and mineral content of the bones. Findings in general for the two groups of mothers and offspring showed certain well-defined similarities and differences.
3. The bitches in Group I, which received sodium carbonate, were not successfully bred. Dental tissue excised after they had been on the alkaline diet for one hundred and thirty days showed profound degeneration of the alveolar bone and roots of the teeth.
4. Two bitches, fed quantitatively on bread, lean beef, and potato before and during pregnancy, had convulsions during gestation and died at the time of littering. The physical condition of both animals appeared to be excellent. Their oral conditions and metabolism differed, the latter being indicated by a difference of 34 per cent in the mineral content of their offspring, although their food intakes were exactly the same and the total weights of their young differed by only 35 grams. Physical, blood, and urine findings during pregnancy were well within the normal range. Dental tissue excised post-mortem showed degenerative changes in the roots of the teeth and alveolar bone, but the degree of involvement differed in the two animals. The diet had no effect upon the number of individuals in the litters, but the puppies were small and poorly nourished, and the total weights markedly less than those for the preceding litters. The percentage of ash in the intact bones of the offspring of one mother varied from 7.8 to 10.4 per cent, with an average of 9.4 per cent. The mineral contents of the fresh body-substance of the two litters were 1.73 and 2.09 per cent. Histologic findings in both bone and dental tissues were markedly different, in certain respects, from those observed for the puppies in Group II.
5. Chemical and histologic findings on two puppies in a litter of eight in Group II were arbitrarily adopted as “normal.” The mother’s early history was unknown. During the last six weeks of gestation she was fed on bread and meat. Surviving members of the litter showed no clinical or x-ray evidence of rickets at the time of weaning. The fresh intact bone of the new-born puppies contained 14.2 and 14.7 per cent of ash. Histologic findings on bone and dental tissues in the two puppies were comparable, and are described.
6. The substitution of bread and meat for a vegetable, raw-milk diet, and of restricted living quarters (with a negligible amount of ultraviolet radiation) for the freedom of a ranch and an abundance of sunshine, apparently arrested an active degenerative process in the alveolar bone of a bitch. Concomitantly, the crowns of the teeth suffered disintegration. A litter born twelve months after the bread and meat diet was instituted had an average of 14.06 per cent of bone ash, the values ranging from 11.1 to 16.1 per cent. The physical condition of the puppies was extremely poor and that of the mother excellent. Histologic findings on bone and dental tissues differed in certain respects, in the various individuals, but on the whole were comparable with those arbitrarily chosen as normal.
7. Alveolar bone from three bitches, two in Group I and one in Group II, that was obtained immediately after the birth of the litters, presented a histologic picture not commonly observed. The mineral constituents of the compact bone and the cementum of the teeth appeared to have been leached out, leaving the corpuscles in various stages of degeneration in a matrix of. fibers. The condition was not observed in biopsy tissue excised twelve months previously from one of the animals, nor in the post-mortem tissue from the two bitches in Group I that were given sodium carbonate, and not successfully bred.
8. The experiments are preliminary and not sufficiently numerous to warrant deductions in regard to foetal calcification. They show, however, that different combinations of common foodstuffs, in the absence of ultraviolet radiation, exert markedly different effects upon the dentition of brood bitches, and upon the dental and skeletal development of their offspring.

The writer wishes to acknowledge, and to express appreciation for, the assistance of Dr. Zera Bolin of the Department of Pathology, University of California Medical School, in examining tissues and interpreting histologic findings.

 

References Cited:

1. Jones, Martha R., and Simonton, F. V. 1926 Proceedings of the Society for Experimental Biology and Medicine, xxiii, p. 734.
2. Gottlieb, B. 1923 Zeitschrift Für Stomatologie, iv, p. 195.
3. Simonton, F. V., and Jones, Martha R. 1927 Journal of the American Dental Association, xiv, p. 984.
4. Wolbach, S. B., and Howe, P. R. 1925 Journal of Experimental Medicine, lii, p. 753.
5. Mellanby, M. 1923 British Dental Journal, xliv,p. 1.
6. Sherman, H. C., and Hawley, E. 1922 Journal of Biological Chemistry, liii, p. 375.
7. McCluggage, H. B., and Mendel, L. B. 1918 Ibid., xxxv, p. 353.
8. Givens, M. H., and Mendel, L. B. 1917 Ibid., xxxi, p. 421.
9. Stehle, R. L. 1917 Ibid., xxi, p. 461.
10. Kochmann, M., and Petzsch, E. 1911 Biochemische Zeitschrift, xxxii, pp. 10 and 27.
11. Seeman, —-1879 Virchow’s Archiv für pathologische Anatomie, lxxvii, p. 299.
12. Zander, —- 1881 Ibid., lxxxiii, p. 377.
13. Scheer, K. 1922 Jahrbuch für Kinderheilkunde, xcvii, p. 130.
14. Flood, R. G. 1926 American Journal of Diseases of Children, xxxii, p. 550.
15. Jones, Martha R. 1924 Journal of the American Medical Association, lxxxii, p. 439.
16. Sohl, A. T., and Sato, A. 1923 Journal of Biological Chemistry, lviii, p. 235.
17. Aub, J. C., Fairhall, L. T., Minot, A. S., and Reznekoff, P. 1925 Medicine, iv, p.1.
18. Zucker, T. F., Johnson, W. C., and Barnett, Marion. 1922 Proceedings of the Society for Experimental Biology and Medicine, xx, p. 20.
19. Jones, Martha R. 1927 American Journal of Physiology, lxxix, p. 694.
    —-and Simonton, F. V. 1926 Proceedings of the Society for Experimental Biology and Medicine, xxiii, p. 734.
    —-and Simonton, F. V. 1928 Journal of the American Dental Association, xv, p. 881.
20. Korenchevsky, V., and Carr, M. 1924 Biochemical Journal, xviii, p. 1308.
21. Dental findings on certain animals in this series of experiments have been reported by Dr. John A. Marshall in the Journal of the American Dental Association (January 1924). In that communication the following statements appear in regard to the diet: “The only alteration was concerned with the proportions of calcium and phosphorus…These elements were, so far as we know, the only variable factors in the whole dietary regimen. Utilizable proteins, as well as vitamins, were given in the form of meat and other fresh foods. The inorganic elements were supplied in the form of McCollum’s salt mixture…” Contrary to these statements, it has never been thought that the calcium-phosphorus ratio of the diet was an important factor in any of the experiments, since bone and dental lesions have occurred on all ratios yet employed, some of which appear to have been optimal, when the diet contained a certain preponderance of basic ions. The composition of all the salt mixtures used was based upon the ash analyses of protein-free milk made according to the directions of Osborne and Mendel with certain modifications, and not McCollum’s as stated.