histological changes in the teeth of rats which differed according as to whether

Transcription

1 J. Physiol. (1946) I04, I2.3II.I A COMPARISON OF THE ACTION OF VITAMIN D ON THE TEETH OF RACHITIC RATS WITH THAT OF ADDITIONAL CALCIUM OR PHOSPHORUS ADDED TO RACHITOGENIC DIETS BY J. T. IRVING, From the Department of Physiology, Medical School, University of Cape Town (Received 2 February 1945) 253 In a previous paper (Irving, 1944a) it was shown that vitamin D caused histological changes in the teeth of rats which differed according as to whether the rats had been on a diet with a high or low Ca: P ratio. No such differences could be observed in the bones, since low-ratio diets do not cause widening of the epiphyses. It appeared to be of interest to compare the changes in the teeth of rachitic rats caused by vitamin D with those induced by altering the Ca : P ratio of the diet to one more favourable for calcification. Other workers (Karelitz & Shohl, 1927; Kramer, Shear & Siegel, 1931; Key & Morgan, 1932; Nicolaysen & Jansen, 1939; Coward & Kassner, 1940) have reported the changes in the bones, and in the blood chemistry, when P is administered to rachitic rats on high Ca: P ratio diets, but the changes in the teeth when this is done, and the effects produced by improving the ratio of a low Ca : P ratio diet, have not been investigated. The following experiments were therefore undertaken. METHODS Animals. As before, young albino rats of the Wistar Institute strain were employed in litters of from six to eight. The conditions under which they were housed were the same as previously reported. When the rats weighed between 50 and 60 g. they were transferred to one of the experimental diets. Diets of the experimental animals. Six experimental diets were employed; they were based on the Steenbock & Black rachitogenic diet no (1925), the Ca and P contents being altered by the addition of various amounts of CaCO3 (B.P.) and Na2HPO4, anhyd. (Merck puriss.). Table 1 gives the composition of the diets, the figures being checked by analysis.* For simplicity, the diets are referred to in the text by the number of the 'Ca : P ratio. The first three were similar to those previously employed. The composition of diet 5-6 varied somewhat from one batch to another. The figures given for * The P content of the basal diet, without CaCO3 or Na2HPO4, was erroneously given as % in the previous paper (Irving, 1944a). This should have been 0-206%.

2 254 J. T. IRVING TABLE 1 Ca: P ratio and no. of diet Ca % P % Description 'Normal' Ca: P ratio High Ca: P ratio Low Ca: P ratio 3-7 ~~1-49 0*40 High Ca : P ratio, intermediate 2 6 1* }fbetween 1 9 and 5;6 this diet are the averages of analyses of several samples. The diets were given slightly moistened with water. When vitamin D was given, it was in the form of irradiated ergosterol in oil (the international standard preparation) and was administered in one dose by mouth using a micrometer syringe. Treatment of animals. In all cases the animals were maintained on diet 5-6 or 0-26 for 28 days. The following procedures were then adopted: (a) Two animals of a litter were kept on the original experimental diet for a further 10 days; the rest of the litter was placed on diet 1-9 for 10 days. All the animals were then killed and examined. (b) One animal was killed. The rest of the litter were transferred to diet 1F9 and were killed and examined at daily intervals. (c) One animal was killed as a control. Half the rest of the litter were transferred to diet 1-9, the other half being given 30 i.u. of vitamin D. The animals were then killed after 1, 2, 4 or 6 days. (d) Animals were transferred to diet 1-9 and killed at 6-hourly intervals up to 30 hr. (e) Animals were transferred to diets 3 7, 3 0 or 2-5 and were killed at daily intervals. Weighing of the rats. The animals were weighed weekly while on the first experimental diet, and three times, at 3-day intervals, after transference to the second experimental diet in procedures (a), (b), (c) and (e). Examination of the rats. Longitudinal sections of the upper incisors were examined in the way previously described. The same method of measuring the time of action of vitamin D or of the new diet was used, but NaF injections were not given. The line test was applied to the lower ends of the radius and ulna (Coward, 1938). Some of the bones were also studied histologically. The lower ends of the radius and ulna were cut longitudinally on the freezing microtome without preliminary decalcification, treated with silver nitrate and in some cases also stained with haematoxylin. RESULTS Animals transferred from diet 5-6 to diet 1.9 All the animals gained weight while on diet 5-6. On changing to diet 1-9, weight was still gained in all rats, save those of one litter on procedure (c). These animals all lost weight after the change of diet, but were making it up

3 CALCIUM AND PHOSPHORUS AND THE TEETH 255 as the experiment proceeded. Careful examination showed that this weight loss made no difference to the results, which were the same as those found in animals gaining weight. Karelitz & Shohl (1927) found that the addition of P to a high Ca: P ratio rachitogenic diet rendered the rats hyperexcitable and liable to attacks of tetany. No such effect was seen in the present experiments. One litter of rats was treated with procedure (a). The control animals had marked rickets, while those transferred to diet 1-9 had, after 10 days, stages of healing in the epiphyses varying from 3 to 6 (Coward, 1938). The teeth of the control animals had all the signs of poor calcification with wide predentin and vascular inclusions. The teeth of the animals transferred to diet 1-9 showed a broad stripe of reactive new dentin in the more distal part, staining deeply with haematoxylin (P1. 1, fig. 3), and, more proximally, a mass of interglobular dentin. The predentin between the reactive area and the old dentin tended to fill in with interglobular dentin. Measurements of the reactive dentin showed that the influence of diet 1-9 had begun within a day of changing to this diet. Three litters of rats were treated with procedure (b). The line test became positive in all cases after 2 days on diet 1-9. The type of response seen in the bones during the early stages was in many respects, however, not like the changes after giving vitamin D (P1. 3, fig. 12). It appeared to be a kind of disorderly healing (P1. 3, fig. 13). Sometimes a separate line would be seen, as in the early stages of healing after vitamin D, but, more often, abnormal downgrowths of calcified material in the metaphysis joined the line to the diaphyseal bone. In several cases, the line was absent or not complete, and the downgrowths were a very notable feature. It was often impossible to grade the healing into one of the usual stages. The pattern of change in the tooth was the same in all cases. After 1 day on diet 1-9, a narrow deeply staining stripe of new reactive dentin was seen distally on both the lingual and labial sides. This stripe widened on the lingual side during successive days and was often striated, so that the stripes could be counted. When this was so, one stripe corresponded to each day's action. On the labial side, the stripe again widened with successive days' action and also extended proximally. At about the time that it first appeared, a coarse sprinkling of calcospherites began throughout the intermediate and proximal predentin (P1. 1, fig. 4). These built up in masses from near the already calcified dentin and spread over to the odontoblast side of the predentin, where they gradually fused to produce a stripe backed by much interglobular dentin and laid down at normal predentin width from the odontoblasts (P1. 1, fig. 5). This stripe joined with that formed more distally. In order to compare this reaction with that produced simultaneously by vitamin D in comparable rats, three litters were treated with procedure (c). The results in the animals transferred to diet 1-9 were the same as those above. Pr'. CIV. 18

4 256 J. T7. IRVING The line test in the animals getting vitamin D did not become positive till after 6 days in two of the litters and after 4 days in the other. The changes in the teeth after vitamin D were the same as previously described (Irving, 1944 a), the teeth of one of the litters reacting after 2 days and those of the other two after 1 day. While the reactions after diet 1-9 on the one hand, and vitamin D on the other, were similar in some respects, they differed in several important details. In particular, the reaction in the intermediate and proximal predentin differed considerably. After vitamin D, the first reaction in this part of the tooth, seen after 4 days, was a very fine peppering of calcospherites in the predentin, which faded off towards the old and already calcified dentin (P1. 1, fig. 6). By 6 days, the calcospherites had fused to form a fine stripe at normal predentin width from the odontoblasts (P1. 2, fig. 7). There, was, however, no coarse mottling of large calcospherites between the stripe and the already calcified dentin. The space was either uncalcified, or else contained a fine sprinkling of very small calcospherites. In addition, the reaction in this part of the tooth was much slower after vitamin D than after diet 19. Three litters of rats were treated with procedure (d). This experiment was undertaken to investigate the very early changes in the teeth and to find out if the teeth reacted before 24 hr. had elapsed after changing the diet, as appeared probable from earlier observations. One rat of each litter was killed after 6 hr., and two thereafter at 6-hourly intervals up to 30 hr. The line test became positive in one litter after 18 hr., in one after 24 hr., and not at all in the third litter. Kramer et al. (1931) and Karelitz & Shohl (1927) have noted that healing may occur in rachitic bones from 12 to 24 hr. after the addition of P to the diet. In all cases but one, a fine hypercalcified stripe occurred after 12 hr. in the distal area on both sides of the tooth. On the labial side this stripe was laid down at about 16,u from the odontoblasts. With the passage of time, this stripe became wider and more distinct and also extended farther and farther proximally. After 24 hr. this stripe on the labial side joined proximally with a line of calcospherites which were laid down in an irregular fashion and- which were the precursors of those which later filled this area in a haphazard manner. These results showed that the previous conclusion was correct, namely, that the tooth reacted within 24 hr. after the change of diet. Animals transferred from diet 5-6 to diets 3 7, 3-0 or 2-5 It was thought that the differences noted between the action of vitamin D and that of diet 1-9 might be due to the fact that the Ca : P ratio of the diet had been so suddenly and so markedly lowered. Diets of Ca: P ratio intermediate between 1-9 and 5-6 were therefore used, the total content of Ca not being altered (procedure (e)). Nine litters of rats were treated in this way, five litters being transferred to diet 3'7, two litters to diet 3 0 and two litters to diet 2-5, all after 28 days on diet 5-6. In some cases the animals were

5 CALCIUM AND PHOSPHORUS AND THE TEETH 257 weighed after transfer to the new diets, and in none was weight lost; in most an increase was found. A careful note was also taken of the food consumption, and no diminution in the amount of food consumed was seen after the change. Two rats from each of two of the litters transferred to diet 3-7 were killed on the 1st, 2nd, 3rd or 4th day after transference. The results showed that this was rather too short a period of observation, so one rat of each of the remaining litters was killed on the 2nd to 8th days after changing to diet 3-7. The line test in the first. two litters had not become positive by the 4th day. In the remaining three litters, the line test had become positive after 8, 6 and 7 days respectively. The healing was still of the same abnormal kind and could not be classified into the ordinary line-test stages. In no case was advanced healing found, even at the end of.the experimental period; the most that was seen was a complete line across the epiphysis, with downgrowths joining it to the diaphysis, which somewhat resembled stage 3 of healing. The response in the teeth was exactly like that described above under procedure (b), with the exception that the intensity of the reaction, as judged by the staining of the newly calcified dentin, was much less marked. The teeth in all cases reacted within 24 hr. by laying down a distal stripe on both lingual and labial sides. On the -labial side the same interglobular dentin response was seen, but in some litters it took rather longer to develop than when the Ca: P ratio of the diet was changed more drastically. P1. 2, fig. 8, shows the building up of interglobular dentin in the tooth of an animal after 4 days on diet 3*7. The reaction was just as with procedure (b). In later stages the whole area filled up with interglobular dentin, but the predentin was still abnormally wide at the end of the experimental period. The action of diet 3-7 thus resembled that of vitamin D in postponing the time of epiphyseal reaction, for it had been previously noted (Irving, 1944a) that at least 4 days separated the time of reaction of the teeth and bones after vitamin D dosage. This was the only particular in which a resemblance was seen; the reaction in the teeth bore little resemblance to that caused by vitamin D. Of the animals on diets 3-0 and 2-5, two from each litter were- killed after 1, 2, 3 or 4 days on the new diet. The line test became positive after 2 days in one litter transferred to diet 3 0 and after 4 days in the other litter; with diet 2-5, the line test became positive after 2 days in one litter and 1 day in the other. Thus, as with diet 3 7, the time of line-test reaction was postponed with diet 3 0, compared to the reaction time with diets 1-9 or 2-5, becoming earlier as the Ca: P ratio of the diet was made more favourable. In all cases, however, the line test had the same peculiar character previously described, and was not like that after vitamin D dosage. The changes in the teeth in the two groups were identical, and very similar to those seen when the animals were transferred to diet 1-9. With both, the teeth reacted within 18-2

6 258 J. T. IRVING 24 hr. of changing the diets. The pattern of change was exactly like that previously described, and, in particular, the coarse interglobular response in the proximal part of the tooth still occurred. It was therefore concluded from these observations that the difference between the action of vitamin D on the teeth, and probably also on the bones, and that of lowering of the Ca: P ratio of the diet, was not due to the sudden and considerable change in the dietary ratio. Animals transferred from diet 026 to diet 1-9 All the animals gained weight over the period on diet Three rats lost weight after transference to diet 1-9 (procedure (b)). The results obtained from these animals were the same as those from animals whose weight increased. Shohl & Wolbach (1936) found that some rats on diets with a Ca: P ratio of 0-25 showed carpopedal spasms and tremors. The animals on diet 0-26 in the present work were somewhat more excitable than normal, but no changes resembling tetany were seen. As was previously stated (Irving, 1944 a), it was also found here that none of the animals on diet 0 26 had widened epiphyseal cartilages, and so it was not possible to assess the effects of changes in the diet on the bones by means of the line test. One litter of rats was treated with procedure (a). The control animals had very badly calcified teeth and many vascular inclusions in the predentin. The animals which had been transferred-to diet 1-9 showed a marked response in the teeth; this took the form of a broad hypercalcified stripe on the distal lingual and labial sides, which was often laid down in incremental lines. From measurements of this stripe it was found that the teeth had reacted to the change in diet within 24 hr. In the intermediate and proximal parts of the labial side the response became more of an interglobular dentin type, with large calcospherites scattered haphazard throughout the predentin. *These tended to coalesce on the odontoblast side to form a stripe which fused distally with the solid stripe already mentioned (Plate 2, fig. 9). The reaction was broken by a series of 'dips' where calcification did not occur, or only occurred to a slight extent; vascular inclusions were found in these parts of the predentin, and apparently inhibited calcification. This inhibition of calcification in the proximity of vascular inclusions has also been noted by Schour, Chandler & Tweedy (1937), who stated that an arcade-like appearance was caused. The response after diet 0 26 was, in general, identical with that already found in animals originally on diet 5-6, except for the arcades in the proximal part of the tooth. In the case of animals on diet 5-6, there were many fewer vascular inclusions and so the arcades were not a prominent feature. Three litters of rats were treated with procedure (b). One day after the change of diet the teeth already showed a fine reactive stripe in the distal part and on both lingual and labial sides. With the passage of time the stripe

7 CALCIUM AND PHOSPHORUS AND THE TEETH 259 on the lingual side extended over the whole length of the tooth and thickened, often being laid down in incremental lines. On the labial side, the stripe also thickened in an incremental way and extended proximally. As it did this, a haphazard scattering of calcospherites developed in the intermediate and proximal predentin, fusing on the odontoblast side to form an irregular stripe, which joined with the distal stripe. In early stages, these calcospherites appeared first near the calcified dentin, and then built up towards the odontoblast side (P1. 2, fig. 10). The arcades already mentioned occurred frequently; but apart from these, which were caused by the large number of vascular inclusions, the reaction in these animals appeared identical with that in the animals first on diet 5-6. Three litters of rats were treated with procedure (c). The teeth of the animals transferred to diet 1-9 reacted in the same way as described in the preceding paragraph. The teeth of the animals given vitamin D behaved as previously described (Irving, 1944a), save that, in the present results, a distal stripe was more prominent on the labial side. One day after vitamin D dosage a fine stripe was visible on the lingual and labial sides in the distal part. On the lingual side this extended proximally with the passage of time and became wider. On the labial side an irregular mass of calcospherites developed in the intermediate and proximal predentin after about 4 days (P1. 2, fig. 11). The distal stripe extended back in later days and blended with these calcospherites. This stripe showed the same arcades as were noted in the other teeth. The reaction in the proximal part of the tooth developed more quickly after vitamin D than after diet 1-9, being, after 6 days, about 2 days more advanced. Apart from this, the reaction after vitamin D was identical with that after changing to diet 1-9. In the previous report (Irving, 1944a) the animals were given 18'4 i.u. of vitamin D, whereas here they had 30 i.u. This accounts for the earlier response of the teeth. Two litters of rats were treated with procedure (d), as it appeared from some of the above results that the teeth had reacted within 24 hr. of the change of diet. This supposition was found to be correct. The lingual side of the tooth reacted 12 hr. after the change of diet; the labial side reacted after 18 hr. in one litter and after 24 hr. in the other. The reaction in both cases was a very narrow hypercalcified stripe seen in the distal area. On the labial side this first appeared at a distance of 9,u from the odontoblasts. This low figure, compared with that of 16,u obtained when animals were transferred from diet 5-6 to diet 19 under similar conditions, is presumably due to the very slow incremental growth rate of the dentin when animals are on diet 0-26 (Irving, 1944a). Since the reaction caused by diet 1-9 resembled so closely that caused by vitamin D after diet 0-26, it was not thought necessary to analyse the reactions further by using procedure (e).

8 260 J. T. IRVING DISCUSSION Several writers (McCollum, Simmonds, Shipley & Park, 1922a; Kramer et al. 1931, and others) have drawn attention to the fact that changes in the diet or treatment of rachitic rats may cause spontaneous healing of rickets to take place. This is usually attributed to a reduction in the food intake causing a loss of weight. From the data cited above it can be concluded that loss of weight was not responsible for the changes described in the present results. Shohl, Bennett & Weed (1928) have shown that variations in the acidity or alkalinity of -a rachitogenic diet, concurrent with the addition of extra P, can affect the response of the animals. The alkalinity of the diets used in the present work was calculated by the method of Sherman & Gettler (1912), save that a valence of 1 8 was attributed to P04, as was done by Shohl et al. (1928), since this is its neutralizing value at the reaction of the body. Calculation showed that diet 1-9 had an alkalinity equivalent to 640 c.c. of 0.1 N. alkali per 100 g., and for diet 5-6 the figure was 600 c.c. of 0-1 N. alkali. Diets 3.7, 3-0 and 2-5 had values intermediate between these two figures. Since these differences were so slight, it was felt that the changes induced by these diets could not be attributed to alterations in the alkalinity of the diets. Shohl et al. (1928) found that the changes caused by extra P were virtually the same if the alkalinity of the diet was 859 or 311 c.c. of 0-1 N. alkali per 100 g. Diet 0-26 had a much lower alkalinity, equivalent to 93 c.c. of 0-1 N. alkali per 100 g. However, since the effects produced when changing from this diet-to diet 1-9 were the same as those found when continuing the animals on diet 0-26 but dosing them with vitamin D, it was again felt that the change in reaction of the diet had little to do with the observed effects. The retrospective type of calcification of predentin, previously described as caused by vitamin D in animals on low Ca: P ratio diets (Irving, 1944 a), also occurs after both high and low Ca: P ratio diets if the Ca and P contents are adjusted to a favourable ratio. The previously calcified dentin is not changed, but the process of calcification which is slowly going on is accelerated and extends to predentin which is nearer to the odontoblasts. How and why this should happen it is at present impossible to say, nor is it easy to picture the mechanisms involved. Presumably the elements concerned in calcification get to the site to be calcified via Tomes's fibres (Lefkowitz, 1943), and, on high or low Ca: P ratio diets, this process is so slow that the incremental growth rate outstrips that of calcification, thus causing wide predentin. This suggestion has been made by Schour & Hoffman (1939). On adjusting the diet, a better supply of these elements is available and in larger amount, and thus this process of calcification is accelerated, predentin laid down as much as 6 to 8 days previously (calculated from Irving, 1944 a) being retrospectively calcified. Vitamin D, acting in conjunction with low Ca: P ratio diets, does the same.

9 CALCIUM AND PHOSPHORUS AND THE TEETH 261 Why the vitamin acting on animals taking high Ca: P ratio diets should not do this, but cause the odontoblasts to begin forming normal predentin anew, leaving the older predentin almost untouched, is quite unexplained. In Text-fig. 1 are shown the three different types of recalcification of malformed predentin which have so far been described. In A is shown that which occurs with high Ca: P ratio diets after vitamin D and in parathyroidectomized rats after vitamin D or parathormone (Schour, Tweedy, Chandler & Engel, 1937). The action of vitamin D on rachitic rats also causes a healing reaction in the epiphyses as shown by the line test. In B is shown the reaction V, ~ ~ ~ ~ 'A b a b a b a A B C Text-fig. 1. Camera lucicta dirawings to illustrate the various types of response in abnormal predentin. a =dentin, b =predentin. The odontoblasts have not been drawn. The diet numbers refer to the Ca: P ratio. A. Animal on diet 5-6. Given 30 i.u. of vitamin D 6 days previously. The new-lv calcified area in the predentin, backed by very fine calcospherites, is shown. B. Animal originally on diet 0 26, changed 4 days previously to diet 1-9. The typical coarse interglobular dentin response is seen. A similar change would have occurred had the animal been dosed w ith vitamin D, or if it had originally been on diet 5-6 and then changed to diet 1-9. C. Animal on diet 0-26 injected with NaF 4 days previously. The arrow points to the hypercalcified line. The indentation in the predentin is a vascular inclusion. described in the present paper, when the Ca : P ratio of the diets is changed, or vitamin D is given to rats on low Ca: P ratio diets. This is accompanied by abnormal epiphyseal healing if the animal is on the high Ca: P ratio diet. In C is shown the change that occurs in animals on low Ca : P ratio diets if F or P is injected (Irving, 1943, 1944b). This is a very short-lived process in which again only the predentin forming at the moment of injection is affected. In all cases where the Ca : P ratio of the first diet was high and that of the second diet was lower or normal, the early stages of the healing process in the epiphyses were not like those in the classical line-test response but fell into the class described as diaphyseal healing by Bills, Honeywell, Wirick & Nussmeier (1931). Such calcification may occur after D medication, but preliminary work in this department (Irving, 1944a) has shown that vitamin D causes

10 262 J. T. IRVING it only in animals with insufficiently developed rickets, as has also been found by McCollum et al. (1922b), Bourdillon, Bruce, Fischmann & Webster (1931), and others. Since all the animals used in the present experiments were strictly comparable as regards the severity of the rickets, and all those dosed with vitamin D showed proper lines, it would appear that this response on adding P to the high Ca : P ratio diet normally occurs after such a procedure. This supposition is supported by the work of Nicolaysen & Jansen (1939) who found that the histological structure of the bones of rachitic rats may differ after the giving of P from that which is found after vitamin D medication. In the previous paper (Irving, 1944 a) it was noted that, after vitamin D dosage, the bones did not respond till at least 4 days had elapsed after the teeth had reacted. It is apparent from the results above that this difference in reaction can be imitated by varying the Ca: P ratio of the second diet. The more unfavourable the ratio of the second diet, the later does the bone react as compared with the tooth. This is what would be expected from previous work (Gaunt, Irving & Thomson, 1939), in which it was shown that under unfavourable conditions for calcification, the teeth were less affected than the bones. The effect of varying the Ca: P ratio upon the healing of rickets has also been studied by Coward & Kassner (1940), who found that the amount of healing in the epiphyses of rachitic rats gradually increased as the Ca P ratio of the diet was lowered. Animals which had become rachitic after a period on a rachitogenic diet were used; subsequently, after 10 days on a diet with a Ca: P ratio of 3-84 or 3-69, no healing was found, but with a diet of Ca: P ratio 3 43, an average degree of healing of 0-08 occurred. With lower Ca: P ratios, still better healing was found. It seems likely (Irvi'ng, 1941) that epiphyses, which show a lower grade of healing after a definite period, have begun to recalcify later than those which have a higher grade of healing. The present results with diet 3-7 are an interesting confirmation of the findings of Gaunt & Irving (1940), that normal teeth were formed on diets with Ca: P ratios between 4 and 0.5, provided that the Ca and P contents were each at least 0 3%. Diet 3-7 caused healing of the teeth in spite of the Ca: P ratio of 3 7, since the P content was 0 4 %. It would appear from the above findings that the early stages in the healing process in the teeth, and probably also in the epiphyses, of animals on high Ca : P ratio diets, differ when vitamin D is given from those which occur when the Ca: P ratio of the diet is adjusted to a normal figure. Kramer et al. (1931) have shown that while vitamin D causes the blood P to rise in rachitic rats, lowering the Ca: P ratio of the diet by adding extra P is followed by a much greater and quicker increase in blood P. The experiments quoted above make it unlikely that the differences in the action of the vitamin, and of the adjustment of the Ca : P ratio, were due to the rather drastic change in the latter, since the same differences existed when the Ca : P ratio was altered much

11 CALCIUM AND PHOSPHORUS AND THE TEETH 263 more slightly. In animals on low Ca: P ratio diets, vitamin D caused a healing change in the teeth identical with that found after the Ca: P ratio of the diet was raised to normal. It would appear at least from the evidence of changes in the teeth, that the underlying mechanisms of the action of vitamin D, and of the adjustment of the Ca : P ratio of the diet, are not identical when the Ca: P ratio is initially high; but that they may be the same when the Ca: P ratio of the diet is initially low. SUMMARY 1. Young rats weighing from 50 to 60 g. were placed on rachitogenic diets of high or low Ca: P ratio for 28 days. Some were then transferred to diets of more normal Ca: P ratio and others were dosed with vitamin D. After a further period, they were killed and their incisor teeth and their bones examined. The histological response in the dentin was such that the time and nature of the action of the new diet or of vitamin D could be compared. 2. Some of the rats on the diet with a Ca : P ratio of 5-6 and showing marked rickets, were transferred to a diet with a Ca: P ratio of 1-9. Healing in the bones was first detected from 18 hr. to 2 days after change of diet, but the pattern of healing was abnormal and unlike that after vitamin D dosage. A reactive response was seen in the teeth after 12 hr. This response, especially in the early stages, was unlike that caused by vitamin D, being an irregular deposit of interglobular dentin affecting the matrix laid down as much as 6-8 days before the diet was changed. 3. Others of the rats on the diet with a Ca: P ratio of 5f6 and showing marked rickets, were transferred to diets with Ca: P ratios of 2-5, 3-0 or 3-7. The time of the line response in the bones was the more delayed the higher the Ca: P ratio, averaging 14, 3 and 6 days respectively after change of diet. The response was of the same abnormal nature. The changes in the teeth were qualitatively the same as those noted in (2) above, and began in all cases within 24 hr. of the change of diet. It was concluded that the differences- in the action of vitamin D and of the adjustment of the Ca: P ratio of the diet mentioned in (2) above, were not due to the considerable change in the Ca : P ratio of the diet. 4. Rats on the diet with a Ca: P ratio of 0-26 were transferred to a diet with a Ca: P ratio of 1-9. As is usual with low Ca: P ratio diets, no widening of the epiphyses was seen. The teeth responded 18 hr. after the change of diet, the appearance of the new calcification closely resembling that after vitamin D dosage. 5. The changes in the teeth of animals initially on high or low Ca: P ratio diets appeared identical when the ratio of the diets was adjusted to a normal one. Vitamin D produced a similar histological change in the teeth of animals on the low Ca : P ratio diet, but a different response in animals on the high Ca : P ratio diet.

12 264 J. T. IRVING The writer is indebted to Dr Helen Schwartz, Department of Chemistry, University of Cape Town, for assistance with the dosing of the animals, and for analysing the diets; to Prof. R. Goetz for taking the microphotographs; and to Mr D. G. Duncan for technical assistance. The expenses of this work were defrayed by grants from the Staff Research Fund, University of Cape Town, and the National Research Council and Board, Union of South Africa; for these, grateful acknowledgement is made. REFERENCES Bills, C. E., Honeywell, E. M., Wirick, A. M. & Nussmeier, M. (1931). J. biol. Chem. 90, 619. Bourdillon, R. B., Bruce, H. M., Fischmann, C. & Webster, T. A. (1931). Spec. Rep. Ser. med. Bes. Coun., Lond., no Coward, K. H. (1938J. The Biological Standardisation of the Vitamin8. London: Bailliere, Tindall and Cox. Coward, K. H. & Kassner, E. W. (1940). Biochem. J. 84, 538. Gaunt, W. E. & Irving, J. T. (1940). J. Physio. 99, 18. Gaunt, W. E., Irving, J. T. & Thomson, W. (1939). J. Hyg., Camb., 39, 91. Irving, J. T. (1941). Nature, Lond., 147, 608. Irving, J. T. (1943). J. dent. Re8. 22, 447. Irving, J. T. (1944a). J. Physio. 103, 9. Irving, J. T. (1944b). Nature, Lond., 154, 149. Karelitz, S. & Shohl, A. T. (1927). J. biol. Chem. 73, 665. Key, K. M. & Morgan, B. G. E. (1932). Biochem. J. 26, 305. Kramer, B., Shear, M. J. & Siegel, J. (1931). J. biol. Chem. 91, 271. Lefkowitz, W. (1943). J. dent. Re8. 22, 287. McCollum, E. V., Simmonds, N., Shipley, P. G. & Park, E. (1922a). Bull. Johns Hopk. Hosp. 33, 31. McCollum, E. V., Simmonds, N., Shipley, P. G. & Park, E. (1922b). J. biol. Chem. 51, 41. Nicolaysen, R. & Jansen, J. (1939). Acta Paediat. 23, 405. Schour, I., Chandler, S. B. & Tweedy, W. R. (1937). Amer. J. Path. 13, 945. Schour, I. & Hoffman, M. M. (1939). J. dent. Re8. 18, 161. Schour, I., Tweedy, W. R., Chandler, S. B. & Engel, M. B. (1937). Amer. J. Path. 13, 971. Sherman, H. C. & Gettler, A. 0. (1912). J. biol. Chem. 11, 323. Shohl, A. T., Bennett, H. B. & Weed, K. L. (1928). J. biol. Chem. 78, 181. Shohl, A. T. & Wolbach, S. B. (1936). J. Nutrit. 11, 275. Steenboch, H. & Black, A. (1925). J. bil. Chem. 64, 263. EXPLANATION OF PLATES 1-3 Figs. 2 to 11 are longitudinal sections of the intermediate part of the labial side of the upper incisor tooth. Magnification in all is x 90. a = dentin, b = predentin, c = odontoblasts, d = pulp. The diet numbers refer to the Ca: P ratio. PLATE 1 Fig. 2. Normal tooth to show the various structures. Fig. 3. Section from an animal on diet 5-6 for 28 days and transferred to diet 1 9 for 10 days. al=dentin formed while on diet 5*6; a =dentin formed while on diet 1 9. Two arcades are shown. These were not very, common in animals on diet 5-6. Fig. 4. Section from an animal on diet 5-6 for 28 days and killed 2 days after transfer to diet 1 9. Note the formation of coarse calcospherites in the predentin.

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14 PLATE- 2 THE JOURNAL OF PHYSIOLOGY, VOL. 104, No. 3 (I 1 C Fig. 7 (I I. c Fig. 8. (1 (1_ i.1 Fig. 9. Fig. 10. a b c Fig. 11.

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16 CALCIUM AND PHOSPHORUS AND THE TEETH 265 Fig. 5. Section from an animal on diet 5-6 for 28 days and killed 6 days after transference to diet 1-9. a' =dentin formed while on diet 5-6; a2= the interglobular dentin and calcified stripe formed under the influence of diet 1-9. Fig. 6. Section from an animal 32 days on diet 5-6, having been dosed with 30 i.u. of vitamin D 4 days before being killed. Note the fine peppering of calcospherites in the predentin. PLATE 2 Fig. 7. Section from an animal 34 days on diet 5-6, and which received 30 i.u. of vitamin D 6 days before being killed. Note the narrow line of new dentin which has formed in the predentin. Fig. 8. Section from an animal 28 days on diet 5-6 and then transferred to diet 3-7 for 4 days. This section shows the interglobular dentin response in the predentin caused by diet 3-7. Fig. 9. Section from an animal 28 days on diet 0-26 and transferred to diet days before being killed. a' =dentin formed while on diet 0-26; a2=dentin and interglobular dentin formed while on diet 1-9. The typical arcades are weu shown. Fig. 10. Section from an animal 28 days on diet 0-26 and killed 5 days after transfer to diet 1 9. The second diet has caused the formation of large calcospherites in the predentin. Compare with P1. 1, fig. 4. Fig. 11. Section from an animal on diet 0-26 for 32 days and given 30 i.u. of vitamin D 4 davs before being killed. Note the interglobular dentin response in the predentin, similar to that seen in Figs. 4 and 10. PLATE 3 Figs. 12 and 13 are longitudinal frozen sections of the lower ends of undecalcified ulnae and radii, treated with AgNO3. Magnification x Fig. 12. Typical early line-test response in a rachitic rat after treatment with vitamin D. Fig. 13. Abnormal epiphyseal response in a rachitic rat caused by transference to diet 3-7.