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An experimental study of certain factors affecting calcification of bone

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An experimental study of certain factors affecting calcification of bone

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Content AN EXPERIMENTAL STUDY OP CERTAIN FACTORS AFFECTING CALCIFICATION OF BONE A Thesis Presented to the Facility of the Department of Biochemistry The University of Southern California School of Medicine In Partial Fulfillment of the Requirements for the Degree Master of Science by Elizabeth Sumner Hu n’ t er February, 1946 This thesis, written by .EJLlzBl).ai:ii...ja...jSuinhBj;..Huatar........................ under the guidance of Far.... Faculty Committee, and approved by a ll its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fu lfill ment of the requirements fo r the degree of Maa.t.9£....o£...&c±9nQa Secretary Faculty Committee ■ . f c y u e j t f f i Chairman The writer gratefully acknowledges her Indebtedness to Or. harry J, Deuel, Jr. and Mrs* Cornelia Johnston for their guidance In this work and to Dr, Caroline Hrubetz for her assistance with some of the laboratory work. TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION ............. 1 II. REVIEW OF THE LITERATURE .............. 6 Literature on the effect of vitamin A on calcifi cation of the bone 6 Literature on the effect of vitamin D in relation to the calcium and phosphorus content of the diet on calcification of the bone. . •••••• 8 Literature on the effect of age and rate of growth on calcification of the bone............... . * 10 III. TECHNIQUE AND RESULTS OF THE INDIVIDUAL EXPERIMENTS. 12 The effects of vitamin A and vitamin D on ash con tent of the bones in vitamin A-deflcient rats. • 12 The effect of age as compared with size on ash content of the bones in normal stock rats. . . . 16 Discussion of results obtained from experiments on the effects of vitamin A and vitamin D on the ash content of the bones in vitamin A-deficient rats •.•••••• ...••••• 32 IV. SUMMARY................. 36 BIBLIOGRAPHY . 38 II ill TABLE I. II. III. IV. V. VI. VII. LIST OP TABLES AND FIGURES PAGE Summary Table of Experiments on Male and Female Rats Receiving $ Carotene and, or Viosterol in Cotton seed Oil or tbs Oil Alone »•....••••••• 17 Summary Table of Experiments on Male Rats Receiving 0 Carotene and, or Viosterol in Cottonseed Oil or the Oil Alone . • * • * . • • • . • • . . . * 19 Summary Table of Experiments on Female Rats Receiving 0 Carotene and, or Viosterol in Cottonseed Oil or the Oil Alone • • • • . • . • . . . • ......... . 21 Summary Table of Experiments on Male and Female Rats Receiving 0 Carotene and, or Viosterol in Cotton seed Oil or the Oil Alone While on an A-free Diet Obtained from SMA, Chagrin Falls, Ohio. ...... 23 Summary Table of Experiments on Male Rats Receiving 0 Carotene and, or Viosterol in Cottonseed Oil or the Oil Alone While on an A-free Diet Obtained from SMA, Chagrin Falls, Ohio. • 24 Summary Table of Experiments on Female Rats Receiving 0 Carotene and, or Viosterol in Cottonseed Oil or the Oil Alone While On an A-free Diet Obtained from SMA, Chagrin Falls, Ohio. ••.•••..••••• 25 Summary Table of Experiments on the Ash Content of Bone from Male and Female Rats Receiving /^Carotene in Cottonseed Oil or the Oil Alone. ••••...• 26 iv TABLE PAGE VIII* Summary Table of Experiments on the Male and Female Rats Receiving the USC Diet and Showing the Effect of Vitamin D independent of the Dosage of ,6 Carotene ............ • ♦ ♦ • 27 IX* Summary Table of Experiments on the Male and Female Rats Receiving the USC Diet and Showing the Effect of Carotene Independent of the Dosage of Vitamin D .......... 26 X* Summary Table of Experiments on Stock Rats Fed Ad Libitum ................... 30 XI* Summary Table of Experiments on Stock Rats with Food Intake Rest to 60 per cent of That of Similar Animals Fed Ad Libitum ••••••••* 31 FIGURE PAGE 1* Relationship of Gain in Weight to Dosage of & Carotene for Animals Receiving Viosterol and or (3 Carotene in Cottonseed Oil • *.••••• 29 CHAPTER I INTRODUCTION There are many known factors affecting calcification of the bone and many theories about their mode of action. All theories of calcification assume as an Important step in the mineralization of bone the production of a supersaturated condition of one or more calcium salts and the subsequent incorporation of the precipitated solid salt In the organic matrix. Besides the physical factors which determine solu bility, without a doubt very definite biological, factors are involved In normal and pathological calcifications. Among these biological factors are cartilage, which has a selective action particularly favorable to calcifica tion, and the enzyme phosphatase. It Is assumed that this enzyme Is needed to hydrolyze the organic esters of phosphoric acid and so, by Increasing the inorganic phosphate ion concen tration to enable a readier precipitation of. a slightly soluble salt of calcium and phosphate. Important chemical factors are vitamin D, Ca++ and PO4 as well as the hormone, parathormone. It has been known for some time that the lack of vitamin D brings about a condition known as rickets. The levels of both calcium and phosphorus in the diet are also very important factors in the production or cure of this disease. There is, however, some divergence of opinion as to 1 2 the correct definition of the term, rickets* It is believed by some to include only the condition in which the epiphyseal cartilage is unable to produce calcification in the growing bone. Cohn, et al. (5) state, Rickets is generally found in conjunction with dietary mineral deficiencies, and in the rat this Is always the case* Any procedure which produces a net deposition of bone will cure rickets, but It Is possible in both man and rat to cure rickets (produce epiphyseal calcification) with vitamin D without Increasing, even sometimes decreasing, the percentage of bone ash in the skeleton as a whole. This condition in which the usually proliferating cartilage fails to produce new calcification at the epiphyseal groove of the long bones of the body is associated not only with vitamin D deficiency but with a low intake of phosphorus or with an abnormal calcium/phosphorus ratio in which the phos phorus intake is relatively low, Sherman and others (20), however, also Include under the term rickets the conditions known as osteoporosis and osteomalacia, which may be produced by a low or relatively low level of calcium Intake and also by Insufficient dietary levels of both calcium and phosphorus* He also maintains, • * * that unless the mineral supply is very deficient, any of the three types of rickets can be prevented by vitamin D, which acts to restore to normal the concentrations of calcium and phosphorus ions in the blood. Parathormone also appears to be an Important factor in normal bone calcification due to its action on blood cal cium* The removal of the parathyroids causes tetany and a 3 drop In the blood calcium. Injection of parathormone into a parathyroldectomized animal restores its health and, at the same time, raises the percentage of calcium in the blood. The increase in plasma calcium is followed by an increased urinary excretion of calcium and inorganic phosphate and a decrease in blood phosphate if the administration is continued. It is believed that this extra calcium is derived from the bones by a withdrawal of the element. Under such conditions of hyper calcemia, the kidney undergoes pathological changes and abnormal deposits of calcium salts accumulate in soft tissues. There is some indication of a possible relation in the action of parathormone and vitamin D. Activated products of ergo- sterol and cholesterol also have the ability to increase serum calcium. Both calciferol (vitamin D^>) and dlhydro- tachysterol are used In parathyroid deficiency. Another vitamin whose effect on bone calcification is being studied, is the antlxerophthalmic factor or vitamin A. There has been considerable, rather contradictory literature on the effect of vitamin A or carotene on the vitamin D requirements of rats with rickets. Tabor, Deutcher and Guerrant (24) found that the addition of carotene to the Steenbock and Black rachitogenic diet appeared to aggravate the production of rickets and retard reca^clfication of the bone more than diets without carotene. Several workers in Germany (25), however, have found that carotene had no direct 4 action on the development or cure of rickets tout independently of dosage worked synergistically with ergosterol in affecting the calcium deposition which accompanied healing* Mellanby, (15) working in England on skeletal changes effecting the nervous system in vitamin A-defielent dogs, believes that the actual bone formed in the vitamin A-deficient animal is protoably not far removed from the normal toone and there is no increase in osteoid tissue* It is the gross arrangement of the toone which is so strikingly changed and it is probable, therefore, that the main effect of vitamin A on toone growth is to control the activity and number of osteoblasts and osteoclasts* In its absence these cells become more active, tout the change seems to toe only in intensity and not in func tion, and the relative increase in their activities remains balanced, so that the total amount of calcified tissue does not greatly differ in the vitamin A-deficlent animal from that in one receiving this vitamin in abundance. Affects of vita min A deficiency must toe considered in relation to basal diets given* Modifications of the basal diets even in the absence of vitamin A affect calcification quantitatively if not quali tatively* In particular, addition of calcium salts and the presence of vitamin D modify the toone overgrowth in vitamin A deficiency* Results under these conditions do show that toone growth is affected toy vitamin A (15, 16)* Another biological factor which influences the calcification of bone is age* Sherman and co-workers (20) in many experiments on rats have found that when there is an adequate level of calcium and phosphorus in the diet the rate of growth as expressed by body weight at a given age is in itself not a disturbing factor in the rate of calcification in normal development. In the normal growing individual of a given sex having the same hereditary and nutritional back ground, age is the predominant determining factor in the Increasing percentage of calcium in the body. In the present series of tests, the influence of size at a particular age has been studied by restricting the growth of animals on an adequate stock diet. Another factor studied was the possible effect of vitamin D on total bone ash of vitamin A-deflcient rats, A few preliminary experiments had indicated that under certain conditions the ash content of bone in the rat is significantly lowered in vitamin A deficiency even when the vitamin D intake Is adequate* CHAPTER II REVIEW OP THE LITERATURE Much has been written in regard to the effects of the many factors affecting the calcification of bone, but only a brief summary of the work of experimenters on problems very closely related to the one at hand will be given* Literature on the effect of vitamin A era calcification of the bone* Much of the work that has been done on the effect of vitamin A on calcification is contradictory* Thoenes (25} working on the effect of the antirachitic and antixerophthalmic factors, found that carotene had no affect on either development or cure of rickets, but that it did assist in affecting the calcium deposition which accompanied healing* Bomskov and Seemann (3), however, found that in rickets the curative action of vitamin D is hindered when excess dosages of vitamin A are given* They also found that in rats on a normal diet large dosages of vitamin A would cause a mineral depletion of the bone* von Euler and Karrer (27), in studying the effects of carotene on rats on a diet relatively free of vitamin A and vitamin D observed a normal growth of the animals even in the absence of vitamin D if the diet contained 0*03 mgm* carotene per day* Even with vitamin D in the diet, the calcification was rather defective in the absence of carotene. 6 7 Tabor, Deutcher and. Guerrant (24), In more recent studies on the effect of carotene Intake on the vitamin D requirement of rats on the Steenbock and Black rachltogenic diet found that the addition of 90 U.S.P. units of vitamin A with or without 5 per cent Wesson oil. aggravated, the produc tion of a ricketie condition in the animals and appeared to retard the recalclflcation of the bone more than diets with out added vitamin A* Their conclusions were drawn from the line test data that they obtained, but these data were not In complete agreement with bone ash determinations or serum analyses of calcium and phosphorus. Mellanby (15, 16), in a long series of studies on the effect of vitamin A on bone growth and the resulting neurolog ical changes in dogs deficient in this factor, found that the bone formed in such animals, was very little different from normal bone. The bone of the vitamin A-deflcient animal was usually thick and cancellous but contained about the same amount of calcium as the thinner, more compact bone of the animal receiving sufficient vitamin A, The effects of vitamin A deficiency were modified by the basal diets given. Under the conditions of his experiments, the calcium in the diet was not high enough for optimal bone formation when growth was rapid, but increasing the calcium content of the diet modified, though it did not prevent, the structural abnormali ties of the vitamin A-deficient animal. Literature on the effect of vitamin D in relation to the calcium and phosphorus content of the diet on calcifica tion of the bone. Since the calcium of the body is localized in the bones and teeth (the skeletal system contains over 99 per cent of the body calcium), the calcium content of the food becomes a very important factor in affecting normal bone calcification. In an extensive study of the calcium content of the rat Sherman and Booher (21) found that Mien calcium was the sole significant variable factor the amount of cal cium in the body of the growing animal, varied with the calcium content of the food, Bachraann, at al. (1) observed that a progressive decrease in the percentage of calcium and phosphorus in the food mixture resulted in a corresponding dimunition in wet weight, dry weight, ash, calcium and phosphorus content of the bone and an increase in the per cent of organic material. Boelter and Greenberg (2) in work on severe calcium deficiency in growing rats found that the per cent of ash and calcium of the bone was only about one half of that found normally. There was a lesser decrease in the phosphorus and magnesium per cent. The effect of vitamin D on calcification is very intimately related to the calcium and phosphorus content of the diet. It Is well known that, in the rat, rickets can be produced on a diet low, or relatively low in respect to calcium and high in phosphorus or vice versa. Under such conditions the antirachitic factor stimulates the proliferat ing cartilage to calcify, although the total hone ash Is not necessarily increased. In studying the roentgenograms of rachitic children, Hess (12) found that elementary yellow phosphorus was Ineffective as a prophylactic or curative agent in ordinary cases of rickets. Although it had no affect upon the failure of proliferating cartilage to calcify, it did in some cases increase the total calcification of the hone. Zucker, Hall and Young (2a) working on rats on a diet deficient only In phosphorus, showed that although the addi tion of vitamin D prevented the anatomical signs of rickets, it did not produce a normal percentage of hone ash; the values were not equal to those of animals of the same age or weight on a stock diet. The addition of phosphorus was required to bring the composition of the hone to normal. When rats were fed on diets adequate in calcium and phosphorus but deficient in vitamin D, Sherman and stiebeling (22) observed an increase in calcification from 53.6 per cent for negative controls to 59.4 per cent for animals receiving the missing factor in whole milk. In a continuation of this work (23), they eliminated any possible effects of fat, lactose or added minerals, hut did not consider the fact that whole milk contains vitamin A as well as vitamin D. 10 In the human subject, adult rickets or osteomalacia is primarily a mineral deficiency due in part to avitaminosis D and responding completely to therapy with calcium, phosphorus and vitamin D. Liu and co-workers (14) in studies on the effect of vitamin J> and calcium levels in osteomalacia on calcium retention in pregnant women, observed that a variable proportion of the total fetal requirement of calcium might be drawn from the maternal store if there was an adequate supply of vitamin D* This might not constitute a serious loss to the mother, but in the absence of the vitamin the mineral loss would be much greater than that imparted to the fetus, if the calcium intake was rather low* In such a case the calcium content of the food would have to be increased for balance to be maintained* Vitamin D, however, appeared to be a more important factor than the actual level of calcium intake in determining retention, provided a reasonable amount of calcium was present* Under conditions of optimum vitamin D nutrition, the ealcium requirement could be lowered. literature on the effect of age and rate of growth on calcifl'cation of the bone* Brlwa and Sherman (4) in studying the calcium content of the normal growing body at a given age found that the rate of growth as expressed by body weight at a given age is in Itself not a disturbing factor in the rate of calcification in normal development* In the normal growing Individual of a given sex, having the same hereditary and nutritional background, ago is the predominant determining factor in the increasing per cent of calcium in the body. Lanford, Campbell and Sherman (13) also observed that neither a more rapid growth rate nor an increase of growth rate by an enrichment of the dietary protein content could augment the rate of increase of calcium In the body. It could, however, be Increased by an enrichment of the calcium content of the food. CHAPTER III technique and results op the INDIVIDUAL EXPERIMENTS The effects of vitamin A. and vitamin D on ash content of the bones In vitamin A-deficient rata. The method of assaying the effect of vitamin A on the. ash content of the bones and any possible synergism between vitamins A and D was essentially the same as the United States Pharmacopeia bioassay method of determining vitamin A* Females were placed on a diet low in vitamin A for several months, then bred and their litters used for the assay. The animals used were albino rats from the stock colony of The University of Southern California. The composition of the vitamin A-low diet was as follows: Whole wheat flour 66 per cent Powdered whole milk 33 per cent Sodium chloride 1 per cent When the litters were fourteen days old they were changed to a diet low in vitamin A and at weaning were placed on an A-free diet. These two diets were essentially the same except for the source of casein. (Casein for the A-free diet is extracted with alcohol.) Casein 18 per cent Salt Mix (Osbome-MendaL) 4 per cent Yeast _ 8 per cent Starch 65 per cent Cottonseed oil 5 per cent (for each kgm. of oil one gm. of tocopherol was added) 12 13 Since the possible effect of vitamin D alone or the effects of varying combinations of the two vitamins was to be studied, no vitamin D was added to the A-free diet and the source of yeast was D-free. (Anheuser Busch, strain G, is entirely satisfactory*) This diet will henceforth be termed the USC diet* The young rats were weaned when they were at least twenty-one and not over twenty-eight days old and weighed between forty and fifty grams* At weaning they were placed five in a cage and were weighed once weekly for two weeks, after which they were placed in small Individual cages and were weighed daily* The rats were kept in separate cages for the remainder of the experiment* When the rats showed a constant weight level for five days or took a sudden drop in weight of more than four grams they were ready for the assay period* They were then divided into groups in such a way as to make equivalent litter and sex distribution of the groups* When the assembling of groups was completed, each group contained the same number of rats with the sex distribution similar and the average weight of the rats in any one group at the start of the assay period did not exceed by more than ten gram3 the weight of those in any other group* The source of vitamin D was a sample of viosterol from the Parke Davis Company containing approximately 11,000 I*U* 14 of vitamin D per gram of oil* Since most fish liver oils contain vitamin D, a vitamin A reference cod liver oil could not be used* Pure carotene was thus selected as the source of vitamin A* The vitamin supplements were given orally by means of a syringe to which a blunt end needle was attached* The supplements were administered daily, six days a week, and were of such concentrations that the animals received the selected dosage in a volume of 0*1 cc. In order to obtain the correct number of units per dose, suitable amounts of viosterol were weighed into a 50 cc* volumetric flash and made up to volume with cottonseed oil containing 0,5 r of ©( tocopherol per 0*1 cc, of oil* The various levels of vitamin D supplement were 0*5 I.U., 2*5 I*U,, 5 I.U. and 10 I.TJ* per 0*1 cc* of supplement. The method of preparing the /3 carotene supplements was that of Deuel et al, (8)* Levels of carotene used in the experiment were 0*3r, 0.6r, l*2r, 2.4rand 9*6r per 0*1 cc* of supplement* The negative controls were given 0*1 cc* of cottonseed oil containing 0,5rof tocopherol daily. Each group of rats receiving a particular level of carotene was subdivided into subgroups which received different levels of vitamin D (Table I), In this way the effect of either of the vitamins could be calculated independently of the other factor (Tables VIII and IX). 15 During the assay period of twenty-eight days the animals were weighed every five days and on the twenty-eighth or final day. The rats were then killed and X-ray photo graphs were taken of the head of the tibia. This was only done as a precautionary measure since it was very unlikely that any of the rats would show a rachitic condition even without vitamin D as the A-free diet had a normal calcium phosphorus ratio. After taking X-ray photographs, both femurs were removed from the animals and freed as completely as possible from adhering tissue. The bones were then tied in cheese cloth and extracted for from twenty-four to forty-eight hours with hot alcohol in a Soxhlet extractor. They were then removed, air dried for several hours, placed in tared cruci bles, and brought to constant weight in a 100° oven. The bones were then ashed in a muffle furnace at approximately 750° and the ash content determined. The results were expressed as per cent of ash in. the dry extracted bone (Tables I, II and III). This same procedure was repeated with fewer groups using another A-free diet that was obtained from SMA, Chagrin Falls Ohio (Tables IV, V and VI). These experiments were carried out because in a few preliminary tests ash values were obtained that were con siderably lower than those obtained from rats on the USC diet. 16 The preliminary experiments consisted of determining the ash content of bones from a few animals taken from an experiment on the affect of stereoisomers on vitamin A potency in which an SMA diet had been used (9) (Table VII), The effect of age as compared with 3lze on ash content of the bones in normal stock rata. In order to determine whether calcification Is dependent on the rate of growth of an animal or whether it is a function of age, another series of tests were made on two groups of rats on a stock diet* The control group received food ad lib while the second group was fed the same diet at a restricted level of 60 per cent of the intake of the first group. The results are recorded In Tables X and XI, TABLE X ■SUMMARY TABLE OF EXPERIMENTS ON MALE AND FEMALE RATS RECEIVING B CAROTENE AND, , OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE* ■■■ I © § -p © o So u © a m ■ ss Vitamin D Dosage 1 Depletion Period Assay Period Age when Killed © o a _ o A m © «s X-Ray Value Age in Days a P 41 ■H © g: c! j Length Final Weight © & . Length Final Weight Gain in Weight © & 22 43 21 92 2 25 74(12) -17(12) 4(12) 69 55.0 ’ 0.5 IU 23 44 20 95 2 23 96 (3) ♦ 4 (3) 2 (3) 66 55.9 14** ... 2.5 IU 23 43 20 92 3 22 72 (3) -15 (3) 4 (3) 65 57.6 14** 5.0 IU 22 43 21 94 2 25 72 (3) -20 (3) 4 (3) 68 55.5 14 0.3 f 24 43 24 94 2 26 103 (6) ♦ 4 (6) 2 (6) 74 54.4 14 0.3 r 0.5 IU 23 44 23 95 3 27 104 (6) ♦ 5 (6) 3 (6) 74 53.8 14 0.3 r 2.5 IU 23 44 22 92 2 26 95 (7) ♦ 6 (7) 2 (7) 72 54.3 14 0.3r 5.0 IU 24 44 24 94 2 28 97 ♦ 3 2 76 58.0 14 0.6lr ... 22 43 21 86 2 28 117 ♦32 2 71 54.6 14 0.6r 0.5 IU 22 42 21 91 2 28 117 ♦26 2 71 56.6 14 0.6r 2.5 IU 22 44 22 96 3 28 112 +26 2 72 54.3 14 0.6r 5.0 IU 22 ( 42 23 90 2 28 110 (7) ♦22 (7) 2 (7) 73 56.9 14 0.6 r .0.0 IU 24 43 23 96 2 28 113 (7) ♦23 (7) 2 (7) 72 57.8 14 U 2r ... 23 42 23 93 3 28 134 ♦42 1 74 55.4 14 1.2 r 0.5 IU 22 42 23 93 3 28 135 ♦42 1 74 55.7 14 I.2r 2.5 IU 23 42 22 92 3 28 133 ♦43 1 73 57.5 14 1.2 r 5.0 IU 22 42 23 92 3 28 139 ♦47 1 74 56.8 14 TABLE. I (Continued) SUMMARY TABLE OP EXPERIMENTS ON MALE AND FEMALE RATS RECEIVING $ CAROTENE AND, OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE / 3 Carotene Dosage Vitamin D Dosage Depletion Period Assay Period it H < D H ■*S t. % Ash of Dry Bone X-Ray Value Age In Days S f i J:§ Length Pinal Weight & & Length Final Weight 3* S'S 33 w . Og!' 0 fr 2.4 r 22 43 23 92 3 28 139 +44 1 73 57.5 14 2*4r 0.5 ICf 25 42 23 87 3 28 141 +55 1 74 57.8 14 2.4r 2.5 IN 22 43 23 91 2 28 145 +54 1 73 59.5 14 2.4r 5.0 IU 22 42 23 91 3 28 144 +52 1 73 59*7 14 9*6r 23 43 26 94 2 28 157 +63 1 75 56.6 14 * Numbers in parentheses indicate the number of animals still alive at the close of the assay period* The averages stated are for the indicated number of animals. The group receiving cottonseed oil alone consisted of twenty-one males and thirteen females at the start of the assay; the group receiving 0.5 I.E. of viosterol consisted of four males and four females; and all other groups consisted of five males and three females. H 00 TABLE II SUMMARY TABLE OP EXPERIMENTS ON MALE RATS RECEIVING & CAROTENE AND, OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE* 1 ■ . ' / © 43 O O W) h « c o r a o o , o j . n Q So B9 45 C O $8 Depleteon Period Assay Period Age when Killed % Ash of Dry Bone § {> h 5 • ..k . - Age In Days $ 5 3 wk H 3 © & at o Length. Pinal Weight 8 if, Length ■p •SS Gain in Weight k ... 24 43 21 93 2 24 75(7) -17(7) 4(7) 68 54.7 14** — M 0.5 IU 24 42 18 88 3 22 84(1) * 4(1) 2(1) 64 52.6 14** ... 2.5 IU 23 42 21 94 3 18 ~<o> (0) -(0) 61 55.3 14** «»«»«» 5.0 IU 23 42 20 95 2 24 55(1) -27(1) 5(1) 66 55.8 14 O.Sr — 24 44 24 91 3 25 95(3) - 4(3) 3(3) 73 53.6 14 0.3r 0.5 IU 23 44 24 95 3 27 96(4) 2(4) 3(4) 74 53.9 14 0.3r 2.5 IU 23 45 22 94 2 25 98(4) t 8(4) 2(4) 70 52,0 14 o.sr 5.0 IU 25 45 23 93 2 28 96 ♦ 2 2 . 76 57.5 14 Q*6r — - 22 44 20 91 2 28 119 *28 2 70 52.6 14 0.6r 0.5 IU 22 42 21 94 2 28 122 *27 2 71 54.8 14 0.6r 2.5 IU 22 45 23 102 3 28 112 *10 2 74 52*1 14 0.6r 5.0 IU 21 42 23 85 2 28 108 ♦23 1 72 55.7 14 0.6r .0.0 IU 23 44 23 99 3 „ 24 110(4) ♦15(4) 2(4) 70 58.3 14 . 1.2r . . . . . . 23 43 22 92 2 28 134 _ *42 . 1 . 74 54.7 14 l*2r 0.5 IU 22 43 22 90 2 28 136 ♦46 1 72 55.3 14 1.2r 2.5 IU 23 42 21 91 3 28 141 ♦50 1 72 56.1 14 1.2t 5.0 IU 22 41 23 90 3 28 139 ♦49 2 73 54.2 14 TABLE II (Continued) SUMMARY TABLE OF EXPERIMENTS ON MALE RATS RECEIVING B CAROTENE AND, OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE $ Carotene Dosage 8 S P •p « •H O -Depletion Period ' ■ ' / -Assay Period g^ 11 t ~ i ©H ■ jfa u © o a ° xs tb t n < h § c ! > & « A Age in Days 0 •H i§ |s c £ > \ XJ • p w £ a s • p ■3 ft S J -H •H © 1 ■ * * r Length Final Weight Gain in Weight & 2*4 r 22 43 23 91 3 28 143 ♦52 1 73 56.8 14 2.4 r 0.5 IU 23 41 22 86 3 28 145 ♦59 1 73 56.4 14 2.4 r 2.5 IU 22 43 22 95 2 28 151 ♦56 1 72 59.1 14 2.4r 5.0 IU 22 41 23 92 3 28 151 ♦59 1 73 60.4 14 9.6r 23 42 24 92 2 28 161 ♦69 1 73 55.4 14 * Numbers in parentheses indicate the number of animals still alive at the close of the assay period# The averages stated are for the indicated number of animals. ■ J H S - The group receiving cottonseed oil alone consisted of twenty-one animals at the start of the assay; the group receiving 0*5 I.U# of viosterol consisted of four animals; and all other groups consisted of five animals. TABLE III SUMMARY TABLE OP EXPERIMENTS ON FEMALE RATS RECEIVING ,8 CAROTENE AND, OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE5 * £Carotene Dosage Vitamin D Dosage ; Depletion Period fAssay Period t Age when Killed % Ash of Dry Bone X-Ray Value Age in Days S 3 t§ H f f l < D C ) Length Final Weight © & Length Final Weight Gain in Weight © « • t 22 43 22 91 2 25 70 (5) -14 (5) 4 (5) 70 56.1 r 14** 0*5 IU 22 46 22 103 2 25 103 (2) » 4 (2) 3 (2) 69 59.2 14** 2.5 IU 22 45 20 88 2 28 72 -15 4 70 61.3 14** 5*0 IU 22 44 22 91 2 26 88 (1) -13 (1) 3 (I) 70 55.0 14 0.3r .... 24 41 24 99 2 28 111 412 2 76 55.8 14 0.3r 0.5 IU 22 43 23 96 2 28 119 ♦13 2 73 53.4 14 0.3r 2.5 IU 24 43 23 87 2 28 89 ? 2 2 72 58.0 14 0.3r 5.0 IU 22 41 27 96 2 28 100 ♦ 4 2 77 58.3 14 0.6r «• ••* >« 22 41 23 77 2 28 114 ♦37 2 72 57.8 14 0.6r 0.5 IU 21 42 22 86 2 28 110 ♦24 2 71 59.4 14 O.Sr 2.5 IU 21 43 21 87 2 28 113 ♦26 2 70 57.8 14 0.6r 5.0 IU 22 42 24 97 3 28 113 (2) ♦22 (2) 2 (2) 73 58.7 14 0.6r 10.0 IU 24 41 22 91 2 28 117 ♦26 2 72 56.9 14 1.2r 22 41 25 94 3 28 132 ♦42 1 75 56.6 14 1.2r 0.5 IU 21 41 25 97 3 28 132 ♦35 1 74 55.5 14 1*2 r 2.5 IU 22 42 25 93 2 28 124 ♦31 1 75 59.9 14 1.2r 5.0 IU 22 42 22 97 3 28 139 ♦42 1 74 61.0 14 TABLE III (Continued) SUMMARY TABLE OP EXPERIMENTS ON FEMALE RATS RECEIVING A CAROTENE AND, ,OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE L . \ © § +3 © SS 8 n o o S qP Vitamin D Dosage Depletion Period Assay Period S 3 © it r1 © H 9A $4 © O S 3 £ m ^.P § > & « - A a Cl < D f > » 9& 5 43 £ a bOS <H 3 s§ Cl Length Pinal Weight 1 Length Pinal Weight 5+3 i3 5S OS 4) Ogs 1 2.4 r 22 44 25 95 2 28 155 ♦40 1 72 58.5 14 2.4 r 0.5 IU 25 42 24 . 87 2 28 154 ♦47 1 74 60.5 14 2*4 r 2.5 IU 22 42 25 84 5 28 154 ♦51 1 74 60.0 14 2.4 r 5.0 IU 22 45 24 90 5 28 152 ♦42 1 74 58.6 14 9.6r 22 44 28 97 5 28 149 ♦52 1 77 58.6 14 Numbers in parentheses indicate the number of animals still alive at the close of the assay period. The averages stated are for the indicated number of animals. The groups receiving cottonseed oil alone consisted of thirteen animals at the start of the assay; the groups receiving 0.5 I.U. of viosterol consisted of four animals; and all other groups consisted of three animals. TABLE IV i SUMMARY TABLE OP EXPERIMENTS ON MALE AND FEMALE RATS RECEIVING / 8 CAROTENE AND, OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE WHILE ON AN A-FREE DIET OBTAINED PROM SMA, CHAGRIN PALLS, OHIO* BCarotene Dosage Vitamin D Dosage Depletion Period . ; vAssay Period Age when Killed Vi © d CQ © £ / © 1. £> & m A . & •H t a © ! * » 5 ^ § Si t § d 43 9 3 • p ■34 & & 1 d 43 W © d Final Weight s'® *H - r i St © I ____ 24 41 19 71 2 23 71 (3) - 2 (3)' 6 (3) 66 49.4^ 14 2.5 IU 24 41 19 66 2 24 64 ♦ 1 5 @8 47.0 14 1.0 r 24 42 20 66 2 28 87 ♦21 3 72 47.5 14 l.o r 2.5 IU 23 42 18 69 2 28 95 ♦26 1 71 49,4 14 * Numbers in parentheses indicate the number of animals still alive at the close of the assay period* The averages stated are for the indicated number of animals. to o* TABLE V SUMMARY TABLE OF EXPERIMENTS ON MALE RATS RECEIVING 0 CAROTENE AND, OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE WHILE ON AN A-FREE DIET OBTAINED FROM SMA, CHAGRIN FALLS, OHIO* < * ■ 0 Carotene Dosage Vitamin D Dosage Depletion Period f 4 Assay Period Age when killed © 0 g _ ° Xlffl © X-Ray Value Age in Days Weight i n Grams Length f j * r l «H © 1 Length Final Weight •H -P S3 bO • r j « H I C O © ap & « * <mm —m m i 24 42 18 72 2 28 71 (3) - 2(3) \ 6(3) 70 47.7 14 m — 2.5 IU 24 41 20 74 2 23 68 (2) - K2) 5(2) 67 47.2 14 l.Oi" 23 42 21 66 2 28 87 ♦20 3 72 49.7 14 1.0 r 2.5 IU 25 42 18 72 2 28 99 ♦27 X 71 50.7 14 * * Numbers In parentheses indicate the number of animals still alive at the close of the assay period. The averages stated are for the indicated number of animals. to TABLE VI SUMMARY TABLE OP EXPERIMENTS ON FEMALE RATS RECEIVING 6 CAROTENE AND, OR VIOSTEROL IN COTTONSEED OIL OR THE OIL ALONE WHILE ON AN A-FREE DIET OBTAINED FROM SMA, CHAGRIN FALLS, OHIO* / 8 Carotene Dosage Vitamin D Dosage * Depletion Period Assay Period Age when Killed u © O f j _ o ,3 a X-Ray Value Age in Days Weight in Grams Length Final Weight © £ Length Final Weight Gain in Weight I D & m*mwm 24 40 20 71 2 16 --(0) — (0) — (OJ 61 ; 51,7 14 2,5 IU 25 41 18 62 2 27 60 (2) ♦2 (2) 5 (2] 69 46,8 14 1.0 r 26 42 19 66 2 28 87 ♦22 3 72 44,5 14 l,Or 2,5 IU 24 42 18 66 2 28 92 ♦27 1 71 47.5 14 * Numbers in parentheses indicate the number of animals still alive at the close of the assay period. The averages stated are for the indicated number of animals. to oj 26 TABLE VII SUMMARY TABLE OP EXPERIMENTS ON THE ASH CONTENT OP BONE PROM MALE AND FEMALE RATS RECEIVING 0 CAROTENE IN COTTONSEED OIL OR THE OIL ALONE Negative’Controls 0 Carbtene [Dose*e 1.2r per day] Rat # % Ash. of Dry Bone Rat # i * Weight in Grains at Close of Assay Period \ c Gain in Weight Dur ing Assay Period % Ash of Dry Bone 4576 41.10 4592 130 44 48.60 4554 47.10 4596 116 24 49.00 4556 34.49 4602 159 64 48.70 4544 38.23 4615 ' 167 64 49.90 4547 45.67 4553 118 32 41.80 4553 38.37 4623 115 29 44.70 4674 42.10 4617 134 46 45.90 4616 41.60 4629 104 33 48.50 4569 49.70 4675 98 18 46.50 4696 41.80 4678 176 78 46.70 4628 41.80 4656 124 36 49.90 4706 43.70 4703 139 54 52.70 4610 28.13 4666 146 45 47.70 4645 43.40 4711 180 83 42.50 4543 42.13 4687 154 53 42.50 4715 4619 4581 4562 45.80 42*20 47.40 36.84 4701 165 65 49.90 Averages 41.55 139 48 48.49 27 TABLE VIII SUMMARY- TABLE OP EXPERIMENTS ON MALE AND FEMALE RATS RECEIVING THE USC DIET AND SHOWING THE EFFECT OP VITAMIN D INDEPENDENT OP THE DOSAGE OF 0 CAROTENE 0 Carotene Dosage Vitamin D Dosage ------ 0.5 IU 2.5 IU 5.0 IU 10 IU 0.3 r 0.6 r 1.2 r 2.4 r 9.6 r Averj 55.0 54.4 54.6 55.4 57.5 56.6 ge % Ash. e 55.9 53.8 56.6 55.7 57.S f Dry Bone 57.6 54.3 54.3 57.5 59.5 (See Table 55.5 58.0 56.9 56.8 59.7 I) 57.8 Average 55.6 55.9 56.6 57.4 28 TABUS IX SUMMARY TABLE OF EXPERIMENTS ON MALE AND FEMALE RATS RECEIVING THE USC DIET AND SHOWING THE EFFECT OF $ CAROTENE INDEPENDENT OF THE DOSAGE OF VITAMIN D Vitamin D & Carotene Dosage Dosage mm-mm mm 0.3 r 0.6 r 1.2 2.4 r 9.6 0.5 IU 2.5 IU 5.0 IU 10.0 IU Ave] 55.0 55. 9 57.6 55.5 >age % Ash 54.4 53.8 54.3 58.0 of* Dry Bo 54.6 56.6 54.3 56.9 57.8 le (See ' 55.4 55.7 57.5 56.8 •able I) 57.5 57.8 59.5 59.7 56.6 Average 55.8' 55.1 56,0 56.3 58.6 GAIN I N WEIGHT I N G R A M S LO G T DO SE RELATIONSHIP OF GAIN IN WEIGHT TO , DOSAGE OF /3CAR0TENE FOR ANIM A LS R E - CEIVING VIOSTERO L AND;OR /3 C A R - . Q TEN E . IN COTTONSEED OIL V IT A M IN D DOSAGE! ' o ' 0.0 TU D - 0:5 IU o =0= 2.5 IU • - X =o= 5.0 IU : ' AVERAGE 30 TABES X SUMMARY TABLE OP EXPERIMENTS ON STOCK RATS FED AD LIB Age In Days Avv Wt. in G&ns. Males ' Av. Wt. In Gas. Females Av. Wt. in Gkns. Males & Ffemales Av. % Ash of Dry Bone Males Av. % Ash of Dry Bone Females Av. % Ash of Dry Bon< Males and Females 28 42*8 37.8 40.3 45.2 44.9 45.1 48 138.4 111.6 125.0 55.9 55.1 55.5 68 168*0 138.3 150.2 59.3 59.3 59.3 88 162.5 141.1 149.7 60 .6 1 60.8 60.7 108 247.2 164.8 206.0 61.3 63.4 62.4 128 259.6 187.2 223.4 62.4 63.0 62.8 148 229.4 178.8 204.1 62.4 64.5 63.4 31 TABLE XI SUMMARY TABLE OF EXPERIMENTS ON STOCK RATS WITH FOOD INTAKE RESTRICTED TO 60 PER CENT OF THAT OF SIMILAR ANIMALS FED AD LIB ! Age in Days Av. Wt. In Gms. Males Av. Wt. in Gms. Females Av. Wt. in (Mis. Males & Females 4 . i Av. % Ash. of Dry Bone Males Av. % Ash of Dry Bone Females Av. % Ash of Dry Ben Males and Females 48 74.0 73.2 73.6 54.0 55.9 55.2 58 91.8 78.7 86.0 54.2 56.8 55.4 68 68.6 63.2 65.9 . 57.6 63.3 60.6 78 77.5 74.7 76.8 63.0 61.2 62.1 88 97.0 83.0 90.0 63.8 62.9 63.4 Discussion of results obtained from experiments on the effeets of vitamin A and vitamin D on the ash content, of the hones in vitamin A-deficient rats* Table I gives a summary of the data obtained on the 202 rats used in the experiment carried out on the USC diet. The results of the negative con trols indicate that the rats were sufficiently depleted in vitamin A at the 3tart of the test and that the basal diet used was essentially vitamin A-free* Figure 1 shows the dosage gain in weight curves for all but two of the groups receiving 0 carotene. The fact that the various points on the curves fall on reasonably straight lines is a proof of a normal response to the carotene administered as well as a confirmation of the concept of this relationship of vitamin A between growth and dosage. The accuracy of the data is also suggested by the fact that the various curves are reasonably parallel. Tables II and III give a summary of the data on male and female rats on the USC diet respectively. It can be seen from these tables that the ash content of the bones of females is generally higher than that of males in corresponding groups. In seventeen of the twenty-two groups the results in females were higher than in males. These results are in accord with others obtained in this laboratory, since It has been found that male rats have a slightly higher water and protein con tent and a lower lipid, ash and calcium content than the females. 33 The X-ray photographs taken of the head of the tihla were compared with a series of such photographs used in this laboratory in vitamin D bioassay3. The series covers a range of increasingly severe rachitic conditions. No rickets is indicated by the number 14 and very severe rickets by the number 1. The results given in Tables I and IV show that none of the animals used exhibited a rachitic condition. Only a few X-rays were made of the animals used in the preliminary experi ment. None of these, however, showed a rachitic condition. Tables VIII and IX give a summary of the ash content of the bone of males and females in the experiment using the USC diet. They show the effect of the dosage of either vitamin independent of the dosage of the other vitamin. Prom these it can be seen that there are no significant differences between the various groups. The slight trend towards a higher ash con tent with increasing dosages of either vitamin has no signifi cance because of the rather wide variation in individual values within each group (See Appendix A) . Table X gives a summary of the data obtained on stock rats of the same strain as the animals used in the various other experiments. Table XI shows the results obtained when the food Intake of similar animals was restricted to 60 per cent of the ad libitum Intake. These results show that regardless of size the average ash content of the bones increases with the age of the animal until a level of 60 to 63 per cent Is obtained. 54 Although there are slight differences In &3h content at the various age levels between animals fed ad libitum and animals in which the food intake was restricted, they have no signifi cance in view of individual variations and in comparison with the striking differences in body weight. For example, the average ash content of rats fed ad libitum at 68 days of age was 59.5 per cent and the average body weight was 168 grams. The average ash content of rats on the restricted food intake at the same age was 60.6 per cent and the average body weight was 68.6 grams. Table I shows an average ash percentage of dry bone of 56*4 per cent for the animals on the USC diet. In comparison with the value of 59.5 per cent for stock animals of the same age, it can be seen that, as with the animals on a restricted food Intake, size had no effect. The animals fed on the two lots of SMA diet, however, show much lower ash values. The data obtained from the preliminary experiment (See Table VII) show an ash value of 41.6 per cent for the negative controls and 48.5 per cent for the animals receiving 1.2 r of /S carotene per day. Table IV gives the data obtained on rats fed on an SMA diet of a different lot number from the diet fed to the rats on the preliminary experiment. Although the various groups of these animals received either both or neither & carotene or vitamin D there Is no significant difference between the results obtained from the various groups. The average of all 35 values Is 48.3 per cent. Results from Tables I and IV Indicate no effect of either vitamin on total calcification of the bone when either the USC diet or one lot of the SMA diet was used, There is, however, a difference between the two levels of calcification obtained. The data obtained in the preliminary experiment would indicate an effect of 73 carotene on the total calcification of the bone. Since, in this last work, an adequate supply of vitamin D was furnished the animals, and in view of the elimination of any effect of size or of vitamin A or vitamin £> when the mineral content of the diet is adequate and the calcium/phosphorus ratio normal, it is possible that under abnormal conditions of mineral Intake vitamin A would in some way assist in Increasing the cal cification of the bone. CHAPTER IV SUMMARY" 1* When the mineral content and calcium/phosphorus ratio of the diet are adequate, neither vitamin A or vitamin D have any significant effect on the ash content of animals deficient In vitamin A* Under such conditions there Is no synergistic effect on ash content shown by these vitamins. 2. A few preliminary experiments on animals on an A-free diet Indicate that /S carotene influences the ash content of the bone. Vitamin A-deficlent animals receiving no 0 carotene had an average ash content of 41.6 per cent and those receiving 1.2 r of 0 carotene per day had an average ash content of 48.5 per cent. The mineral composition of the diet used in these experiments was not known. 3. Size has no effect on the increasing ash content of bone in stock animals receiving a diet adequate in all essen tials but whose growth has been retarded by restricting their food intake to 60 per cent of that of similar animals fed ad libitum. 36 BIBLIOGRAPHY BIBLIOGRAPHY Bachmann, G., J. Haldi, W. Wynn, and G. Ensor, "The Effects Produced by Decreasing the Calcium and Phosphorus Intake on Calcium and Phosphorus Absorp tion and Deposition and on Various Bodily Constituents of the Rat,1 * Journal of Nutrition, v. 20, p, 145 (1940). . Boelter, M. and D, Greenberg, “Severe Calcium Deficiency in Growing Rats II* Changes in Chemical Composition," Journal of Nutrition, v, 21, p, 75, (1941), Bomskov, C. and G. Seemann, “The Effect of Vitamin A on Mineral Metabolism," Zeltschrift ffir Geschicte Experi mental Medicine, v,_8§, P* ?7l (1933). Briwa, K. E. and H* C, Sherman, "The Calcium Content of the Normal Growing Body at a,Given Age," Journal of Nutrition, v. 21, p. 155 (1941). Cohn, W. F*, E* T. Cohn and J* G. Aub, "Calcium and Phosphorus Metabolism: Clinical Aspects," Annual Review of Biochemistry, v. XI, p. 415 (1942), Coward, Katharine, Biological Standardization of the Vitamins. Baltimores William Wood and Company, 1938. Deuel, H. J., Jr., L. F. Hallman, E. Movitt, F. Mattson, and E. Wu, "Studies of the Comparative Nutritive Value of Fats II. _ The Comparative Composition of Rats Fed Different.Diet," Journal of Nutrition, v. 27, p. 335 (1944). . “ “ Deuel, H. J., Jr., C. Johnston, E. Sumner, A. Polgar, and L. Zechmeister, "Stereochemical Configuration and Provitamin A Activity I. All Trans- Carotene and Neo- Carotene U," Archives of Biochemistry, v. 5, p. 107 (1944). , Deuel, H. J., Jr., C. Johnston, E. Sumner, A. Polgar, W. A. Shroeder, and L. Zechmeister, "Sterochemical Configuration and Provitamin A Activity II. All Trans- Carotene and Pro Carotene," Archives of Biochemistry, v. 5, p. 365 (1944). „ Eddy, W. H., and G* Dalldorf, Vitamins. Baltimore: Williams Wand Wilkins Company, 1944. 39 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Harrow, Benjamin, Textbook of Biochemistry. Philadelphia and London: W. B. Saunders Company, 1943. Hess, A. P., "Effect of Elementary Phosphorus in Rickets,1 * American Journal of Diseases of Children, v. 41, p r i O T T d s s r r : -------------------------------- Lanford, C. S., H. T. Campbell, and H. C. Sherman, "Influence of Different Nutritional Conditions Upon the Level of Attainment in the Normal Increase of Calcium in the Growing Body," Journal of Biological Chemistry, v, 137, p. 627 (1941). Liu, S. H., H. I. Chu, H. C. Hou, H. C. Chao and S. H. Gheu, “Calcium and Phosphorus Metabolism in Osteomalacia XI. The Pathogenic Role of Pregnancy and Relative Impor tance of Calcium and Vitamin D Supply,” Journal of Clinical Investigation, v. 20, p. 255 (1941). Mellsncy, E., “Skeletal Changes Affecting the Nervous System Produced in Young Dogs Deficient in Vitamin A,” Journal of Physiology, v. 99, p. 467 (1941). Melianby, E., "The Effect of Bone Dysphasia (Overgrowth) on Cranial.Nerves in Vitamin A Deficient Animals," Journal of Physiology, V. 101, p. 408 (1943). Reed, G. I., H* C. Struck, and I. E* Steck, Vitamin D: Chemistry, Physiology, Pharmacology, Pathology, jfeperimental and Cllnical Invea't'ipations'. Chicago: 'tfhe University of Chicago Press, 1939. Rosenberg, H. R., Chemistry and Physiology of the Vitamins. New Yorks Interscience Publications, 19¥2. Schmidt, C. L. A., and D. M. Greenberg, “Occurrence, Trans port and Regulation of Calcium, Magnesium and Phosphorus in the Animal Organism," Physiological Reviews, v. 15, p. 297 (1935). Sherman, H. C., Chemistry of Food and Nutrition. New York: The Macmillan Company,“T941. Sherman, H. C. and L. E. Booher, "The Calcium Content of the Body in Relation to That of the Food," Journal of Biological Chemistry, v. 93, p. 93 (1931). Sherman, H. C., and H. K. Stiebeling, "Quantitative Studies of Response to Different Intakes of Vitamin D,“ Journal of Biological Chemistry, v. 83, p. 497 (1929).. 40 23. Sherman, H. G. and H. K. Stiebeling, "The Relation of Vitamin D to Deposition of Calcium in Bone,” Proceed- ings of the Society for Experimental Biology and ^ H c H e ~ . ‘ ^ 7 p 7 355 Tl929)7 --------------- 24. Tabor, F. S., R. A. Deutcher, and M. B. Guerrent, "The Effect of Vitamin A (Carotene) Intake on the Vitamin D Requirement of Rats in the Production and Cure of Rickets,1 1 Journal of Mutritlon, v. 12, p. 39 (1936). 25. Thoenes, F., "The Metabolic Effects of Antirachitic and An t ixarophthalmic Factor in Rats -with Experimental Rickets," Jahrbuchen Kinderheilken, v. 139, p. 200 (1933). . “ 26. von Euler, H., B. von Euler, and M. Rydbon, "Antirachitic and Growth Promoting Action in Rats of Blood Sterols and Steryl Phosphate,” Biochemische Zeitshrift, v. 199, p. 276 (1928). - 27. von Euler, B., H. von Euler, and P. Karrer, "Observations on Epiphyses and Liver Extracts of Rats Fed on Caro- tenoids,” Biochemishe Zeitshrift, v. 209, p. 240 (1929). „ 28. Zucker, T., L. Hall and M. Young, "Growth and Calcification on a Diet Deficient in Phosphate but Otherwise Adequate," Journal of nutrition, v. 22, p. 139 (1941). UMI Number: EP41278 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be. noted. Also, if material had to be removed, a note will indicate the deletion. UMI Dissertation ft*b®rts»g UMI EP41278 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

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Creator Hunter, Eizabeth G. Sumner (author)

Core Title An experimental study of certain factors affecting calcification of bone

School School of Medicine

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 1946-02

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Deuel, H.J. (committee chair), Morehouse, Margaret G. (committee member), Wingell, Richard J. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-775368

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