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Rickets in rats by iron feeding
RICKETS 1N RATS BY IRON FEEDING J. F. BROCK, M.D.* AND L. K .
DIAMOND, M . D .
BOSTON, )/IASS. INTRODUCTION
HE studies reported here were mldertaken in pursuance of some T ideas arising out of preliminary experiments bearing upon the problem of the absorption ~f iron. ~ It was found that the dialysis of ferric ammonium c~trate across cellophane membranes was markedly impeded by the addition to the solution of the secondary and tertiary sodium phosphates, even though there was no precipitate. This effect was considered to be due to the formation of colloidal ferric phosphate. Other soluble iron salts used in these dialysis experiments all formed precipitates with secondary and tertia~T sodium phosphate under the conditions of the experiment, and tile passage of the iron salts was naturally greatly impeded. The supposition that these principles might be applicable to. conditions in the intestinal tract suggested the possibility that iron and phosphorus might, by forming insoluble compounds in the intestinal tract, each hinder the absorption of the other. It was decided, therefore, to find out whether in the case of rats the addition of iron to a nonrachitogenic diet might so interfere with the absorption of phosphorus as to render that diet r achitogenic. EXPERIMENTAL
Young rats were obtained from the Albino Supply Company of Philadelphia. They were of the type called by the suppliers " vitamin rats," recently weaned, and weighing between 35 and 50 grams. The rats were divided into groups of five to a cage, all being exposed to the same amount of daylight coming through a plain glass window facing north. Particulars are given in TaMe I of the various diets used. The C,o~trol D~et used is one which has repeatedly been shown to be nonraehitogenie to rats. The Steenbook Raehitoge~de Diet is a h i g h calcium, low p h o s p h o r u s diet, which has repeatedly been shown to be raehitogenic to r a t s raider conditions similar to those which were used for our animals. Eavh o f the remaining diets consists o f the contro~ diet p~u~ the va~m~s vubsCanve~ mentio~e& F r o m the Departments of Pediatrics and 2r I-farvard Medical School, and the Thorndike Memorial Laboratory, Second and F o u r t h Medical Services ( H a r v a r d ) of the Boston City H o s p i t a l *Leeverhulme Research Scholar of the Royal College of 1Jhysicians, London. 449.
BROCK A N D D I A M O N D :
R I C K E T S I N RATS B Y IRON F E E D I N G
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444
THE JOUI~NAL OF PEDIATRICS
Tl~e 2~errio Chloric~e Diet contains iron (9.14 gin.) suificient to combine chemically as ferric phosphate with 5.06 gm. of phosphorus, or as ferrous phosphate with 3.37 grams of phosphorus. I r o n in the above q u a n t i t y was added to the control diet on the assumption that, by f o r m i n g in the intestinM tract insoluble ferric or f e r r o u s phosphate, it m i g h t interfere with the absorption of phosphorus. The, A~mno~iv/~ Chloride Dive contains the same weight of chlorine as is contai~e4 in the ferric chloride present in the previeus diet. I t was intended to eliminat(~ the possibility t h a t a n y rachitogeade effect of added ferric chloride m i g h t be due to alterations in the hydrogen ion concentration of the contents of the intestinal tract or to the chlorine radical. The Fe~'rio Chloride a~z~ Ade~ec~ 1~hosphoeua Diet contains i n addition to the phosphorus already in the ferric chloride diet a further allowance of phosphorus, s~ffficient to combine as ferric phosphate with all the added iron~ and t h u s provide the same amount of " u n b o u n d " phosphorns as the control diet. The F err~o Ammoniu~r~ Ci;tr~te, F e r r o ~ C'h~oride, Reduce~ Iroqv, a~nd Orya~dv Iron Diets consist each of the control diet with the addition of the salt named, in a m o u n t sufficient to yield about the same weight of metallic iron as is contained in the, ferric chloride diet.
The diets were made up in the following manner. Each of the salts was finely ground in a m o r t a r and intimately mixed in a large bowl with d r y corn meal and wheat g l u t e n . The mixture, after the addition of sufficient water to make it cohesiv% was rolled out upon a piece of smooth p a p e r which had been greased with olive off, cut into small sections, and baked until crisp in a oven. In the ease of ferric chloride and ferric ammonium citrate, these salts were dissolved in the amount of water which had to be added to the mixture. W a t e r and food ad libi~um were supplied fresh each day. The control diet and the ammonium chloride diet were very well taken and the rats gained weight well and remained sleek and healthy. The Steenbock rachitogenic diet, the ferric chloride diet, the ferric chloride with added phosphate diet, the ferric ammonium citrate diet, and the ferrous chloride diet were moderately well taken. The animals appeared in good health but gMned little weight. The reduced iron diet was less well taken and the animals lost weight. The o rga.nie iron diet was obviously hard and unpalatable to the rats. Although the animMs on this diet appeared to be in moderately good health, they lost a great deal of weight. In spite of loss of weight in the last two groups all the animals obviously increased in skeletM stature. There was no noticeable diminution of activity n o r weakness o~ the extremities. The rats were weighed individually at the start of the experiment, and again from time to time throughout the duration of the experiment. A f t e r ten days or a fortnight they were lightly anesthetized with ether and radiographed to detect the development of rickets. A t the termination of the experiment, the animals were anesthetized, decapitated and as much blood drained out of the body as possible. The blood from each group was pooled, and the serum phosphorus estimated by the method of Fiske and SubbarowY Each carcass was then radio-
BROCK
AND
DIAMOND:
RICKETS
IN
RATS
BY
.~r
IRON
445
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THE
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OF P E D I A T R I C S
graphed. One knee joint and two or three costochondral junctions were taken f r o m each animal for microscopic examination. Sections were made f r o m the principal organs of the body to determine if there was evidence of disease. RESULTS
The results of these experiments are smnmarized in Table II. Tile first f o u r groups in the table comprise the p r e l i m i n a r y experiment in which the effect of added iron in the f o r m of ferric chloride was studied. The five rats on the control diet showed no rickets in twenty-two days, whereas the four rats on the Steenbock rachitogenic diet all showed gross m a n i f e s t a t i o n s of rickets at the end of the same period of time. The
iFig. 1 . - - C o n t r o l d i e t ; n o r i c k e t s ; s e c t i o n o f c o s t o e h o n d r a l j u n c t i o n
(X104).
five rats fed on the ferric chloride diet all presented the manifestations of rickets to a degree at least as m a r k e d as those on the Steenbock rachitogenie diet. None of the rats fed on the ammonium chloride diet showed any manifestations of rickets. Groups 5, 6, and 7 were repetitions of Groups 1, 3, and 4, respectively, and confirmed the results previously obtained with the control diet, the Steenboek rachitogenic diet, and the a m m o n i u m chloride diet. Group 8 was fed on a diet which contained ferric chloride in an amount which has been shown in Group 2 to produce rickets. B u t in this ease the ferric chloride was " c o v e r e d " by an amount of added phosphate sufficient chemically to combine with all the iron1 as ferric phosphate. None of these rats showed a n y manifestations of rickets. Groups 9 and 10 were fed on a diet to which had been added, respectively, ferric a m m o n i u m citrate and ferrous chloride in amounts equiva-
BROCK AND DIAMOND:
RICKETS IN RATS BY IRON FEEDING
447
Fig. 2.--Steenbock r a e h i t o g e n i c d i e t ; active r i c k e t s ; section of costoehondral junction ( X 1 0 4 ) .
Fig. 3.--lWerric chloride d i e t ; a c t i v e r i c k e t s ; section of costochondral junction (X104).
lent in metallic iron content to t h a t of the ferric chlorfde diet. All the r a t s in these two groups showed rickets almost as severe as t h a t of the members of the ferric chloride group although the diets were not as well t a k e n and the rats failed to gain weight.
448
THE
, J O U R N A L OF P E D I A T R I C S
The members of Group 11, fed on a diet containing ferrum reductum, took their diet very poorly and lost weight. In Group 12, which received a diet containing organic iron, the loss of weight was even more pronouneed. In these two groups the gross evidences of rickets were lacking. These findings are quite compatible with the well-known requirement of growth for the production of experimental rickets. In fact, it has been shown that cessation of growth resulting from starvation may readily heal the lesions in animals previously rachitic, a The degree of rickets present in the roentgenograms of each group was assessed by Dr. Edward C. Vogt, ~ without prejudice of previous knowledge of the diets. His reports as shown in Column 7 of Table II stated that severe active rickets was present in the members of Groups 4 and
:Fig. 4 . - - R o e n t g e n o g r a m s of lower extremity. Left: Control; no :Ferric c h l o r i d e d i e t ; a d v a n c e d a c t i v e r i c k e t s .
rickets.
Right:
7 (Steenbock raehitogenic diet), Group 2 (ferric chloride diet), Group 9 (ferric ammonium citrate diet), and Group 10 (ferrous chloride diet). CHEMICAL ANALYSES
In Column 6 of Table II are summarized the estimations of serum phosphorus on the pooled blood from each group. The serum phosphorus of all the groups showing no evidence of rickets (Groups 1, 3, 5, 6, 8, 12) varied between 5.6 and 7.8 rag. per cent. That of all the groups showing gross evidence of rickets (Groups 2, 4, 7, 9, 10) wa~ between 3.7 and 2.5 rag. per cent. Group 11 which failed, to show gross evidence of rickets showed nevertheless a decrease of the phosphorus to 3.6 rag. per cent. * W e zare g r e a t l y i n d e b t e d t o D r . : E d w a r d C. V o g t , l ~ o e n t g e n o l o g i s t to t h e I n f a n t s ' a n d C h i l d r e n ' s H o s p i t a l s , f o r h i s a s s i s t a n c e irL t h e t a k i n g " a n d i n t e r p r e t a t i o n of t h e roentgenograras.
BROCK AND DIAMOND:
R I C K E T S IN R A T S B Y I R O N F E E D I N G
IIISTOLOGIC
449
EXAMINATION
The sections of the principal organs showed no evideaee of any morbid processes. Sections of the knee joint, including tlle lower end of the femur and the upper end of the tibia, and of the costochondral junctions were sub-
F i g . 5 . - - l ~ o e n t g e n o g r a m s of l o w e r e x t r e m i t y . Steenboek diet; advanced
Left: Control; active rickets
no r i c k e t s ,
IRight:
Fig. 6.~Roentgenograrns of lower extremity. Left: Ferrous chloride diet; advanced active rickets. :Right: Iron arr~monium citrate diet; advanced active rickets.
mitted to Dr. Sidney Farber ~ for examination without any indication as :to their source, ttis report, summarized in Column 8 of Table II, corroborates the findings in the roentgenograms. Severe active rickets was present in Groups 4 and 7 (Steenbock rachitogenic diet), Group 2 (ferric chloride diet), Group 9 (ferric ammonium citrate diet), and and
eWe appreciate the cooperation of Dr. Sidney Farber, Pathologist Children's Hospitals, in reviewing the pathologic sections.
to the Infants'
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Group 10 (ferrous chloride diet). In his opinion, the rickets in tbo members of Group 2 (ferric chloride diet) was more severe than that found in the members of Groups 4 and 7 (Steenbock rachitogenic diet). In Groups 9 and 10 (ferric ammonium citrate diet and ferrous chloride diet), the rickets was slightly less severe. The members of Group 11 (ferrum reduetum diet), which had taken their food poorly, had lost weight and had shown no gross manifestations of rickets, nevertheless, presented evidence 02 having typical rickets, in a moderate degree, by microscopic examination of sections (from the bones of the lower extremities and from the eostochondral junctions). There was no microscopic evidence Of rickets in Group 12 (organic iron diet) where the loss of body weight had been so great. There was no qvalitative difference between the rickets produced by these various dietsP DISCUSSION
A high ratio of Ca to P in the diet has been utilized for a decade in the production of experimental rickets. This principle underlies the Steenbock raehitogenic diet No. 2965 used in Groups 4 and 7. It is believed that the high ratio of calcium to phosphorus in the intestinal tract interferes sufficiently with the absorption and utilization of phosphorus to make this diet rachitogenic. The knowledge that elements other than calcium when added to the diet may produce rickets is not new. In 1908 StoeltzneP reported that feeding of strontium to animals led to the development of a rachitic process. This has been confirmed several times. An elaborate investigation by Lehnerdt s in 1910 on puppies and young rabbits showed that strontium produced a marked increase in osteoid tissue in the bones, which he called strontium sclerosis. More recently in 1922, Ship]ey, Park et al., G by adding 2.2 per cent of strontium carbonate to an otherwise normal diet, produced in rats histologic changes of the raehitic type. These abnormalities could not be prevented by the addition of cod liver oil or liberal amounts of viosterol. Many of the rats developed incomplete or total paralyses of the extremities. No relatioi1 has ever been demonstrated between strontium and the natural occurrence of infantile rickets. The close relationship of magnesium to calcium in the production and especially in the healing of rickets has been known for some time. In 1932, HSrste 7 produced in rats rickets of the low phosphorus type by the addition of magnesium carbonate to a nonrachitogenic diet. He showed that the rickets might be regarded as depende~lt upon the ratio of magnesium plus calcium to phosphorus. * W e a r e g r e a t l y i n d e b t e d to Dr. S. ~ u r t W o l b a c b , P r o f e s s o r of P a t h o l o g y , H a r v a r d ,~r School, for allowing us to quote him regarding the rickets produced in these experiments by the ferric chloride diet as histologically typical of low phospi]ortlS rickets, and indistinguishable from that produced by the Steenbock diet.
BROCK AND DIAMOND:
RICKETS IN RATS BY IRON FEEDING
~51
In 1931, Branion, Ouyatt, and Kay* reported briefly the production of bone lesions similar to rickets in young rats by the replacement of the calcium earbonate in Steenboek's rachitogenie diet No. 2965 with an equivalent amount of beryllium carbonate. They have expanded these observations more recently (1933) and have shown that the administratiou of cod liver oil, viosterol, or ultraviolet light in generous amounts does not prevent the onset of this type of rickets. 9 They mention that sections'of the bones of their animals show striking differences from the appearances usually encountered in low phosphorus rickets. Cox, Dodds, et al., 1~ in an excellent, brief report, have shown that the addition of ferrie or soluble aluminum salts to the diet of guinea pigs and rabbits interferes with the absorption of phosphorus from the intestinal tract, and claim that this effect is produced by the precipitation of phosphorus in the intestinal traet as ferric and almninum phosphates. WMtner, in 1927, published in the German literature evidenee that reduced iron added to a nonraehitogenie diet produced rachitic-like changes ir~ rats. In the investigations described in this paper, it has been established that the addition of large amounts of ferric chloride to a nonrachitogenic diet can render that diet raehitogenie to rats. The chlorine radical of the ferric chloride has been eliminated as the rachitogenic agent by the use of a control diet containing the same weight-of chlorine in the form of ammonium chloride. It has been shown that this "ferric chloride rickets" can be prevented by the addition of excess phosphate sufficient theoretically to. combine with M1 the ferric ehIoi~de as ferric phosphate. Ferrous chloride and ferric ammonium citrate have been shown to produce the same degree of rickets when added to the control diet in amounts equal in metMlie iron content to the ferric chloride used in the ferrie ehloride diet. Partial evidence has been adduced that reduced iron behaves in the same way. The rickets produced by the addition to the control diet of the aforementioned amounts of iron was, in these experiments, indistinguishable by roentgenogram, by ehemieal 'studies, and by microscopic examination of the tissues from the rickets produced by Steenboek's raehitogenic diet No. 2965. With these facts established, it is interesting to speculate briefly as to the mechanism of the production of " i r o n rickets." The experiments were based on the assumption that iron might enter into chemical combination, in the intestinal tract, with phosphorus, to form compounds which are relatively difficult for absorption. There is considerable support in the results for the view that this actually happens. The serum phosphorus was low in all the members of the raehitie groups. The fact that the ammonium chloride diet gave negative results eliminates the possibility that the addition of ferric chloride produces
452
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rickets by its effect on the hydrogen ion concentration, of the intestinal contents. That it is actually the iron radical, which is responsible for the rickets, is shown by the similar effect of the other iron compounds used. The prevention of the raehitogenie effect of the ferric chloride by the addition o.f extra phosphate in amount sufficient, theoretically, to " h o l d " all the iron as ferric phosphate is very striking evidence in favor of the simple nonabsorption interpretation. The data presented in the literature by other investigators suggest a similar simple nonabsorption explanation. The striking figures of Cox, Dodds, et al. 1~ supply convincing evidence that ferric salts administered to rabbits and guinea pigs call seriously interfere with the process of absorption of phosphorus. ]in the experimental rickets induced by the addition to normal diets of beryllium, strontium, and high ratios of calcium and mag-nesium to phosphorus, it has been suggested that these elements also. interfere with the absorption of phosphorus from the intestinal tract. However, this theory has not been satisfactorily proved as yet. And on the evidence so far available, it cannot be claimed that the simple nonabsorption mechanism is the only factor responsible for the production of "iron rickets." Strontium and beryllium are not in common use as medicinal agents and, therefore, can have no relation to the occurrence of human rickets. Iron, however, especially in the form of the ammonium citrate salt, is now widely used in large doses as a medieinal agent, and the question arises whether it might occasionally be causative of infantile rickets. In considering this possibility it must here be emphasized that the amounts of iron used in these experiments on rats are, in relation to body weight, approximately twenty times the maximum dose of iron usually administered to a child. This fact makes it seem unlikely that the results of these experiments possess clinical significance. Large doses of iron ha~e now been used for many years in both adults and children without raising any clinical suspicions of disturbed phosphorus metabolism. It is conceivable, however, that long continued use of large doses of iron in a child on a diet somewhat low in phosphorus might lead over a prolonged time to the same result as a diet containing a considerable reduction below the optimum in phosphorus. Typical rickets might not appear, but later in life evidence of malnutrition of the bones might develop, as for example, the transverse lines seen occasionally in the roentgenograms of long bones. The relationship of iron therapy to phosphorus and calcium metaholism is being" studied by means of human balance experiments and will be reported upon at a later date. Likewise, the possible effect of phosphorus on the absorption of iron and the metabolism of iron in the anemia present in infantile rickets are being investigated.
BROCK AND DIAMOND: RICKETS IN RATS
BY
IRON FEEDING
453
SUMMARY
1. Rickets has been produced in rats by the addition of ferric chloride to a normal, nonraehitogenic diet. 2. The rickets so produced, as judged by roentgenograms, by microscopic studies, and by chemical studies, was qualitatively similar to, but more severe than that produced by Steenbock's rachitogenic diet No. 2965. 3. The addition of phosphorus to this ferric chloride diet prevented the occurrence of rickets. 4. The chlorine radical of the ferric chloride has been eliminated as the raehitogenie factor by negative results from the addition of ammonium chloride to the nonraehitogenic diet. 5. Similar raehitic changes have been produced by the addition o~ other iron compounds to the nonrachitogenie diet. 6. The experimental and clinical significance of these findings has been discussed. REFERENCES 1. Brock, J. F., and Taylor, ~. H . L . : I n Press. 2. Fiske, C. tt., and Subbarow, u g. Biochem. 66: 2, 1925. 3. MeCollum, E. V.; Simmonds~ N.; Shipley~ P. G.; and Park~ E. 2k.: I3ull. J o h n s Hopkins 1-Iosp. 33: 31, 1922. 4. Stoeltzner, I-t.: Bioehena. Ztsehr. 12: 119, 1908. 5. Lehnerdt, 1%: Beitr. z. path. Anat. u. z. allg. Path. 47: 215, 1910. 6. Shipley, P. G.; Park, E. A.; MeCollum, E. V.; Simmonds, N.; and Kinney, E. iV[.: Bull. J o h n s ~ o p k l n s Kosp. 33: 216, 1922. 7. tISrste, G. 2r : Lk[onatsehr. f. ]~2inderh. 5~:: 203, 1932. 8. ]Jrannion, I-I. D.; Guyatt~ I3. L.; and t~ay~ I-I. D.: J. Biochem. 92: xi~ 1931. 9. Guyatt, B. L.; Kay, H. D.; and B r a n n i e n , H. D.: J. N u t r i t i o n 6: :313, 1933. 10. Cox, G. J . ; ])odds, M. L ; ~ i g m a n , t[. B. i and iY[urphy, F . J . : Y. Bi0ehem. 92: xi~ 1931 11. WaRner, K . : Bioehem. Ztsehr. 188: B81; 1927.
DIAMOND, M . D .
BOSTON, )/IASS. INTRODUCTION
HE studies reported here were mldertaken in pursuance of some T ideas arising out of preliminary experiments bearing upon the problem of the absorption ~f iron. ~ It was found that the dialysis of ferric ammonium c~trate across cellophane membranes was markedly impeded by the addition to the solution of the secondary and tertiary sodium phosphates, even though there was no precipitate. This effect was considered to be due to the formation of colloidal ferric phosphate. Other soluble iron salts used in these dialysis experiments all formed precipitates with secondary and tertia~T sodium phosphate under the conditions of the experiment, and tile passage of the iron salts was naturally greatly impeded. The supposition that these principles might be applicable to. conditions in the intestinal tract suggested the possibility that iron and phosphorus might, by forming insoluble compounds in the intestinal tract, each hinder the absorption of the other. It was decided, therefore, to find out whether in the case of rats the addition of iron to a nonrachitogenic diet might so interfere with the absorption of phosphorus as to render that diet r achitogenic. EXPERIMENTAL
Young rats were obtained from the Albino Supply Company of Philadelphia. They were of the type called by the suppliers " vitamin rats," recently weaned, and weighing between 35 and 50 grams. The rats were divided into groups of five to a cage, all being exposed to the same amount of daylight coming through a plain glass window facing north. Particulars are given in TaMe I of the various diets used. The C,o~trol D~et used is one which has repeatedly been shown to be nonraehitogenie to rats. The Steenbook Raehitoge~de Diet is a h i g h calcium, low p h o s p h o r u s diet, which has repeatedly been shown to be raehitogenic to r a t s raider conditions similar to those which were used for our animals. Eavh o f the remaining diets consists o f the contro~ diet p~u~ the va~m~s vubsCanve~ mentio~e& F r o m the Departments of Pediatrics and 2r I-farvard Medical School, and the Thorndike Memorial Laboratory, Second and F o u r t h Medical Services ( H a r v a r d ) of the Boston City H o s p i t a l *Leeverhulme Research Scholar of the Royal College of 1Jhysicians, London. 449.
BROCK A N D D I A M O N D :
R I C K E T S I N RATS B Y IRON F E E D I N G
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444
THE JOUI~NAL OF PEDIATRICS
Tl~e 2~errio Chloric~e Diet contains iron (9.14 gin.) suificient to combine chemically as ferric phosphate with 5.06 gm. of phosphorus, or as ferrous phosphate with 3.37 grams of phosphorus. I r o n in the above q u a n t i t y was added to the control diet on the assumption that, by f o r m i n g in the intestinM tract insoluble ferric or f e r r o u s phosphate, it m i g h t interfere with the absorption of phosphorus. The, A~mno~iv/~ Chloride Dive contains the same weight of chlorine as is contai~e4 in the ferric chloride present in the previeus diet. I t was intended to eliminat(~ the possibility t h a t a n y rachitogeade effect of added ferric chloride m i g h t be due to alterations in the hydrogen ion concentration of the contents of the intestinal tract or to the chlorine radical. The Fe~'rio Chloride a~z~ Ade~ec~ 1~hosphoeua Diet contains i n addition to the phosphorus already in the ferric chloride diet a further allowance of phosphorus, s~ffficient to combine as ferric phosphate with all the added iron~ and t h u s provide the same amount of " u n b o u n d " phosphorns as the control diet. The F err~o Ammoniu~r~ Ci;tr~te, F e r r o ~ C'h~oride, Reduce~ Iroqv, a~nd Orya~dv Iron Diets consist each of the control diet with the addition of the salt named, in a m o u n t sufficient to yield about the same weight of metallic iron as is contained in the, ferric chloride diet.
The diets were made up in the following manner. Each of the salts was finely ground in a m o r t a r and intimately mixed in a large bowl with d r y corn meal and wheat g l u t e n . The mixture, after the addition of sufficient water to make it cohesiv% was rolled out upon a piece of smooth p a p e r which had been greased with olive off, cut into small sections, and baked until crisp in a oven. In the ease of ferric chloride and ferric ammonium citrate, these salts were dissolved in the amount of water which had to be added to the mixture. W a t e r and food ad libi~um were supplied fresh each day. The control diet and the ammonium chloride diet were very well taken and the rats gained weight well and remained sleek and healthy. The Steenbock rachitogenic diet, the ferric chloride diet, the ferric chloride with added phosphate diet, the ferric ammonium citrate diet, and the ferrous chloride diet were moderately well taken. The animals appeared in good health but gMned little weight. The reduced iron diet was less well taken and the animals lost weight. The o rga.nie iron diet was obviously hard and unpalatable to the rats. Although the animMs on this diet appeared to be in moderately good health, they lost a great deal of weight. In spite of loss of weight in the last two groups all the animals obviously increased in skeletM stature. There was no noticeable diminution of activity n o r weakness o~ the extremities. The rats were weighed individually at the start of the experiment, and again from time to time throughout the duration of the experiment. A f t e r ten days or a fortnight they were lightly anesthetized with ether and radiographed to detect the development of rickets. A t the termination of the experiment, the animals were anesthetized, decapitated and as much blood drained out of the body as possible. The blood from each group was pooled, and the serum phosphorus estimated by the method of Fiske and SubbarowY Each carcass was then radio-
BROCK
AND
DIAMOND:
RICKETS
IN
RATS
BY
.~r
IRON
445
F]~EDING
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446
THE
JOURNAL
OF P E D I A T R I C S
graphed. One knee joint and two or three costochondral junctions were taken f r o m each animal for microscopic examination. Sections were made f r o m the principal organs of the body to determine if there was evidence of disease. RESULTS
The results of these experiments are smnmarized in Table II. Tile first f o u r groups in the table comprise the p r e l i m i n a r y experiment in which the effect of added iron in the f o r m of ferric chloride was studied. The five rats on the control diet showed no rickets in twenty-two days, whereas the four rats on the Steenbock rachitogenic diet all showed gross m a n i f e s t a t i o n s of rickets at the end of the same period of time. The
iFig. 1 . - - C o n t r o l d i e t ; n o r i c k e t s ; s e c t i o n o f c o s t o e h o n d r a l j u n c t i o n
(X104).
five rats fed on the ferric chloride diet all presented the manifestations of rickets to a degree at least as m a r k e d as those on the Steenbock rachitogenie diet. None of the rats fed on the ammonium chloride diet showed any manifestations of rickets. Groups 5, 6, and 7 were repetitions of Groups 1, 3, and 4, respectively, and confirmed the results previously obtained with the control diet, the Steenboek rachitogenic diet, and the a m m o n i u m chloride diet. Group 8 was fed on a diet which contained ferric chloride in an amount which has been shown in Group 2 to produce rickets. B u t in this ease the ferric chloride was " c o v e r e d " by an amount of added phosphate sufficient chemically to combine with all the iron1 as ferric phosphate. None of these rats showed a n y manifestations of rickets. Groups 9 and 10 were fed on a diet to which had been added, respectively, ferric a m m o n i u m citrate and ferrous chloride in amounts equiva-
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Fig. 2.--Steenbock r a e h i t o g e n i c d i e t ; active r i c k e t s ; section of costoehondral junction ( X 1 0 4 ) .
Fig. 3.--lWerric chloride d i e t ; a c t i v e r i c k e t s ; section of costochondral junction (X104).
lent in metallic iron content to t h a t of the ferric chlorfde diet. All the r a t s in these two groups showed rickets almost as severe as t h a t of the members of the ferric chloride group although the diets were not as well t a k e n and the rats failed to gain weight.
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The members of Group 11, fed on a diet containing ferrum reductum, took their diet very poorly and lost weight. In Group 12, which received a diet containing organic iron, the loss of weight was even more pronouneed. In these two groups the gross evidences of rickets were lacking. These findings are quite compatible with the well-known requirement of growth for the production of experimental rickets. In fact, it has been shown that cessation of growth resulting from starvation may readily heal the lesions in animals previously rachitic, a The degree of rickets present in the roentgenograms of each group was assessed by Dr. Edward C. Vogt, ~ without prejudice of previous knowledge of the diets. His reports as shown in Column 7 of Table II stated that severe active rickets was present in the members of Groups 4 and
:Fig. 4 . - - R o e n t g e n o g r a m s of lower extremity. Left: Control; no :Ferric c h l o r i d e d i e t ; a d v a n c e d a c t i v e r i c k e t s .
rickets.
Right:
7 (Steenbock raehitogenic diet), Group 2 (ferric chloride diet), Group 9 (ferric ammonium citrate diet), and Group 10 (ferrous chloride diet). CHEMICAL ANALYSES
In Column 6 of Table II are summarized the estimations of serum phosphorus on the pooled blood from each group. The serum phosphorus of all the groups showing no evidence of rickets (Groups 1, 3, 5, 6, 8, 12) varied between 5.6 and 7.8 rag. per cent. That of all the groups showing gross evidence of rickets (Groups 2, 4, 7, 9, 10) wa~ between 3.7 and 2.5 rag. per cent. Group 11 which failed, to show gross evidence of rickets showed nevertheless a decrease of the phosphorus to 3.6 rag. per cent. * W e zare g r e a t l y i n d e b t e d t o D r . : E d w a r d C. V o g t , l ~ o e n t g e n o l o g i s t to t h e I n f a n t s ' a n d C h i l d r e n ' s H o s p i t a l s , f o r h i s a s s i s t a n c e irL t h e t a k i n g " a n d i n t e r p r e t a t i o n of t h e roentgenograras.
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EXAMINATION
The sections of the principal organs showed no evideaee of any morbid processes. Sections of the knee joint, including tlle lower end of the femur and the upper end of the tibia, and of the costochondral junctions were sub-
F i g . 5 . - - l ~ o e n t g e n o g r a m s of l o w e r e x t r e m i t y . Steenboek diet; advanced
Left: Control; active rickets
no r i c k e t s ,
IRight:
Fig. 6.~Roentgenograrns of lower extremity. Left: Ferrous chloride diet; advanced active rickets. :Right: Iron arr~monium citrate diet; advanced active rickets.
mitted to Dr. Sidney Farber ~ for examination without any indication as :to their source, ttis report, summarized in Column 8 of Table II, corroborates the findings in the roentgenograms. Severe active rickets was present in Groups 4 and 7 (Steenbock rachitogenic diet), Group 2 (ferric chloride diet), Group 9 (ferric ammonium citrate diet), and and
eWe appreciate the cooperation of Dr. Sidney Farber, Pathologist Children's Hospitals, in reviewing the pathologic sections.
to the Infants'
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Group 10 (ferrous chloride diet). In his opinion, the rickets in tbo members of Group 2 (ferric chloride diet) was more severe than that found in the members of Groups 4 and 7 (Steenbock rachitogenic diet). In Groups 9 and 10 (ferric ammonium citrate diet and ferrous chloride diet), the rickets was slightly less severe. The members of Group 11 (ferrum reduetum diet), which had taken their food poorly, had lost weight and had shown no gross manifestations of rickets, nevertheless, presented evidence 02 having typical rickets, in a moderate degree, by microscopic examination of sections (from the bones of the lower extremities and from the eostochondral junctions). There was no microscopic evidence Of rickets in Group 12 (organic iron diet) where the loss of body weight had been so great. There was no qvalitative difference between the rickets produced by these various dietsP DISCUSSION
A high ratio of Ca to P in the diet has been utilized for a decade in the production of experimental rickets. This principle underlies the Steenbock raehitogenic diet No. 2965 used in Groups 4 and 7. It is believed that the high ratio of calcium to phosphorus in the intestinal tract interferes sufficiently with the absorption and utilization of phosphorus to make this diet rachitogenic. The knowledge that elements other than calcium when added to the diet may produce rickets is not new. In 1908 StoeltzneP reported that feeding of strontium to animals led to the development of a rachitic process. This has been confirmed several times. An elaborate investigation by Lehnerdt s in 1910 on puppies and young rabbits showed that strontium produced a marked increase in osteoid tissue in the bones, which he called strontium sclerosis. More recently in 1922, Ship]ey, Park et al., G by adding 2.2 per cent of strontium carbonate to an otherwise normal diet, produced in rats histologic changes of the raehitic type. These abnormalities could not be prevented by the addition of cod liver oil or liberal amounts of viosterol. Many of the rats developed incomplete or total paralyses of the extremities. No relatioi1 has ever been demonstrated between strontium and the natural occurrence of infantile rickets. The close relationship of magnesium to calcium in the production and especially in the healing of rickets has been known for some time. In 1932, HSrste 7 produced in rats rickets of the low phosphorus type by the addition of magnesium carbonate to a nonrachitogenic diet. He showed that the rickets might be regarded as depende~lt upon the ratio of magnesium plus calcium to phosphorus. * W e a r e g r e a t l y i n d e b t e d to Dr. S. ~ u r t W o l b a c b , P r o f e s s o r of P a t h o l o g y , H a r v a r d ,~r School, for allowing us to quote him regarding the rickets produced in these experiments by the ferric chloride diet as histologically typical of low phospi]ortlS rickets, and indistinguishable from that produced by the Steenbock diet.
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In 1931, Branion, Ouyatt, and Kay* reported briefly the production of bone lesions similar to rickets in young rats by the replacement of the calcium earbonate in Steenboek's rachitogenie diet No. 2965 with an equivalent amount of beryllium carbonate. They have expanded these observations more recently (1933) and have shown that the administratiou of cod liver oil, viosterol, or ultraviolet light in generous amounts does not prevent the onset of this type of rickets. 9 They mention that sections'of the bones of their animals show striking differences from the appearances usually encountered in low phosphorus rickets. Cox, Dodds, et al., 1~ in an excellent, brief report, have shown that the addition of ferrie or soluble aluminum salts to the diet of guinea pigs and rabbits interferes with the absorption of phosphorus from the intestinal tract, and claim that this effect is produced by the precipitation of phosphorus in the intestinal traet as ferric and almninum phosphates. WMtner, in 1927, published in the German literature evidenee that reduced iron added to a nonraehitogenie diet produced rachitic-like changes ir~ rats. In the investigations described in this paper, it has been established that the addition of large amounts of ferric chloride to a nonrachitogenic diet can render that diet raehitogenie to rats. The chlorine radical of the ferric chloride has been eliminated as the rachitogenic agent by the use of a control diet containing the same weight-of chlorine in the form of ammonium chloride. It has been shown that this "ferric chloride rickets" can be prevented by the addition of excess phosphate sufficient theoretically to. combine with M1 the ferric ehIoi~de as ferric phosphate. Ferrous chloride and ferric ammonium citrate have been shown to produce the same degree of rickets when added to the control diet in amounts equal in metMlie iron content to the ferric chloride used in the ferrie ehloride diet. Partial evidence has been adduced that reduced iron behaves in the same way. The rickets produced by the addition to the control diet of the aforementioned amounts of iron was, in these experiments, indistinguishable by roentgenogram, by ehemieal 'studies, and by microscopic examination of the tissues from the rickets produced by Steenboek's raehitogenic diet No. 2965. With these facts established, it is interesting to speculate briefly as to the mechanism of the production of " i r o n rickets." The experiments were based on the assumption that iron might enter into chemical combination, in the intestinal tract, with phosphorus, to form compounds which are relatively difficult for absorption. There is considerable support in the results for the view that this actually happens. The serum phosphorus was low in all the members of the raehitie groups. The fact that the ammonium chloride diet gave negative results eliminates the possibility that the addition of ferric chloride produces
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rickets by its effect on the hydrogen ion concentration, of the intestinal contents. That it is actually the iron radical, which is responsible for the rickets, is shown by the similar effect of the other iron compounds used. The prevention of the raehitogenie effect of the ferric chloride by the addition o.f extra phosphate in amount sufficient, theoretically, to " h o l d " all the iron as ferric phosphate is very striking evidence in favor of the simple nonabsorption interpretation. The data presented in the literature by other investigators suggest a similar simple nonabsorption explanation. The striking figures of Cox, Dodds, et al. 1~ supply convincing evidence that ferric salts administered to rabbits and guinea pigs call seriously interfere with the process of absorption of phosphorus. ]in the experimental rickets induced by the addition to normal diets of beryllium, strontium, and high ratios of calcium and mag-nesium to phosphorus, it has been suggested that these elements also. interfere with the absorption of phosphorus from the intestinal tract. However, this theory has not been satisfactorily proved as yet. And on the evidence so far available, it cannot be claimed that the simple nonabsorption mechanism is the only factor responsible for the production of "iron rickets." Strontium and beryllium are not in common use as medicinal agents and, therefore, can have no relation to the occurrence of human rickets. Iron, however, especially in the form of the ammonium citrate salt, is now widely used in large doses as a medieinal agent, and the question arises whether it might occasionally be causative of infantile rickets. In considering this possibility it must here be emphasized that the amounts of iron used in these experiments on rats are, in relation to body weight, approximately twenty times the maximum dose of iron usually administered to a child. This fact makes it seem unlikely that the results of these experiments possess clinical significance. Large doses of iron ha~e now been used for many years in both adults and children without raising any clinical suspicions of disturbed phosphorus metabolism. It is conceivable, however, that long continued use of large doses of iron in a child on a diet somewhat low in phosphorus might lead over a prolonged time to the same result as a diet containing a considerable reduction below the optimum in phosphorus. Typical rickets might not appear, but later in life evidence of malnutrition of the bones might develop, as for example, the transverse lines seen occasionally in the roentgenograms of long bones. The relationship of iron therapy to phosphorus and calcium metaholism is being" studied by means of human balance experiments and will be reported upon at a later date. Likewise, the possible effect of phosphorus on the absorption of iron and the metabolism of iron in the anemia present in infantile rickets are being investigated.
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SUMMARY
1. Rickets has been produced in rats by the addition of ferric chloride to a normal, nonraehitogenic diet. 2. The rickets so produced, as judged by roentgenograms, by microscopic studies, and by chemical studies, was qualitatively similar to, but more severe than that produced by Steenbock's rachitogenic diet No. 2965. 3. The addition of phosphorus to this ferric chloride diet prevented the occurrence of rickets. 4. The chlorine radical of the ferric chloride has been eliminated as the raehitogenie factor by negative results from the addition of ammonium chloride to the nonraehitogenic diet. 5. Similar raehitic changes have been produced by the addition o~ other iron compounds to the nonrachitogenie diet. 6. The experimental and clinical significance of these findings has been discussed. REFERENCES 1. Brock, J. F., and Taylor, ~. H . L . : I n Press. 2. Fiske, C. tt., and Subbarow, u g. Biochem. 66: 2, 1925. 3. MeCollum, E. V.; Simmonds~ N.; Shipley~ P. G.; and Park~ E. 2k.: I3ull. J o h n s Hopkins 1-Iosp. 33: 31, 1922. 4. Stoeltzner, I-t.: Bioehena. Ztsehr. 12: 119, 1908. 5. Lehnerdt, 1%: Beitr. z. path. Anat. u. z. allg. Path. 47: 215, 1910. 6. Shipley, P. G.; Park, E. A.; MeCollum, E. V.; Simmonds, N.; and Kinney, E. iV[.: Bull. J o h n s ~ o p k l n s Kosp. 33: 216, 1922. 7. tISrste, G. 2r : Lk[onatsehr. f. ]~2inderh. 5~:: 203, 1932. 8. ]Jrannion, I-I. D.; Guyatt~ I3. L.; and t~ay~ I-I. D.: J. Biochem. 92: xi~ 1931. 9. Guyatt, B. L.; Kay, H. D.; and B r a n n i e n , H. D.: J. N u t r i t i o n 6: :313, 1933. 10. Cox, G. J . ; ])odds, M. L ; ~ i g m a n , t[. B. i and iY[urphy, F . J . : Y. Bi0ehem. 92: xi~ 1931 11. WaRner, K . : Bioehem. Ztsehr. 188: B81; 1927.