Skeletal Effects of Magnesium Deficiency in Normal, Ovariectomized, and Estrogen-treated Rats RITA BOGOROCH AND LEONARD F. BELANGER Department of Histology and Embryology, Faculty of Medicine, University of Ottawa, Ottawa, Canada K I N 6 N 5
ABSTRACT The effects of magnesium deficiency in ovariectomized and estrogen-treated rats were examined in histological sections of bones and various soft tissues. The changes observed in the femora of intact rats deprived of magnesium for three weeks were: 1. a general increase in diaphyseal thickness, 2. the presence of localized fibrous or bony-like masses in subperiosteal and metaphyseal sites, and 3. the occurrence, although rare, of endosteal hyperplasia. In ovariectomized, magnesium-deprived animals, the incidence and location of fibrous masses were similar to that in the femora of magnesium-deficient intact rats; however, no increase in diaphyseal thickness was noted. Daily injections of 25 pg estradiol caused a reduction of the frequency of skeletal hyperplasia from 80% to 20%, as well as a reduction in femoral diaphyseal thickness. Estradiol hormone administration also brought about a marked alleviation of the dermal and neural manifestations of magnesium deficiency, but, at the same time, caused an exacerbation of renal calcinosis. Earlier investigators recognized that prolonged magnesium deficiency leads to a decrease in body weight (Leroy, '26), to the formation of "brittle bones" (Kruse et al., '32; Watchorn and McCance, '37; Duckworth et al., '40) and to osseous magnesium depletion (Duckworth and Godden, '41; Blaxter, '56; Larvor et al., '65; Martindale and Heaton, '64; Hunt, '71). More recently, Hunt and Belanger ('72) observed that, concurrent with the general skeletal growth retardation, medullary trabeculae of endosteal origin and localized bone hyperplasia along the linea aspera of the femur also occurs. The mechanism by which this hyperplasia develops is still unknown. It is known, however, that no medullary bone of endosteal origin occurs in magnesium deficient rats that were previously parathyroidectomized (Hunt and Belanger, '72). Furthermore, it was also shown that the cellular composition of the hyperplasia along the linea aspera is also modified i n the parathyroidectomized animal (Hunt and Bklanger, '72). I n intact rats, the hyperplastic lesion consists mainly of fibroblasts interspersed with collagen fibers. In parathyroidectomized rats. the ANAT. REC., 183: 4 3 7 4 4 8 .
lesion also contains cartilage i n addition to fibrous tissue. The administration of parathormone to parathyroidectomized rats causes a regression of the cartilage component in favor of the fibrous portion. The fact that parathormone plays a role in modifying both the size and cellular composition of skeletal lesions in magnesium deficient rats suggested that other hormones which affect bone growth and development might also modify the bone hyperplasia seen in magnesium deprived animals. There has, as yet, been no report on the combined effects of estrogens and magnesium deficiency on rat bone. This paper, therefore, examines the effect of ovariectomy and estrogen treatment on the growth of bone and the occurrence of skeletal lesions in magnesium deficient rats. In addition, in order to determine whether hormone treatment modifies nonosseous tissues in these animals, a summary of some morphological extra-skeletal effects of the magnesium deficiency is also included. Received July 16, '73. Accepted Apr. 21, '75. 1 Present address: Environmental Health Centre, National Health and Welfare, Tunney's Pasture, Ottawa, Canada. K1A OL2.
437
438
RITA BOGOROCH A N D LEONARD BlLANGER MATERIALS AND METHODS
Fifty rats of the Charles River Caeserian Derived Strain (CRCD) weighing 110 to 120 g. were used in each of two series of experiments. In Series I, one-half the number of rats were bilaterally ovariectomized (gonadectomized) ; the other half were left intact, All animals were fed a standard nutritionally adequate diet (Rockland chow pellets) for 35 days. After this period of time, the rats were divided into six groups and treated as follows for another period of 21 days: Group 1
2
3 4
5 6
Intact rats Control diet Intactrats Magnesium deficient diet Gonadectomized ($) Control diet Gonadectomized Control diet estrogen supplement Gonadectomized Magnesium deficient diet Gonadectomized Magnesium deficient diet estrogen supplement
(g)
+
+
(6) (6)
!Mg(+)l [Mg(-
)I
+> I + >I +[Mg( [El [Mg(
[Mg( -)I IMg( -)I
+ [El
In Series II, groups of 25 intact and 25 gonadectomized rats were fed the experimental magnesium deficient diet for 24 days. The control and experimental diets (in pellet form) were obtained from General Biochemicals, Chagrin Falls, Ohio and were provided ad libitum to the rats. The magnesium deficient diet contained 0.925 to 1.2 mg. magnesium per 100 g. of dry weight of diet; the control magnesium supplemented diet, 265 mg. per 100 g. dry eight.^ Estrogen supplement was administered intramuscularly as 17-p-estradiol in corn oil at a dose of 25 pg per day.4 The physical condition and behaviour of the animals was assessed daily and the severity of symptoms (dermal hyperemia of ears and paws, dermal lesions, irritability, etc. ) were graded from 0 (no symptoms) to (e.g., all rat ears were intensely hyperemic). Body weight was recorded twice weekly throughout the experimental period (fig. 1). All animals were killed with chloroform. Tibiae and femora were fixed in AFA (ethanol, 75 parts; formaldehyde, 20 parts; glacial acetic acid, 5 parts) for 24 hours and then demineralized in 10% NazEDTA (ethylenediaminetetraacetic acid) by the
++++
drip process method (Bklanger et al., ’65). After three weeks in EDTA, each bone was bisected longitudinally in the sagittal plane, dehydrated through graded ethanol solutions, and embedded in paraffin. The longitudinal sections (approximate thickness 7 of the bones were stained with phloxine, methylene blue and azure (PMBA). Sections of kidney and skin were also prepared and examined for lesions. The severity of renal calcinosis in each kidney was graded on a scale from 0 to according to the size of the calculus and the number of tubules involved. Measurement of bone thickness. A magnified ( X 73) image of each histological longitudinal section was projected on a screen and outlines of the magnified outer contours of the bone section, bone marrow space, epiphyseal plate, and limits of anterior and posterior diaphyses were traced onto graph paper, so that the long axis of the bones constituted the vertical axis. The width of the anterior diaphysis was obtained by averaging measurements made at five chosen levels along the bone shaft. The selected horizontal levels on the drawing of the femur were at distances of 330, 430, 530, 630, and 730 mm. from the distal extremity of the bone and corresponded to 4.6-10 mm. from the distal end of the actual bone section. The average width and standard error of the diaphysis in each treatment group were calculated and compared to those of other groups by the application of Student’s “t” test. Widths of the tibia1 diaphysis and of the epiphyseal plate were also assessed from these tracings.
++++
RESULTS
Weight gain All growing rats, whether intact (fig. 1, curve 6 ) or ovariectomized (curve 2), 2 In these experiments the animals in the various groups were not pair fed and the amount of diet consumed by each rat was not monitored daily. Generally, however, the animals on the magnesium deficient diet consumed less food per day than did the control rats. 3 The magnesium analyses of the diet were obtained from General Biochemicals and were verified by absorption spectrophotometry by Dr. Barbara Hunt. 4To 12.5 mg estradiol was added 2 ml absolute ethanol and 25 ml Mazola corn oil. When the estradiol was completely dissolved, the ethanol was evaporated and another 25 ml of corn oil was added. The pharmacologic dose of 25 pg estradiol was used for the purpose of studying the combined effect of large doses of this estrogen and magnesium deficiency in rats.
439
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY
EFFECT
OF OVARIECTOMY A N D MAGNESIUM DEFICIENCY ON THE WEIGHT OF RATS
310 300 290
-
280 270 260
-
250 v)
240-
t-
210-
*0
200-
Z
190-
-
intact == o v a r i e c t o m i z e d
'lo'
Ib
1'5
=
20
ovari:::~~~;e,d,
25
3b
=-=-* m g d e f i c i e n t -I= m g d e f i c i e n t - 0
3'5
4b
mg d e f i c i e n t
I
4'5
5b
5'5
60
DAYS Fig. 1 Effect of ovariectomy a n d magnesium deficiency on t h e weight of rats.
gained less weight after three weeks on a Mg( -) diet than did the respective control rats fed a M g ( S ) diet (curves 5 and 1, respectively). On the other hand, when the normal increment of weight gain was greatly reduced by hormonal treatment, rats on the Mg(-) diet gained as little weight as did the control rats fed the Mg( +) diet. As illustration, the administration of estradiol to ovariectomized animals caused a n initial weight loss; after three weeks on a Mg(+) diet, these animals showed only a minimal weight gain (curve 4 ) . The final weight of these and of similarly treated rats fed a Mg(-) diet (curve 3 ) was identical. Severity of extra-skeletal lesions and symptoms A summary of the effect of ovariectomy and of estradiol on the severity of extra-
skeletal lesions and symptoms in rats fed a Mg(-) diet is presented in table 1. Ovariectomy appeared to have reduced the number and severity of some lesions (e.g., renal calcinosis) while, at the same time, it increased the severity of others (e.g., skin). In general, where ovariectomy of the magnesium-deficient rat ameliorated a specific symptom or lesion, estradiol often caused a n exacerbation of the pathology, and vice versa. Skeletal effects Diaphyseal thickness variations Femoral diaphyseal thickness in rats fed the M g ( + ) diet was not significantly altered by either ovariectomy and estradiol replacement or by ovariectomy (fig. 2 ) , columns 1, 2, and 3 respectively). However, the thickness was increased by 30% in intact rats fed a Mg(-) diet (column 1.
I
440
RITA BOGOROCH A N D LEONARD BRLANGER TABLE 1
Effect of magnesium deficiency and estradiol on the severity of extra-skeletal lesions and symptoms Group
Intact
Renal calcinosis
Diet
++
Mg(-)
6
Mg( - )
$+E
Mg(-)
+ +++
Myeloid hyperplasia
Dermal lesions
Dermal hyperemia
++ ++
++ ++++
0
0
++ ++++ +
Irritability
++ +++ +
WIDTH OF ANTERIOR PORTION OF FEMORAL DIAPHYSIS
75r
50.8 51.8
53.1 45.7
65.8
47.8
23.3 2 1 . 7 5 4 . 0 + l . l
47.2 +2.1
l-
(Y
I
-
0 X
E
E Z -
I I-
n -
5 "
.i n t a c t \
6
G
+E V
/\
G +E
V
It /
mgk)
mg(+) Fig. 2
intact
Width of anterior portion of femoral diaphysis.
4). Ovariectomy completely inhibited this hyperplastic effect of magnesium deficiency, but estradiol therapy did not counteract the inhibitory effect of the surgery (column 5). Tibia1 diaphyseal thickness also was altered by the magnesium deficiency as illustrated by a comparison of figures 3 and 4. Both the tibia1 bone wall and the periosteum ( P ) of magnesium deficient rats (fig. 4 ) were considerably thicker than those of rats fed the Mg(+) diet (fig. 3 ) . Furthermore, in magnesium deficient rats the tibia appeared to be a preferential site for the growth of fibrous trabecular medullary bone (fig. 4, MB) of endosteal origin.
Localized hyperplastic lesions Subperiosteal localized masses along the linea aspera of the femora were observed in all groups of magnesium deficient rats. Histologically, this femoral hyperplasia consisted of fibrous tissue and bone. It was found that such masses occurred in approximately 80% of all rats, regardless of whether the animals were intact or ovaritomized. The treatment of similar rats with large doses of estradiol, however, substantially reduced the frequency of the femoral subperiosteal lesions to about 20%. The occurrence of medullary bone in the tibiae of such rats was also considerably less frequent. 2.
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY
Localized endosteal lesions were also observed, but only in a few animals. One single large mass of endosteal bone was recorded (fig. 6). Round fibrous nodules located under the epiphyseal plate (fig. 7) were seen in three instances. In each case, they were free of either cartilage or bone. Other bone changes Other skeletal changes noted were the usual characteristic sequelae of magnesium deficiency. The length of the long bones was reduced (the average femur length," 25.38 0.16 mm, in rats on the Mg(--) diet was significantly less ( p < 0.001) than the average,' 26.82 0.08 mm, in Mg(+) fed rats); the epiphyseal plate was reduced in thickness by 25 to 50% and was poorly stained; bone trabeculae and spicules were shorter, wider, and somewhat irregular in shape; osteocytes were mostly small and elongated and osteocytic osteolysis appeared to be considerably reduced. In rats ovariectomized for 35 days, the epiphyseal plate was thicker than that of control rats and it contained a larger number of chondrocytes. Estradiol administration resulted in a 50% reduction from the control in the size of the plate and an increased azurophilia. A variable number of coarse, elongated spicules were present in the metaphyses of these animals. Magnesium deficiency did not appreciably alter the thickness of the plate in estrogen treated animals, but, as in all magnesium deficient rat bone, the epiphyseal plate also stained poorly with azure. 3.
*
*
DISCUSSION
Magnesium deficiency The results of these studies cmfirm the previously reported observations on the skeletal and extra-skeletal effects of magnesium deficiency in intact rats (Belanger, '58); Clark and Belanger, '67; Hunt, '71; Hunt and BClanger, '72). In addition to the general skeletal growth retardation, the formation of medullary trabeculae of endosteal origin and the subperiosteal hyperplasia described by Hunt and BClanger ('72), the present study also reveals other sites of skeletal hyperplasia at the endo-
44 1
steum and under the epiphyseal plate (figs. 4, 6, 7). One further observation of interest is the 30% increase in the general thickness of the femoral shaft of the magnesium deficient rat. The finding of an increased diaphyseal bone width was unexpected since it was thought that a severe nutritional magnesium deficiency would deprive osteoblasts of the function of a variety of enzymes dependent on magnesium for activation and thus would inhibit osteoblastic activity. Although not assessed by the more reliable labeling methods, osteoblastic activity did not appear, in these experiments, to have been substantially decreased. Furthermore, the femoral periosteal membrane showed signs of hyperplasia. In magnesium deficiency, the enzymes of bone resorption would also be deprived by the loss of magnesium ion, and this would contribute to a reduction in bone resorption. In the presence of normal osteoblastic activity, this decreased resorption would lead to an increase in bone width. The increase in width of the diaphysis in the femur (figs. 2, 5) and in the tibia (figs. 3, 4 ) is probably due to a combination of increased accretion and decreased resorption of bone. Recent studies by BClanger et al. ('72) suggested that the cells in the hyperplastic periosteal membrane were likely to be potential osteoblasts. This was based on the observation that, contrary to expectation, alkaline phosphatase was present in substantial quantity throughout the hyperplastic membrane. As an increased source of osteogenic cells, the hyperplastic periosteum would then account for one aspect of the increased bone width (i.e., accretion). The second aspect, that of decreased bone resorption, might, in fact, be a consequence of the inhibition of the normal rate of resorption in the femora of the magnesium deficient animal. Support for this concept was first presented by Clark and Belanger ('67) who reported a marked decrease in the phenomenon of osteocytic osteolysis in magnesium deficient rats. Osteocytic osteolysis also appeared to be considerably reduced in the present experiments. The relative contributions of increased accretion and 5 We are grateful to Dr. Barbara Hunt for the measurement of the lengths of the bones.
442
RITA BOGOROCH AND LEONARD BELANGER
decreased resorption to the final width of the diaphysis i n magnesium deficient animals awaits to be assessed. It has been known for some time that the skeletal magnesium content is decreased in magnesium deprived animals. I n a n anlysis of the mineral content of the long bones of young rats deprived of magnesium for three weeks, Hunt (unpublished) found that the magnesium content decreased 44% (from 680 mg./100 g. bone ash in control rats to 380 mg./100 g. ash in the deficient rats), whereas in adult animals the decrease, even after eleven weeks of magnesium deprivation, was never greater than 28% (Smith and Nisbet, ’72). At the same time, in young rats the calcium content increased from 37.4 g./100 g. bone ash to 42.4 g./lOO g. ash in the magnesium deprived animals (Hunt, unpublished); in adult rats no significant change in calcium content was noted (Smith and Nisbet, ’72). The large disparity between the Ca/Mg ratios in the long bones of control and magnesium deficient rats reflects the depletion of magnesium, probably from the surface of the bone. Indeed, the skeletal loss of magnesium has been explained by a specific process of depletion taking place by a n exchange process at the surface of the bone crystals (Blaxter, ’56). According to Blaxter, the whole skeleton may be involved since “in infant calves virtually the whole of the skeletal magnesium participates in the depletion and translocation process.” The large disparity between the Ca/Mg ratios in the long bones of control and magnesium deficient rats may also result from the breakdown of limited specific portions of bone and replacement by a newlyformed skeleton that would contain a n adequate amount of calcium but much less magnesium than normal. It is known that in sheep deprived of dietary calcium (Martin and Peirce, ’34) demineralization of the skeleton is not general. Trabeculae of the cancellous ends of long bones are more affected than the cortical shafts. The vertebrae, ribs and skull are also more rapidly depleted than the bones of the extremities (Duckworth and Hill, ’53). I n the experiments by Hunt referred to above and in the present series, the magnesium
deficient rats contained a greater amount than normal of bone i n their skeletons. A large amount of this bone, such as the medullary portion, was of the trabecular type. This trabecular bone has a rapid turnover rate (BClanger and Migicovsky, ’63). I n magnesium deficient rats the rapid turnover of trabecular bone and the replacement by adequate amounts of calcium and subnormal amounts of magnesium could also account for the shift in Ca/Mg ratios.
Ovariectomy and magnesium deficiency There appears to be no positive correlation between the weight gain of ovariectomized animals and bone growth. As expected, the bones of ovariectomized animals were longer than in intact rats (Silberberg and Silberberg, ’71). The diaphyses appeared thinner, but by measurement were not statistically different from those of intact animals. During the first week that the rats were fed the magnesium deficient diet no change in the increments of weight gain and in bone growth was detected. From then on, however, weight gain (fig. 1) and bone growth were rapidly curtailed. Although the widths of the femoral diaphyses were not appreciably altered by the magnesium deficiency, the subperiosteal hyperplasia was equal in frequency and, in some ovariectomized magnesium deprived rats, was more extensive than in intact deficient rats. Estrogen administration and magnesium deficiency A dose of 0.5-1 pg estradiol per 100 g. rat has been reported as “the minimal amount necessary to induce a significant increase in phosphofructokinase” ( Singhal et al., ’67a,b). If this dose is considered as physiologic, the dose of estradiol used in the present experiments was 10-20 times the normal physiologic requirements. This large dose was chosen intentionally because it causes rapid maturing of bone, As expected, a s soon as estradiol administration was initiated, the rats treated with this hormone no longer gained weight at the same rate as their non-treated ovariectomized counterparts. Instead, there was
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY
a n immediate loss of weight and only a minimal weight gain during the following weeks of the experimental period. After three weeks the final weight of the estradiol treated rats was approximately 15% less than the weight of untreated animals. Magnesium deficient rats treated with estradiol showed the same 15% final weight difference from the untreated magnesium deficient ovariectomized rats. Thus, when initiated i n combination with large doses of estradiol, magnesium deficiency per se did not alter the weight gain pattern of these rats. As for the combined effect of estrogen and magnesium deficiency on bone, it is not clear as to why the femoral diaphyses from rats treated with estradiol and fed a magnesium deficient diet did not show the hyperplastic response seen in untreated rats fed the same diet. Nor does there seem to be a simple explanation a s to why the incidence of subperiosteal hyperplasia in estradiol treated magnesium deficient rats is only 25% of that which occurs in untreated deficient animals. The maturation effect on bone of the large dose of estrogen appears to be implicated in the reduction of the lesions but the mechanism by which this reduction occurs remains to be elucidated. A possible speculation is that this dose of estradiol inhibits cell proliferation of the periosteal fibroblasts, as would a large dose of other steroid drugs. With respect to extra-skeletal lesions and symptoms, it is of interest to note that, despite its adverse effect on renal pathology, the administration of estradiol alleviated to a great extent both cutaneous and nervous disorders characteristic of the magnesium deficiency (table 1 ). It is clear from these studies that estrogen (estradiol) and ovariectomy, which affect bone maturation (Urist et al., '48), can also modify the incidence and severity of skeletal lesions and extra-skeletal lesions and symptoms. This potential benefit of estrogen therapy is now under investigation. ACKNOWLEDGMENT
The authors are indebted to the Medical Research Council of Canada for financial support (Grant MT 799).
443
LITERATURE CITED Belanger, L. F., D. Copp and M. A. Morton 1965 Demineralization with EDTA by constant replacement. Anat. Rec., 153: 41-47. BQlanger, L. F., B. J. Hunt and R. Narbaitz 1972 Alkaline phosphatase in hyperplastic bone lesions of rats fed a magnesium-deficient diet. In: Histochemistry and Cytochemistry. T. Takeuchi, K. Ogawa and S. Fujita, eds. Nakanishi Printing Co. Ltd., Kyoto, Japan, pp. 41-50. BBlanger, L. F., and B. B. Migicovsky 1963 Bone cell formation and survival in H3-thymidine-labeled chicks under various conditions. Anat. Rec., 145: 385-390. Blaxter, K. L. 1956 The magnesium content of bone in hypomagnesemic disorders of livestock. In: Ciba Found. Symp. on Bone Structure and Metabolism. G. E. W. Wolstenholme and M. O'Conner, eds. Little, Brown & Co., Boston, pp 117-134. Clark, I., and L. F. Belanger 1967 The effects of alterations in dietary magnesium on calcium, phosphate and skeletal metabolim. Calc. Tiss. Res., I : 204-218. Duckworth, J., and W. Godden 1941 The lability of skeletal magnesium reserves. The influence of rates of bone growth. Biochem. J., 35: 816-823. Duckworth, J., W. Godden and G. M. Warnock 1940 The effect of acute magnesium deficiency on bone formation in the rat. Biochem. J., 34: 97-108. Duckworth, J. and R. Hill 1953 The storage of elements in the skeleton. Nutr. Abstr. Rev., 23: 1-17. Hunt, B. J. 1971 Age and magnesium deficiencv in the rat with emuhasis on bone and muscie magnesium. Arne;. J. Physiol., 221: 1809-1817. Hunt, B. J., and L. F. BBlanger 1972 Localized, multiform, subperiosteal hyperplasia and generalized osteomyelosclerosis in magnesium-deficient rats. Calc. Tiss. Res., 9: 17-27. Kruse, H. D., E. R. Orent and E. V. McCollum 1932 Studies on magnesium deficiency in animals. I. Symptomatology resulting from m a g nesium deprivation. J. Biol. Chem., 96: 519539. Larvor, P., A. Girard and M. Brochart 1974 Etude de l a carence experimentale en mag&sium chez le veau. Ann. Biol. Anim., 4: 371382. Leroy, J. 1926 NCcessitQ du magnesium pour l a croissance de la souris. C. R. SOC. Biol. (Paris), 94: 431-433. Martin, C. J., and A. W. Peirce 1934 Studies on the phosphorus requirements of sheep. Coun. Sci. Ind. Res., Aust. Bull. No. 77, 1-46. Martindale, L., and F. W. Heaton 1964 Maenesium deficiency in the adult rat. Biochem. J., 92: 119-126. Silberberg, M., and R. Silberberg 1971 Steroid hormones and bone. In: The Biochemistry and
444
RITA BOGOROCH AND LEONARD BELANGER
Physiology of Bone. Second ed. G. H. Bourne, ed. Academic Press, New York and London, pp. 401-484. Singhal, R. L., J. R. E. Valadares and G. M. Ling 1967a Estrogen-induced increase in phosphohexose isomerase activity in the rat uterus Metab. Clin. Exp., 16: 271-278. 1967b Metabolic control mechanisms in mammalian systems. I. Hormonal induction of phosphofructokinase in the rat uterus. J. Biol. Chem., 242: 2593-2598.
Smith, B. S. W., and D. I. Nisbet 1972 Biochemical and pathological studies on magnesium deficiency in the rat. 11. Adult Animals. J. Comp. Path., 82: 3746. Urist, M. R., A. M. Budy and F. C. McLean 1948 Species differences in the reaction of the mammalian skeleton to estrogens. Proc. SOC.Exp. Biol. and Med., 68: 324-326. Watchorn, E., and R. A. McCance 1937 Subacute magnesium deficiency in rats. Biochern. J., 31 : 1379-1390.
PLATE 1 EXPLANATION OF FIGURES
3 A portion of tibial diaphysis and proximal metaphysis in a normal (intact) control rat. Phloxine-Methylene Blue-Azure stain. x 43. 4 A comparable portion of tibia of a normal rat fed a magnesiumdeficient diet for 21 days. Notice periosteal hyperplasia ( P ) , diaphyseal thickness and medullary bone (MB). Phloxine-Methylene Blue-Azure stain. x 43. 5
A portion of femoral diaphysis of a normal rat fed a magnesiumdeficient diet for 21 days. In this particular instance, the wall is uniformly twice as thick as that in the control. Phloxine-Methylene Blue-Azure stain. x 43.
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY Rita Bogoroch and Leonard F. BBlanger
PLATE 1
445
PLATE 2 EXPLANATION OF FIGURES
6
Local hyperplasia, consisting mainly of bone growing from the endosteal surface of the tibial diaphysis in a normal rat fed the magnesium-deficient diet for 21-days. Phloxine-Methylene Blue-Azure stain. x 43.
'7 Fibrous nodule located under the greater trochanteric epiphyseal plate in the femur of a normal rat fed the magnesium-deficient diet for 21 days. Phloxine-Methylene Blue-Azure stain. x 43.
446
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY Rita Bogoroch and Leonard F. Belanger
PLATE 2
447
ABSTRACT The effects of magnesium deficiency in ovariectomized and estrogen-treated rats were examined in histological sections of bones and various soft tissues. The changes observed in the femora of intact rats deprived of magnesium for three weeks were: 1. a general increase in diaphyseal thickness, 2. the presence of localized fibrous or bony-like masses in subperiosteal and metaphyseal sites, and 3. the occurrence, although rare, of endosteal hyperplasia. In ovariectomized, magnesium-deprived animals, the incidence and location of fibrous masses were similar to that in the femora of magnesium-deficient intact rats; however, no increase in diaphyseal thickness was noted. Daily injections of 25 pg estradiol caused a reduction of the frequency of skeletal hyperplasia from 80% to 20%, as well as a reduction in femoral diaphyseal thickness. Estradiol hormone administration also brought about a marked alleviation of the dermal and neural manifestations of magnesium deficiency, but, at the same time, caused an exacerbation of renal calcinosis. Earlier investigators recognized that prolonged magnesium deficiency leads to a decrease in body weight (Leroy, '26), to the formation of "brittle bones" (Kruse et al., '32; Watchorn and McCance, '37; Duckworth et al., '40) and to osseous magnesium depletion (Duckworth and Godden, '41; Blaxter, '56; Larvor et al., '65; Martindale and Heaton, '64; Hunt, '71). More recently, Hunt and Belanger ('72) observed that, concurrent with the general skeletal growth retardation, medullary trabeculae of endosteal origin and localized bone hyperplasia along the linea aspera of the femur also occurs. The mechanism by which this hyperplasia develops is still unknown. It is known, however, that no medullary bone of endosteal origin occurs in magnesium deficient rats that were previously parathyroidectomized (Hunt and Belanger, '72). Furthermore, it was also shown that the cellular composition of the hyperplasia along the linea aspera is also modified i n the parathyroidectomized animal (Hunt and Bklanger, '72). I n intact rats, the hyperplastic lesion consists mainly of fibroblasts interspersed with collagen fibers. In parathyroidectomized rats. the ANAT. REC., 183: 4 3 7 4 4 8 .
lesion also contains cartilage i n addition to fibrous tissue. The administration of parathormone to parathyroidectomized rats causes a regression of the cartilage component in favor of the fibrous portion. The fact that parathormone plays a role in modifying both the size and cellular composition of skeletal lesions in magnesium deficient rats suggested that other hormones which affect bone growth and development might also modify the bone hyperplasia seen in magnesium deprived animals. There has, as yet, been no report on the combined effects of estrogens and magnesium deficiency on rat bone. This paper, therefore, examines the effect of ovariectomy and estrogen treatment on the growth of bone and the occurrence of skeletal lesions in magnesium deficient rats. In addition, in order to determine whether hormone treatment modifies nonosseous tissues in these animals, a summary of some morphological extra-skeletal effects of the magnesium deficiency is also included. Received July 16, '73. Accepted Apr. 21, '75. 1 Present address: Environmental Health Centre, National Health and Welfare, Tunney's Pasture, Ottawa, Canada. K1A OL2.
437
438
RITA BOGOROCH A N D LEONARD BlLANGER MATERIALS AND METHODS
Fifty rats of the Charles River Caeserian Derived Strain (CRCD) weighing 110 to 120 g. were used in each of two series of experiments. In Series I, one-half the number of rats were bilaterally ovariectomized (gonadectomized) ; the other half were left intact, All animals were fed a standard nutritionally adequate diet (Rockland chow pellets) for 35 days. After this period of time, the rats were divided into six groups and treated as follows for another period of 21 days: Group 1
2
3 4
5 6
Intact rats Control diet Intactrats Magnesium deficient diet Gonadectomized ($) Control diet Gonadectomized Control diet estrogen supplement Gonadectomized Magnesium deficient diet Gonadectomized Magnesium deficient diet estrogen supplement
(g)
+
+
(6) (6)
!Mg(+)l [Mg(-
)I
+> I + >I +[Mg( [El [Mg(
[Mg( -)I IMg( -)I
+ [El
In Series II, groups of 25 intact and 25 gonadectomized rats were fed the experimental magnesium deficient diet for 24 days. The control and experimental diets (in pellet form) were obtained from General Biochemicals, Chagrin Falls, Ohio and were provided ad libitum to the rats. The magnesium deficient diet contained 0.925 to 1.2 mg. magnesium per 100 g. of dry weight of diet; the control magnesium supplemented diet, 265 mg. per 100 g. dry eight.^ Estrogen supplement was administered intramuscularly as 17-p-estradiol in corn oil at a dose of 25 pg per day.4 The physical condition and behaviour of the animals was assessed daily and the severity of symptoms (dermal hyperemia of ears and paws, dermal lesions, irritability, etc. ) were graded from 0 (no symptoms) to (e.g., all rat ears were intensely hyperemic). Body weight was recorded twice weekly throughout the experimental period (fig. 1). All animals were killed with chloroform. Tibiae and femora were fixed in AFA (ethanol, 75 parts; formaldehyde, 20 parts; glacial acetic acid, 5 parts) for 24 hours and then demineralized in 10% NazEDTA (ethylenediaminetetraacetic acid) by the
++++
drip process method (Bklanger et al., ’65). After three weeks in EDTA, each bone was bisected longitudinally in the sagittal plane, dehydrated through graded ethanol solutions, and embedded in paraffin. The longitudinal sections (approximate thickness 7 of the bones were stained with phloxine, methylene blue and azure (PMBA). Sections of kidney and skin were also prepared and examined for lesions. The severity of renal calcinosis in each kidney was graded on a scale from 0 to according to the size of the calculus and the number of tubules involved. Measurement of bone thickness. A magnified ( X 73) image of each histological longitudinal section was projected on a screen and outlines of the magnified outer contours of the bone section, bone marrow space, epiphyseal plate, and limits of anterior and posterior diaphyses were traced onto graph paper, so that the long axis of the bones constituted the vertical axis. The width of the anterior diaphysis was obtained by averaging measurements made at five chosen levels along the bone shaft. The selected horizontal levels on the drawing of the femur were at distances of 330, 430, 530, 630, and 730 mm. from the distal extremity of the bone and corresponded to 4.6-10 mm. from the distal end of the actual bone section. The average width and standard error of the diaphysis in each treatment group were calculated and compared to those of other groups by the application of Student’s “t” test. Widths of the tibia1 diaphysis and of the epiphyseal plate were also assessed from these tracings.
++++
RESULTS
Weight gain All growing rats, whether intact (fig. 1, curve 6 ) or ovariectomized (curve 2), 2 In these experiments the animals in the various groups were not pair fed and the amount of diet consumed by each rat was not monitored daily. Generally, however, the animals on the magnesium deficient diet consumed less food per day than did the control rats. 3 The magnesium analyses of the diet were obtained from General Biochemicals and were verified by absorption spectrophotometry by Dr. Barbara Hunt. 4To 12.5 mg estradiol was added 2 ml absolute ethanol and 25 ml Mazola corn oil. When the estradiol was completely dissolved, the ethanol was evaporated and another 25 ml of corn oil was added. The pharmacologic dose of 25 pg estradiol was used for the purpose of studying the combined effect of large doses of this estrogen and magnesium deficiency in rats.
439
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY
EFFECT
OF OVARIECTOMY A N D MAGNESIUM DEFICIENCY ON THE WEIGHT OF RATS
310 300 290
-
280 270 260
-
250 v)
240-
t-
210-
*0
200-
Z
190-
-
intact == o v a r i e c t o m i z e d
'lo'
Ib
1'5
=
20
ovari:::~~~;e,d,
25
3b
=-=-* m g d e f i c i e n t -I= m g d e f i c i e n t - 0
3'5
4b
mg d e f i c i e n t
I
4'5
5b
5'5
60
DAYS Fig. 1 Effect of ovariectomy a n d magnesium deficiency on t h e weight of rats.
gained less weight after three weeks on a Mg( -) diet than did the respective control rats fed a M g ( S ) diet (curves 5 and 1, respectively). On the other hand, when the normal increment of weight gain was greatly reduced by hormonal treatment, rats on the Mg(-) diet gained as little weight as did the control rats fed the Mg( +) diet. As illustration, the administration of estradiol to ovariectomized animals caused a n initial weight loss; after three weeks on a Mg(+) diet, these animals showed only a minimal weight gain (curve 4 ) . The final weight of these and of similarly treated rats fed a Mg(-) diet (curve 3 ) was identical. Severity of extra-skeletal lesions and symptoms A summary of the effect of ovariectomy and of estradiol on the severity of extra-
skeletal lesions and symptoms in rats fed a Mg(-) diet is presented in table 1. Ovariectomy appeared to have reduced the number and severity of some lesions (e.g., renal calcinosis) while, at the same time, it increased the severity of others (e.g., skin). In general, where ovariectomy of the magnesium-deficient rat ameliorated a specific symptom or lesion, estradiol often caused a n exacerbation of the pathology, and vice versa. Skeletal effects Diaphyseal thickness variations Femoral diaphyseal thickness in rats fed the M g ( + ) diet was not significantly altered by either ovariectomy and estradiol replacement or by ovariectomy (fig. 2 ) , columns 1, 2, and 3 respectively). However, the thickness was increased by 30% in intact rats fed a Mg(-) diet (column 1.
I
440
RITA BOGOROCH A N D LEONARD BRLANGER TABLE 1
Effect of magnesium deficiency and estradiol on the severity of extra-skeletal lesions and symptoms Group
Intact
Renal calcinosis
Diet
++
Mg(-)
6
Mg( - )
$+E
Mg(-)
+ +++
Myeloid hyperplasia
Dermal lesions
Dermal hyperemia
++ ++
++ ++++
0
0
++ ++++ +
Irritability
++ +++ +
WIDTH OF ANTERIOR PORTION OF FEMORAL DIAPHYSIS
75r
50.8 51.8
53.1 45.7
65.8
47.8
23.3 2 1 . 7 5 4 . 0 + l . l
47.2 +2.1
l-
(Y
I
-
0 X
E
E Z -
I I-
n -
5 "
.i n t a c t \
6
G
+E V
/\
G +E
V
It /
mgk)
mg(+) Fig. 2
intact
Width of anterior portion of femoral diaphysis.
4). Ovariectomy completely inhibited this hyperplastic effect of magnesium deficiency, but estradiol therapy did not counteract the inhibitory effect of the surgery (column 5). Tibia1 diaphyseal thickness also was altered by the magnesium deficiency as illustrated by a comparison of figures 3 and 4. Both the tibia1 bone wall and the periosteum ( P ) of magnesium deficient rats (fig. 4 ) were considerably thicker than those of rats fed the Mg(+) diet (fig. 3 ) . Furthermore, in magnesium deficient rats the tibia appeared to be a preferential site for the growth of fibrous trabecular medullary bone (fig. 4, MB) of endosteal origin.
Localized hyperplastic lesions Subperiosteal localized masses along the linea aspera of the femora were observed in all groups of magnesium deficient rats. Histologically, this femoral hyperplasia consisted of fibrous tissue and bone. It was found that such masses occurred in approximately 80% of all rats, regardless of whether the animals were intact or ovaritomized. The treatment of similar rats with large doses of estradiol, however, substantially reduced the frequency of the femoral subperiosteal lesions to about 20%. The occurrence of medullary bone in the tibiae of such rats was also considerably less frequent. 2.
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY
Localized endosteal lesions were also observed, but only in a few animals. One single large mass of endosteal bone was recorded (fig. 6). Round fibrous nodules located under the epiphyseal plate (fig. 7) were seen in three instances. In each case, they were free of either cartilage or bone. Other bone changes Other skeletal changes noted were the usual characteristic sequelae of magnesium deficiency. The length of the long bones was reduced (the average femur length," 25.38 0.16 mm, in rats on the Mg(--) diet was significantly less ( p < 0.001) than the average,' 26.82 0.08 mm, in Mg(+) fed rats); the epiphyseal plate was reduced in thickness by 25 to 50% and was poorly stained; bone trabeculae and spicules were shorter, wider, and somewhat irregular in shape; osteocytes were mostly small and elongated and osteocytic osteolysis appeared to be considerably reduced. In rats ovariectomized for 35 days, the epiphyseal plate was thicker than that of control rats and it contained a larger number of chondrocytes. Estradiol administration resulted in a 50% reduction from the control in the size of the plate and an increased azurophilia. A variable number of coarse, elongated spicules were present in the metaphyses of these animals. Magnesium deficiency did not appreciably alter the thickness of the plate in estrogen treated animals, but, as in all magnesium deficient rat bone, the epiphyseal plate also stained poorly with azure. 3.
*
*
DISCUSSION
Magnesium deficiency The results of these studies cmfirm the previously reported observations on the skeletal and extra-skeletal effects of magnesium deficiency in intact rats (Belanger, '58); Clark and Belanger, '67; Hunt, '71; Hunt and BClanger, '72). In addition to the general skeletal growth retardation, the formation of medullary trabeculae of endosteal origin and the subperiosteal hyperplasia described by Hunt and BClanger ('72), the present study also reveals other sites of skeletal hyperplasia at the endo-
44 1
steum and under the epiphyseal plate (figs. 4, 6, 7). One further observation of interest is the 30% increase in the general thickness of the femoral shaft of the magnesium deficient rat. The finding of an increased diaphyseal bone width was unexpected since it was thought that a severe nutritional magnesium deficiency would deprive osteoblasts of the function of a variety of enzymes dependent on magnesium for activation and thus would inhibit osteoblastic activity. Although not assessed by the more reliable labeling methods, osteoblastic activity did not appear, in these experiments, to have been substantially decreased. Furthermore, the femoral periosteal membrane showed signs of hyperplasia. In magnesium deficiency, the enzymes of bone resorption would also be deprived by the loss of magnesium ion, and this would contribute to a reduction in bone resorption. In the presence of normal osteoblastic activity, this decreased resorption would lead to an increase in bone width. The increase in width of the diaphysis in the femur (figs. 2, 5) and in the tibia (figs. 3, 4 ) is probably due to a combination of increased accretion and decreased resorption of bone. Recent studies by BClanger et al. ('72) suggested that the cells in the hyperplastic periosteal membrane were likely to be potential osteoblasts. This was based on the observation that, contrary to expectation, alkaline phosphatase was present in substantial quantity throughout the hyperplastic membrane. As an increased source of osteogenic cells, the hyperplastic periosteum would then account for one aspect of the increased bone width (i.e., accretion). The second aspect, that of decreased bone resorption, might, in fact, be a consequence of the inhibition of the normal rate of resorption in the femora of the magnesium deficient animal. Support for this concept was first presented by Clark and Belanger ('67) who reported a marked decrease in the phenomenon of osteocytic osteolysis in magnesium deficient rats. Osteocytic osteolysis also appeared to be considerably reduced in the present experiments. The relative contributions of increased accretion and 5 We are grateful to Dr. Barbara Hunt for the measurement of the lengths of the bones.
442
RITA BOGOROCH AND LEONARD BELANGER
decreased resorption to the final width of the diaphysis i n magnesium deficient animals awaits to be assessed. It has been known for some time that the skeletal magnesium content is decreased in magnesium deprived animals. I n a n anlysis of the mineral content of the long bones of young rats deprived of magnesium for three weeks, Hunt (unpublished) found that the magnesium content decreased 44% (from 680 mg./100 g. bone ash in control rats to 380 mg./100 g. ash in the deficient rats), whereas in adult animals the decrease, even after eleven weeks of magnesium deprivation, was never greater than 28% (Smith and Nisbet, ’72). At the same time, in young rats the calcium content increased from 37.4 g./100 g. bone ash to 42.4 g./lOO g. ash in the magnesium deprived animals (Hunt, unpublished); in adult rats no significant change in calcium content was noted (Smith and Nisbet, ’72). The large disparity between the Ca/Mg ratios in the long bones of control and magnesium deficient rats reflects the depletion of magnesium, probably from the surface of the bone. Indeed, the skeletal loss of magnesium has been explained by a specific process of depletion taking place by a n exchange process at the surface of the bone crystals (Blaxter, ’56). According to Blaxter, the whole skeleton may be involved since “in infant calves virtually the whole of the skeletal magnesium participates in the depletion and translocation process.” The large disparity between the Ca/Mg ratios in the long bones of control and magnesium deficient rats may also result from the breakdown of limited specific portions of bone and replacement by a newlyformed skeleton that would contain a n adequate amount of calcium but much less magnesium than normal. It is known that in sheep deprived of dietary calcium (Martin and Peirce, ’34) demineralization of the skeleton is not general. Trabeculae of the cancellous ends of long bones are more affected than the cortical shafts. The vertebrae, ribs and skull are also more rapidly depleted than the bones of the extremities (Duckworth and Hill, ’53). I n the experiments by Hunt referred to above and in the present series, the magnesium
deficient rats contained a greater amount than normal of bone i n their skeletons. A large amount of this bone, such as the medullary portion, was of the trabecular type. This trabecular bone has a rapid turnover rate (BClanger and Migicovsky, ’63). I n magnesium deficient rats the rapid turnover of trabecular bone and the replacement by adequate amounts of calcium and subnormal amounts of magnesium could also account for the shift in Ca/Mg ratios.
Ovariectomy and magnesium deficiency There appears to be no positive correlation between the weight gain of ovariectomized animals and bone growth. As expected, the bones of ovariectomized animals were longer than in intact rats (Silberberg and Silberberg, ’71). The diaphyses appeared thinner, but by measurement were not statistically different from those of intact animals. During the first week that the rats were fed the magnesium deficient diet no change in the increments of weight gain and in bone growth was detected. From then on, however, weight gain (fig. 1) and bone growth were rapidly curtailed. Although the widths of the femoral diaphyses were not appreciably altered by the magnesium deficiency, the subperiosteal hyperplasia was equal in frequency and, in some ovariectomized magnesium deprived rats, was more extensive than in intact deficient rats. Estrogen administration and magnesium deficiency A dose of 0.5-1 pg estradiol per 100 g. rat has been reported as “the minimal amount necessary to induce a significant increase in phosphofructokinase” ( Singhal et al., ’67a,b). If this dose is considered as physiologic, the dose of estradiol used in the present experiments was 10-20 times the normal physiologic requirements. This large dose was chosen intentionally because it causes rapid maturing of bone, As expected, a s soon as estradiol administration was initiated, the rats treated with this hormone no longer gained weight at the same rate as their non-treated ovariectomized counterparts. Instead, there was
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY
a n immediate loss of weight and only a minimal weight gain during the following weeks of the experimental period. After three weeks the final weight of the estradiol treated rats was approximately 15% less than the weight of untreated animals. Magnesium deficient rats treated with estradiol showed the same 15% final weight difference from the untreated magnesium deficient ovariectomized rats. Thus, when initiated i n combination with large doses of estradiol, magnesium deficiency per se did not alter the weight gain pattern of these rats. As for the combined effect of estrogen and magnesium deficiency on bone, it is not clear as to why the femoral diaphyses from rats treated with estradiol and fed a magnesium deficient diet did not show the hyperplastic response seen in untreated rats fed the same diet. Nor does there seem to be a simple explanation a s to why the incidence of subperiosteal hyperplasia in estradiol treated magnesium deficient rats is only 25% of that which occurs in untreated deficient animals. The maturation effect on bone of the large dose of estrogen appears to be implicated in the reduction of the lesions but the mechanism by which this reduction occurs remains to be elucidated. A possible speculation is that this dose of estradiol inhibits cell proliferation of the periosteal fibroblasts, as would a large dose of other steroid drugs. With respect to extra-skeletal lesions and symptoms, it is of interest to note that, despite its adverse effect on renal pathology, the administration of estradiol alleviated to a great extent both cutaneous and nervous disorders characteristic of the magnesium deficiency (table 1 ). It is clear from these studies that estrogen (estradiol) and ovariectomy, which affect bone maturation (Urist et al., '48), can also modify the incidence and severity of skeletal lesions and extra-skeletal lesions and symptoms. This potential benefit of estrogen therapy is now under investigation. ACKNOWLEDGMENT
The authors are indebted to the Medical Research Council of Canada for financial support (Grant MT 799).
443
LITERATURE CITED Belanger, L. F., D. Copp and M. A. Morton 1965 Demineralization with EDTA by constant replacement. Anat. Rec., 153: 41-47. BQlanger, L. F., B. J. Hunt and R. Narbaitz 1972 Alkaline phosphatase in hyperplastic bone lesions of rats fed a magnesium-deficient diet. In: Histochemistry and Cytochemistry. T. Takeuchi, K. Ogawa and S. Fujita, eds. Nakanishi Printing Co. Ltd., Kyoto, Japan, pp. 41-50. BBlanger, L. F., and B. B. Migicovsky 1963 Bone cell formation and survival in H3-thymidine-labeled chicks under various conditions. Anat. Rec., 145: 385-390. Blaxter, K. L. 1956 The magnesium content of bone in hypomagnesemic disorders of livestock. In: Ciba Found. Symp. on Bone Structure and Metabolism. G. E. W. Wolstenholme and M. O'Conner, eds. Little, Brown & Co., Boston, pp 117-134. Clark, I., and L. F. Belanger 1967 The effects of alterations in dietary magnesium on calcium, phosphate and skeletal metabolim. Calc. Tiss. Res., I : 204-218. Duckworth, J., and W. Godden 1941 The lability of skeletal magnesium reserves. The influence of rates of bone growth. Biochem. J., 35: 816-823. Duckworth, J., W. Godden and G. M. Warnock 1940 The effect of acute magnesium deficiency on bone formation in the rat. Biochem. J., 34: 97-108. Duckworth, J. and R. Hill 1953 The storage of elements in the skeleton. Nutr. Abstr. Rev., 23: 1-17. Hunt, B. J. 1971 Age and magnesium deficiencv in the rat with emuhasis on bone and muscie magnesium. Arne;. J. Physiol., 221: 1809-1817. Hunt, B. J., and L. F. BBlanger 1972 Localized, multiform, subperiosteal hyperplasia and generalized osteomyelosclerosis in magnesium-deficient rats. Calc. Tiss. Res., 9: 17-27. Kruse, H. D., E. R. Orent and E. V. McCollum 1932 Studies on magnesium deficiency in animals. I. Symptomatology resulting from m a g nesium deprivation. J. Biol. Chem., 96: 519539. Larvor, P., A. Girard and M. Brochart 1974 Etude de l a carence experimentale en mag&sium chez le veau. Ann. Biol. Anim., 4: 371382. Leroy, J. 1926 NCcessitQ du magnesium pour l a croissance de la souris. C. R. SOC. Biol. (Paris), 94: 431-433. Martin, C. J., and A. W. Peirce 1934 Studies on the phosphorus requirements of sheep. Coun. Sci. Ind. Res., Aust. Bull. No. 77, 1-46. Martindale, L., and F. W. Heaton 1964 Maenesium deficiency in the adult rat. Biochem. J., 92: 119-126. Silberberg, M., and R. Silberberg 1971 Steroid hormones and bone. In: The Biochemistry and
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RITA BOGOROCH AND LEONARD BELANGER
Physiology of Bone. Second ed. G. H. Bourne, ed. Academic Press, New York and London, pp. 401-484. Singhal, R. L., J. R. E. Valadares and G. M. Ling 1967a Estrogen-induced increase in phosphohexose isomerase activity in the rat uterus Metab. Clin. Exp., 16: 271-278. 1967b Metabolic control mechanisms in mammalian systems. I. Hormonal induction of phosphofructokinase in the rat uterus. J. Biol. Chem., 242: 2593-2598.
Smith, B. S. W., and D. I. Nisbet 1972 Biochemical and pathological studies on magnesium deficiency in the rat. 11. Adult Animals. J. Comp. Path., 82: 3746. Urist, M. R., A. M. Budy and F. C. McLean 1948 Species differences in the reaction of the mammalian skeleton to estrogens. Proc. SOC.Exp. Biol. and Med., 68: 324-326. Watchorn, E., and R. A. McCance 1937 Subacute magnesium deficiency in rats. Biochern. J., 31 : 1379-1390.
PLATE 1 EXPLANATION OF FIGURES
3 A portion of tibial diaphysis and proximal metaphysis in a normal (intact) control rat. Phloxine-Methylene Blue-Azure stain. x 43. 4 A comparable portion of tibia of a normal rat fed a magnesiumdeficient diet for 21 days. Notice periosteal hyperplasia ( P ) , diaphyseal thickness and medullary bone (MB). Phloxine-Methylene Blue-Azure stain. x 43. 5
A portion of femoral diaphysis of a normal rat fed a magnesiumdeficient diet for 21 days. In this particular instance, the wall is uniformly twice as thick as that in the control. Phloxine-Methylene Blue-Azure stain. x 43.
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY Rita Bogoroch and Leonard F. BBlanger
PLATE 1
445
PLATE 2 EXPLANATION OF FIGURES
6
Local hyperplasia, consisting mainly of bone growing from the endosteal surface of the tibial diaphysis in a normal rat fed the magnesium-deficient diet for 21-days. Phloxine-Methylene Blue-Azure stain. x 43.
'7 Fibrous nodule located under the greater trochanteric epiphyseal plate in the femur of a normal rat fed the magnesium-deficient diet for 21 days. Phloxine-Methylene Blue-Azure stain. x 43.
446
SKELETAL EFFECTS OF MAGNESIUM DEFICIENCY Rita Bogoroch and Leonard F. Belanger
PLATE 2
447
