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The protein and lipid constituents of skeletal muscle subcellular fractions isolated from chicks fed a vitamin D-deficient diet for 3 weeks and chicks replated with cholecalciferol (vitamin D3) were analyzed. Administration of the sterol markedly altered the protein composition of mitochondria. The changes were localized in the inner membranes and consisted of a modification of the relative amounts of proteins of approximate mol wt of 83,000, 58,000, 42,000, and 34,000. In addition, treatment with vitamin D3 modified the distribution pattern of components of the actomyosin contractile complex. An increase in actin and troponin C was particularly noticeable. No differences between rachitic and treated animals were detected in the protein composition of sarcoplasmic reticulum membranes and postmicrosomal soluble fraction. A significant increase in the phospholipid content of sarcoplasmic reticulum (P less than 0.05), and to a lesser extent of mitochondria, was observed in repleted chicks. The relative proportions of individual phospholipids, however, were not changed. Injection of an acute dose of cholecalciferol to chicks less severely depleted in vitamin D significantly stimulated the incorporation of 32PO4 in vivo to muscle homogenates, mitochondria, and sarcoplasmic reticulum (P less than 0.05). As the increases in specific activities of sarcoplasmic inorganic P and membrane lipid P were similar whereas that of serum remained unchanged, the results are compatible with the idea that vitamin D3 stimulates phosphate fluxes across muscle membranes. The sterol produced minor modifications in the fatty acid composition of sarcoplasmic reticulum (P less than 0.05).

Four-week-old male Wistar rats were fed a low phosphorus diet without vitamin D and were injected with 1% lead acetate solution twice intravenously. Their tibias were then taken and decalcified in 5% formic acid solution saturated with hydrogen sulphate, in the cross-section of which the deposition of lead sulphate was observed under light microscopy as two concentric lines. The rate of matrix formation was calculated by measuring the distance of the two lines and compared with electron microscopic findings of the same sample. These results showed a decrease of the matrix formation rate and disturbance of mineralization in a low phosphorus diet. In the cortex of rickets, "resorption cavities" of various sizes were observed, and it was presumed that "resorption" came to the front with the progress of rachitogenic change, although whether resorption itself could be accelerated is still dubious.

Forty-eight infants of birthweight less than 1000 g who survived for more than 28 days, had wrist X-rays to prospectively determine the incidence of radiological rickets. Twelve infants (25%) had normal X-rays throughout, 10 infants (21%) showed osteopenia and 26 infants (54%) had classical changes of rickets of which 8 (17% of the total) had spontaneous fractures. There was poor correlation between peak values of serum alkaline phosphatase and the radiological changes.

The influence of anabolic steroid on the process of production of rickets in vitamin-D deficient animals as well as its influence on healing of rickets with vitamin-D therapy was studied. Long-Evans strains of female rats of two age groups (6 weeks and 4 weeks) were given rachitogenic diet and were divided into five sub-groups-(I) control receiving vitamin-D from the 1st day, (II) rachitic group, (III) receiving vitamin-D from 36th day, (IV) receiving anabolic hormone from the beginning of the experiment and (V) receiving both vitamin-D and anabolic hormone from 36th day of treatment. It was observed grossly as well as histologically and radiographically that: (1) the anabolic steroid minimizes the rachitic changes in the bones of vitamin-D deficient rats, (2) in rachitic animals, a combination therapy of anabolic hormone and vitamin-D results in comparatively greater calcification of osteoid matrix and better healing and remodelling of bone than with vitamin-D alone.

A histochemical and topo-optical study was made of the changes of the osteocyte capsule (OC) of the cortical and spongious bone layers of the tibia and vertebra of rats with hypervitaminosis-D and rickets. In hypervitaminosis-D the number of stained OCs was found to decrease. After treatment with high doses of vitamin D, a broadening of the OCs was noted in the area of demineralizing bone. Under the polarization microscope a decrease occurred in the proportion of birefringent OCs, more marked in birefringence than in staining. Since bone demineralization due to vitamin D was not followed by a rise in the number of osteoclasts, it is concluded that osteocytic activity has an important role in demineralization as shown by the broadened OC. In rachitic rats considerably fewer OC took the stain and showed birefringence than in the controls. Thus, alteration of the OC structure disturbs the nutrition of bone tissue and thereby aggravates the pathological process.

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.

Growth plate morphometry and measurements of serum chemistry were correlated to clarify the pathogenesis of hypocalcemic and hypophosphatemic avian rickets. Accumulation of proliferating and maturing cartilage in hypocalcemic chicks is accompanied by increased length and increased variation in length of perforating epiphyseal vessels, decreased number and abnormal arrangement of marrow spaces, an increased proportion of cells to blood vessels in marrow spaces, and a change in the distribution but not the total number of DNA-synthesizing chondrocytes per unit width of growth plate. Accumulation of hypertrophic cartilage in hypophosphatemic rickets is accompanied by no change in length, distribution, or number of perforating epiphyseal vessels, elongation but no change in number or arrangement of marrow spaces, an increase in the relative proportion of blood vessels to cells in the marrow spaces, and no change in distribution but a decrease in total number of DNA-synthesizing chondrocytes per unit width of growth plate. Both types of rickets have decreased amounts of calcified cartilage. These results provide further evidence that hypocalcemia and hypophosphatemia cause morphologically distinct types of rickets in birds. The data indicate that the thickness of the proliferating and maturing region and hence the distance of the hypertrophic zones from the epiphysis are anatomically and temporally related to length of perforating epiphyseal vessels and serum calcium levels. They indicate that in hypocalcemic rickets accumulation of proliferating and maturing cartilage is unlikely to be the result of increased chondrocyte replication and that the relative rates of chondrocyte hypertrophy and resorption of hypertrophic cartilage by marrow are equal. They support the concept that delayed chondrocyte hypertrophy is the major cause of growth plate thickening in hypocalcemic rickets. Data presented in this study, when considered together with data from the literature on hypophosphatemic rickets, support the long-held concept that growth plate thickening in this disease is caused primarily by a decreased rate of resorption of hypertrophic cartilage by marrow relative to the rates of chondrocyte proliferation, maturation, and hypertrophy. The data further support the concepts that growth of cartilage into marrow is a biphasic process including longitudinal growth effected mainly by blood vessels, and resorption of the lateral walls of marrow spaces effected mainly by marrow cells, and that it is the latter phase that is defective in hypophosphatemia.

Reported are the pathologic features of 17 mesenchymal tumors documented as causing osteomalacia or rickets. Although these tumors were histologically polymorphous, they were classifiable into four morphological groups. In the first group there were ten unique tumors showing mixed connective tissue features and containing variably prominent vascular and/or osteoclast-like giant-cell components. Tumors of this group also displayed focal microcystic changes, osseous metaplasia, and/or poorly developed cartilaginous areas. The cartilaginous areas sometimes showed considerable dystrophic calcification. With one exception, all tumors of this group occurred in soft tissue and demonstrated benign clinical behavior. The single malignant tumor originated in bone, recurred locally, and metastasized to lung. The tumors comprising the remaining three groups (six tumors) occurred in bone, demonstrated benign clinical behavior, and were grouped according to their close resemblance to tumors known to occur in bone, that is osteoblastoma-like (four tumors), nonossifying fibroma-like (two tumors), and ossifying fibroma-like (one tumor).

Studied were mass disease outbreaks on eight farms in calves intensively fed concentrate mixtures that were rich in phosphorus compounds. Clinically, there were selling and deformations of the joints with pains. The tarsal and carpal joints were chiefly involved. Kyphosis of the backbone, spastic paresis of the posterior part of the body as well as tetaniform spasms in the initial stage of the disease were also observed. Morphologically, the diseased animals manifested erosions and thinning of the joint cartilage, tearing of the Achilles tendon, and increased amounts of the joint fluid. The histologic study revealed edema of the cartilage cells, disorders in the structure of the hyalin cartilage, formation of cavities, and broadening of the lumen of the Haversian canals. In most cases the blood serum presented hyperphosphatemia (up to 15.67 mg% inorganic P) and relative or absolute hypocalcemia (up to 4.47 mg% Ca). On the farm both hypocalcemia and hypophosphatemia were observed. Good prophylactic results were obtained through correct Ca:P ratios in the diet that contained calcium additives as well as by the injection of vitamin D solutions, etc. It is believed that these cases should be referred to a distinctive form of rickets of a characteristic clinical course' the inadequate Ca:P ratios in the rations playing the main etiologic role. Subsidiary causes are probably vitamin D deficiency, restricted movement of the animals, and mechanical traumata caused by the animal's own bodyweight.

This paper reviews recent work in the authors' laboratories that has led to new observations and thoughts concerning the mineralization of normal and rachitic chick growth cartilage. The proximal tibial growth cartilages of normal and rachitic chicks were rapidly frozen and prepared for SEM and biochemical studies. Using a scanning microfluorimetric technique we showed that at the mineralization front of normal and rachitic cartilage there is an abrupt change in chondrocyte metabolism. Thus cells in this region exhibited an increase in NADH and oxidative metabolism. In rickets, there was a decrease in the reduced pyridine nucleotide content of each of the zones. The reversal in chondrocyte metabolism was not due to low oxygen tension. SEM observations indicated that this region of cartilage was well supplied with vascular channels; moreover, mineral was first seen deposited in matrix in close proximity to the blood supply. Indeed these vascular channels appeared to be a basic architectural feature of normal cartilage, although disorganized in the rachitic state. The morphological studies also showed that gaps existed in the continuity of the mineral phase in normal cartilage. Although the rachitic cartilage does mineralize, discontinuities in the mineral distribution are much more severe, with the general failure of fusion of adjacent mineral clusters. These structures would serve as pathways for transport of nutritional factors and gases to chondrocytes that are distant from the vascular channels. Observation of hypertrophic cells reinforced the view that some osteoblasts represented a terminal stage in the maturation of chondrocytes.

Day-old broiler chicks were fed a vitamin D-deficient diet for 30 days. Gross, microscopic, and radiographic examinations of the proximal tibiotarsus were done at 2, 3, and 4 weeks of age. In 2-week-old chicks, there was variable lengthening and disorganization of the proliferating-prehypertrophied zone (P-PHZ), resorption of cartilage spicules in the degenerating hypertrophied zone and primary spongiosa, and lengthening of bone spicules in the secondary spongiosa; many bone surfaces were lined with increased osteoid. In 3-week-old chicks, there was a tendency for the P-PHZ to decrease in relative length and for cartilage spicules to increase in length. In 4-week-old chicks, however, there was marked lengthening of the P-PHZ, resorption of cartilage spicules, and replacement of the metaphysis with irregularly oriented islands of woven bone, osteoid, and loose fibrous connective tissue.

Using routine transmission electron microscopy and light and electron microscopic techniques for the histologic demonstration (localization) of catalase (a peroxisomal enzyme), peroxisomes in chick duodenal epithelial cells were identified and studied. In these cells, peroxisomes were seen to be small, ovoid structures, delimited by a single unit membrane. They were concentrated in the supranuclear cytoplasm in initimate association with the smooth endoplasmic reticulum. As demonstrated histochemically, the heterogeneous matrix of these organelles was catalase positive. In addition, most of the larger peroxisomes revealed central nucleoids; however, the smaller peroxisomes were generally anucleoid. It thus appears that two classes of peroxisomes exist in chick intestinal absorptive cells: (1) small, anucleoid microperoxisomes, and (2) larger, nucleoid-containing peroxisomes. In addition to the above morphological characteristics, both peroxisome types were numerous in normal and vitamin-D-replete tissues, but were conspicuously decreased or absent from the apical cytoplasm of rachitic epithelial cells. From these observations it is hypothesized that these organelles may be involved in the overall vitamin-D response of the small intestine.

To examine whether either of the two known active vitamin D metabolites 1,25(OH)2D3 or 24,25(OH)2D3 could reverse the mineralization defect induced by 1-hydroxyethylidene-1,1-bis phosphonate (EHDP), a model of EHDP-induced rickets was used. Rats at the age of 31 days were injected for 10 consecutive days with EHDP (10 mg/kg). Other littermates were treated with a combination of EHDP and either 1,25(OH)2D3 or 24,25(OH)2D3 or were treated following 10 days of EHDP, with either of the vitamin D metabolites for an additional 72 hr. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscope studies of growth plate cartilage and bone. EHDP-treated rats revealed signs of typical rickets, manifested by widened growth plates and impaired bone mineralization. Transmission electron microscope (TEM) examination revealed matrix vesicles distributed throughout the growth plate; however, there appeared to be an arrest of the spread of the crystals at the provisional zone of calcification. Treatment with either 1,25(OH)2D3 or 24,25(OH)2D3 failed to reverse the rachitic condition of the animals. Serum calcium blood levels were elevated in the 1,25(OH)2D3 and EHDP-treated group. 1,25(OH)2D3 and 24,25(OH)2/D3 further increased the already elevated serum alkaline phosphatase levels observed in EHDP rats, although the increase observed with 1,25(OH)2D3 was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

A model of low-phosphate, vitamin D-deficient rachitic rats was used to compare the effects of 1 alpha(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3 on cartilage and bone. The rats were maintained for 3 weeks on a high-calcium, low-phosphate, vitamin D-deficient diet, during which period they developed severe rickets. The rachitic rats were injected for 2 or 3 consecutive days with a physiologic dose of either metabolite. Other littermates were given a single dose of 50,000 IU of cholecalciferol in combination with a normal diet. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscopic studies of the growth plate cartilage and bone. Treatment with 1 alpha (OH)D3 or with 1,25(OH)2D3, in spite of increasing Ca and P levels in the Cfl, induced only partial healing of the rickets. In contrast, 24,25(OH)2D3 or vitamin D with a normal diet resulted in complete morphologic and biochemical healing of the rickets. Transmission electron microscopic (TEM) studies have shown partial mineralization of the wide hypertrophic zone of the growth plate following treatment with 1 alpha(OH)D3 or with 1,25(OH)2D3. Mineralization was more complete with 24,25(OH)2D3 treatment. The results of this study emphasize the importance of 24,25(OH)2D3 for normal endochondral bone formation and mineralization.

Lung mechanics was studied at 50 days of age in 7 rachitic rats born from mothers deprived of vitamin D. They were compared with 7 control rats raised in the same conditions but fed a diet supplemented with vitamin D. The animals were anesthetized, tracheotomized, and paralyzed. Quasi-static pressure-volume curves of the respiratory system and of the lungs were obtained. Body weight of the rachitic rats was within the range of the control rats, but dry lung weight (LW) was significantly lower (p less than 0.01). Lung volumes in absolute terms and when normalized for LW were significantly lower than in the control rats. Chest wall compliance (Ccw) of the rachitic rats was within the range of values of the control rats, except for 2 animals with an infinite Ccw. Analysis of the pressure-volume curves of the lungs of the rachitic rats compared with those in the control animals showed a significant decrease in lung compliance (CL) and in CL/LW (p less than 0.01), indicating a decrease in lung distensibility. The more severe the rickets (according to microradiographic criteria of the tibia), the lower the CL/LW. It is speculated that decrease in lung distensibility may be related to abnormal lung growth caused by disturbed alveolar formation and lung connective tissue development. These abnormalities could be due to vitamin D deficiency acting on the growing lung, as on the growing bones, by a mechanism involving proteoglycans.

Rachitic rats, maintained on a diet adequate in Ca and P, were treated daily with varying amounts of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. At low levels (1 ng/day) 1,25-(OH)2D3 sustained a healing response equivalent to that of 25-hydroxyvitamin D3 (100 ng/day) or the parent vitamin. Above 5 ng/day administration of 1,25-(OH)2D3 resulted in an accumulation of osteoid, giving a histological appearance similar to vitamin D deficiency. The effects of 1,25-(OH)2D3 on bone did not correlate with changes in plasma Ca or inorganic phosphorus; doses that were effective in raising bone ash and reducing the amount of osteoid failed to normalize plasma Ca, whilst the amount of sterol required to normalize plasma Ca was excessive in terms of the effect on bone. 1,25-(OH)2D3 did not stimulate any of the histological parameters of bone resorption. We conclude that 1,25-(OH)2D3 can effectively heal the bone lesions of vitamin D deficiency, but that, at high concentrations, the sterol can inhibit mineralization. Furthermore, these results question the accepted role of 1,25-(OH)2D3 as a regulator of bone resorption in vivo.

Defective mineralization of bone and cartilage is the classical histological finding in vitamin D deficiency. Whether this represents a direct effect on mineral deposition or is a consequence of the decreased calcium and phosphorus levels that result from impaired intestinal absorption is not clear. A method has been developed in which vitamin D-deficient rats have plasma calcium and phosphorus levels maintained in the normal range by continuous infusion. Histomorphometric analysis of undecalcified tibiae from these animals was compared with that of rats given vitamin D. Epiphyseal growth plate thickness, trabecular osteoid volume, and mean osteoid seam width were not increased. Moreover, the administration of two time-spaced courses of tetracycline revealed that the mineralization rate and the time interval between apposition and subsequent mineralization of osteoid (mineralization lag time) were identical to those in rats treated with vitamin D. Trabecular bone volume was increased (osteosclerosis) in the vitamin D-deficient rats. In vitamin D-deficient controls without infusions, the osteosclerosis was mostly osteoid, whereas the excess bone was well mineralized in the vitamin D-deficient rats infused with calcium and phosphorus. Osteosclerosis in vitamin D-deficient animals may result from both decreased bone resorption and increased osteoid apposition. This study provides firm evidence that vitamin D is not essential for mineralization in young growing rats. Decreased availability of calcium and phosphorus thus may be the sole basis of the mineralization defect seen in vitamin D deficiency.

The mandibular condylar cartilages from normal and rachitic weanling rats were stained with ruthenium red to demonstrate the distribution of proteoglycan. In the rachitic animal there was an increased concentration of ruthenium-red positive granules in the pericellular region of chondrocytes from the extended hypertrophic zone when compared with the controls. These aggregates may represent unmodified proteoglycan which excludes calcium and thereby prevents matrix calcification.

Young boars supplemented orally with 800, 500,300 and 100 IU of vitamin D2/kg of feed and control boars not supplemented with vitamin D2 for four months developed mineralization in the left atrial endocardium, lamina muscularis mucosae of the fundic stomach and other sites. Since low levels of supplementation with vitamin D2 did not eliminate the lesions, the levels of vitamin D2 added appeared not to be involved in the pathogenesis. All boars had mild hypercalcemia throughout the experiment, and phosphorus levels in sera were lower in all animals receiving than in those not receiving calciferol. Mild morphological lesions of rickets developed in several boars not receiving supplemental vitamin D2 and in boars supplemented with 100 an 300 IU/kg of feed.

In the present study, we have evaluated the role of calcium and phosphorus concentrations in serum on the mineralization of bone in the absence of vitamin D. This was accomplished by feeding mother rats and subsequently their pups vitamin D-deficient diets varying in calcium, phosphorus, and lactose content. After 5-7 wk on these diets, serum concentrations of 25-hydroxyvitamin D [25(OH)D] and 1,25-hydroxyvitamin D [1,25(OH)2D] were undetectable. Rats fed a vitamin D-deficient diet containing 0.44% calcium and 0.3% phosphorus showed a serum calcium of 4.9-5.9 mg/dl and a serum phosphorus of 7.3-8.2 mg/dl; rickets (wide epiphysial plates) had developed as well as osteomalacia (wide osteoid seams). Rats maintained on a vitamin D-deficient diet containing 3% calcium and 0.65% phosphorus had normal serum calcium, low serum phosphorus, and severe rickets, but osteomalacia was not seen. Rats fed a diet containing 20% lactose, 4% calcium, and 1% phosphorus showed normal serum calcium, somewhat low serum phosphorus, normal serum PTH, normal width of the epiphysial plate, normal volume density of trabecular bone, and normal volume density of osteoid seams. These data confirm the findings of others, using a different experimental model, that serum calcium and phosphorus concentrations are the determining factors in mineralization defects and not the absence of 25(OH)D or 1,25(OH)2D. In these rats thyroparathyroidectomy is well tolerated, which makes for an ideal model for the study of the effects of calcium-regulating hormones on bone histology, cytology, and biochemistry.

The effect of Strontium (Sr) and Manganese (Mn) was studied on freshly isolated chondrocytes obtained from costal cartilage of young male rats. Cells isolated from rats rendered rachitic by oral exposure to Sr or Mn exhibited a decreased synthesis of glycosaminoglycans and collagen as judged by incorporation of 35SO4 and 3H proline. In vitro exposure of chondrocytes from untreated (control) animals to Sr or Mn at concentrations comparable to those found in cartilage from Sr and Mn rachitic rats, lowered the O2 consumption in both groups, but only Sr had any influence on the other parameters studied.

Plasma levels of the vitamin D metabolites were related to changes in bone morphology during the development of rickets in rats deprived of phosphorus and vitamin D. Weanling rats were studied at 1, 3, and 5 wk after onset of diets deficient in phosphorus or in both phosphorus and vitamin D. Bone histology and morphometry were carried out and measurements were made of 45Ca and 32P absorption, serum Ca and P, and plasma 25(OH)D3, 24,25(OH)2D3 and 1,25(OH)2D3. After 1 wk of vitamin D restriction, the plasma levels of 25(OH)D3 and 24,25(OH)2D3 were non-detectable (less than 0.5 and less than 0.8 ng/ml). The plasma 1,25(OH)2D3 level was elevated at 1 wk (105.5 pg/ml) and fell to 19 pg/ml by 5 wk. At 1 wk mild rachitic lesions in epiphyseal cartilage were observed despite the elevated 1,25(Oh)D3 level. Serum Ca and P levels and values for 45CA and 32P absorption decreased and the severity of the rickets increased with the fall in plasma levels of 1,25(OH)2D3. In Vitamin D replete, phosphate deficient rats the epiphyseal cartilage was normal throughout the 5 wk study period. Our results provide further evidence that physiological levels of 1,25 (OH)2 D3 will not prevent rickets without adequate plasma concentrations of either 25(OH)D3 or 24,25(OH)2D3.

This study involved comparison of the distribution and integrity of perforating epiphyseal and marrow vessels with the stage of development and integrity of chondrocytes and the distribution of insoluble calcium in the proximal tibial growth plate of 3-week-old vitamin-D3-deficient hypocalcemic chicks and 3-week-old D3-deficient chicks 12, 36, 72, and 120 hours after an oral dose of 10,000 units vitamin D3. The aim was to clarify the mechanisms responsible for chondrocyte hypertrophy and cartilage calcification in the avian growth plate. Within 12 hours after administration of vitamin D3, serum calcium levels rose to normocalcemic levels. The following morphologic changes were first recognizable at the times indicated. Distal portions of previously elongated perforating epiphyseal vessels and adjacent proliferative and maturing chondrocytes underwent necrosis by 12 hours. Chondrocyte necrosis was not preceded by hypertrophy. By 36 hours, vascular and chondrocyte necrosis involved large portions of the thickened proliferating and maturing zone, and perforating epiphyseal vessels were shortened to a normal length. By 72 hours, chondrocyte hypertrophy and calcification resumed around the shortened epiphyseal vessels. By 120 hours, marrow had removed the necrotic cartilage, and morphologically normal growth plate was restored, with perforating epiphyseal and marrow vessels, both ending in a narrow hypertropic cartilage zone. The results indicate that proximity of chondrocytes to perforating epiphyseal vessels is necessary for their viability, but loss of these vessels does not cause hypertrophy. Since hypertrophy and calcification both occur in the proximity of perforating epiphyseal vessels in normocalcemic animals but not in hypocalcemic animals, it is likely that the vessels influence hypertrophy and calcification by delivering calcium to chondrocytes.

Oral administration of manganese to young rats results in poorly mineralized primary spongiosa and an irregularly thickened growth plate with a histologic resemblance to that in vitamin D-deficiency rickets. In the present study, the rachitic lesions were characterized by stereologic methods at the light microscopic level. With increasing doses of Mn in the diet, the animals developed rachitic lesions of increasing severity, i.e., the total height of the growth plate and the relative volume of the hypertrophic zone increased. The experimental animals developed hypophosphatemia, which was dependent on the Mn dose. The observed serum concentrations of Mn and phosphorus are compatible with the idea that MnHPO4 is precipitated in the gut, leaving only small amounts of Mn and phosphate available for absorption. Furthermore, the severity of the rachitic lesions were inversely correlated to the concentration of phosphate in serum. The most important pathomechanism in Mn rickets is phosphate depletion, which per se causes similar rachitic changes, even though Mn also seems to have other effects. Starvation caused a decrease in the height of the growth plate and in the volume fraction of the hypertrophic zone, thus changes contrary to the rachitic lesions.

Two field cases of field rickets in turkey poults were studied. The primary observation was lameness, and the sole necropsy finding of significance was hypomineralization of bone. This finding was confirmed radiographically; thin cortices and wide growth plates were noted. Microscopically, there was failure of osteoid mineralization, failure of orderly progression from cartilage to bone in the growth plate, the formation of bony trabeculae perpendicular to the long axis of the bone, and fibroplasia in marrow spaces. On a routine serological screen for mycoplasma and several viruses, there were no differences between affected and normal poults from the same flock.

Measurements were made of duodenal calcium-binding protein (CaBP) on rats during development of rickets and, subsequently, following vitamin-D2 treatment. Results showed a poor inverse correlation between duodenal CaBP and rickets. In rats fed a phosphate-deficient rachitogenic diet, duodenal CaBP concentration finally fell below detectable limits, but CaBP was still readily measurable 2 weeks after rickets was clearly established. Following a massive dose of vitamin D2 (50 000 I.U.) to rachitic animals, CaBP was formed. However, a small dose of vitamin D2 (500 I.U. daily for 4 days) was insufficient to demonstrate CaBP synthesis than vitamin-D treatment alone. The rachitogenic diet supplemented with phosphate, which caused osteoporosis but not rickets, inhibited CaBP synthesis. The results suggest that nutritional deficiencies from the rachitogenic diet, in addition to vitamin-D deficiency, inhibited CaBP synthesis.

The structure of human dentine affected by familial hypophosphataemic rickets was investigated by polarized light and electron microscopy. Within the calcospherites both radially-orientated and collagen-orientated crystals were present, in approximately equal numbers. This was so, but less distinctly, in the calcospherites of normal dentine. It is concluded that dentine mineralizes entirely by formation of calcospherities which coalesce. It is proposed that matrix vesicles are probably responsible for the initiation of mineralization, and that growth of the calcospherites may depend on scavenging of inorganic pyrophosphate by the first-formed crystals. Growth of these crystals is thus poisoned but the resulting removal of inhibition allows deposition of further crystals by precipitation and by nucleation on collagen. The rachitic structure of dentine appeared to be due to slow calcospherite growth, probably as a result of the depressed serum phosphate characteristic of the disease.

The submicroscopic organization and activity of acid phosphatase and catalase in lysosomes and peroxisomes of the rat hepatocytes were studied with experimental rachitis. It is determined that total and free activity of acid phosphatase in the liver tissue and certain lysosomes with rachitis increases whereas the catalase activity in the tissue and certain peroxisomes decreases. Permeability of the lysosome and peroxisome membranes rises for enzymes and cations. The process of peroxisome differentiation with rachitis is disturbed: there appear peroxisomes containing the marginal plates, that is not peculiar to the redont peroxisomes.

When rachitic chicks are given 1,25-dihydroxyvitamin D3 in amounts as low as 50 ng/bird, the appearance of the duodenal mucosa is altered within 2 h of the administration of the hormone. The changes are most readily apparent on scanning electron microscopy and include: a more plump appearance of villi with loss of furrows and pits on their surfaces, elongation of villi and a smoother, more uniform microvillus surface. These changes occur within 2 h of the administration of the hormone and persist for as long as 24 h. The morphological change precedes the increase in calcium absorption induced by 1,25-dihydroxyvitamin D3 in the intestine. These observations suggest that 1,25-dihydroxyvitamin D3 may play an important part in maintenance of the structure of the duodenal mucosa.

The common marmoset, a New World monkey, requires a large amount of vitamin D3 to maintain its normal growth. This monkey is reported to have an end-organ resistance to 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). In this study, the bone morphology of marmosets fed a high vitamin D3 diet (intake of vitamin D3, 110 IU/day/100 g of body weight) was compared by X-ray and histological examinations with that of rhesus monkeys (Old World monkey) fed a normal diet (intake of vitamin D3, 5 IU/day/100 g of body weight). Three of 20 marmosets were found by X-ray examination to have osteomalacic changes in their bones despite the high daily intake of vitamin D3, whereas none of the 5 rhesus monkeys showed any signs of osteomalacia. Osteomalacic marmosets had distinct increases in osteoid surface, relative osteoid volume, and active osteoclastic bone resorption, whereas non-osteomalacic marmosets had no increase in osteoid tissues in their bones. None of the marmosets, either osteomalacic or non-osteomalacic, was hypercalcemic despite the extremely high circulating levels of 1 alpha,25(OH)2D3. However, the serum 25-hydroxyvitamin D3 (25OHD3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) levels were significantly lower in the osteomalacic than in the non-osteomalacic marmosets. These results suggest that the marmoset is likely to exhibit osteomalacic bone changes despite the high daily intake of vitamin D3. These changes resemble those in vitamin D-dependent rickets, type II.

Oral administration of manganese (Mn) to young rats results in morphologic changes in the growth plate histologically resembling rickets. Previous investigations have indicated that one important factor in the development of Mn rickets is phosphate depletion, because of the precipitation of insoluble manganese phosphate in the gut. In the present study, the morphologic and biochemical changes in rickets induced by phosphate (P) depletion per se as well as in florid and healing Mn rickets were investigated. Rachitic changes were induced in young rats by giving dietary Mn (2%) or by phosphate depletion (0.02% P) for 25 days. The rachitic changes of the proximal tibial growth plate were quantitated with the use of stereologic methods. In addition, the growth plates were dissected into one upper and one lower part and proteoglycans were separately extracted with 4 M guanidine hydrochloride and purified by ultracentrifugation. In rats given phosphate in more than equimolar amounts relative to Mn, the rachitic changes were abolished. When rats with florid Mn rickets were given standard rat food, containing 1% P, rapid healing ensued. Moreover, similar rachitic changes were obtained by phosphate depletion alone. Proteoglycans from the lowermost tissue portion of rachitic growth plates readily formed aggregates with hyaluronic acid and also contained larger chondroitin sulphate chains than controls. Upon healing, these changes tended to normalize. The data support the concept that phosphate depletion is crucial in the pathogenesis of Mn rickets. Also, it seems that the biochemical changes in different forms of rickets are of a similar kind, which further underlines the importance of proteoglycans in enchondral bone formation.

We examined collagens of both normal and vitamin D-deficient chick epiphyseal growth cartilage. Special emphasis was placed on the study of Type X collagen, a recently described product of hypertrophic chondrocytes. Scanning electron microscopy of the epiphyseal growth cartilage of vitamin D-deficient chickens showed an enlarged growth cartilage with a disorganized extracellular matrix. The cartilage collagens were solubilized by proteolytic digestion and disulfide bond reduction of both normal and rachitic growth tissues. Sequential extraction with neutral salt and acetic acid buffers followed by pepsin digestion at 4 degrees C solubilized about 12% of normal tissues and about 7% of collagen from rachitic growth cartilage. Treatment of the pepsin-resistant collagens with neutral salt-dithiothreitol buffer under nondenaturing conditions and a subsequent pepsin digestion increased the yield of solubilized collagen to greater than 95% of the total tissue collagen. Results of the biochemical studies showed a marked increase in the relative proportion of Type X collagen (from 5.6 to 27.9%), a corresponding decrease in the proportions of Types II and IX collagens, and a moderate increase in Type XI collagen in rachitic cartilage. Amino acid analysis indicated that there were no differences in the Types II and X collagens of normal and rachitic cartilage. However, an abnormality in the relative proportions of the CNBr peptides of Type X collagen was detected in the rachitic cartilage. We suggest that the increase in collagen in the rachitic state may reflect increased levels of Type X collagen synthesis by cells in the hypertrophic region. It is likely that in rickets the overproduction of Type X collagen may be a compensatory mechanism by which the hypertrophic chondrocyte attempts to provide a maximum area of calcifiable matrix for the calcium-depleted serum.