Cherk S. Tam

Metabolic Bone Disease in Patients Receiving Long-Term Total Parenteral Nutrition

We have prospectively investigated calcium and bone metabolism in 16 patients receiving total parenteral nutrition for periods ranging from 7 to 89 months. In 12 patients, bone biopsies at 6 to 73 months after the start of parenteral nutrition showed osteomalacia. Plasma 25-hydroxyvitamin D levels were normal in all patients. Seven persons developed hypercalcemia, and 10 had hypercalciuria with a negative calcium balance. Serum phosphorus was normal and plasma parathyroid hormone level, normal or decreased. Three patients with the severest form of the disease had vitamin D withdrawn from their solutions. Subsequently, urinary calcium decreased, and serum calcium became normal; two persons reverted to a positive calcium balance. Thus, patients receiving total parenteral nutrition may develop metabolic bone disease characterized by osteomalacia, hypercalcemia, hypercalciuria, and a negative calcium balance. This may be caused by both defective mineralization and increased bone resorption induced by vitamin D, its metabolites, or another unrecognized factor.

A Possible Role of Vitamin D in the Genesis of Parenteral-Nutrition-Induced Metabolic Bone Disease

Patients receiving long term parenteral nutrition may develop metabolic bone disease. In all 11 patients studied, histologic studies of bone showed excessive unmineralized bone tissue despite normal plasma 25-hydroxyvitamin D levels. Three patients also had bone pain and fractures and severe urinary loss of calcium and phosphate. Withdrawal of vitamin D from parenteral nutrition solutions was associated with improved histologic findings of bone in all patients, shown by a decrease in osteoid tissue and an increase in tetracycline uptake. In the three patients with symptoms, bone pain subsided, fractures healed, and urinary loss of calcium and phosphate decreased. Thus, vitamin D may be a factor in the genesis of parenteral nutrition-induced metabolic bone disease.

Making Rats Rise to Erect Bipedal Stance for Feeding Partially Prevented Orchidectomy-Induced Bone Loss and Added Bone to Intact Rats

The objectives of this study were to investigate the different effects on muscle mass and cancellous (proximal tibial metaphysis [PTM]) and cortical (tibial shaft [TX]) bone mass of sham‐operated and orchidectomized (ORX) male rats by making rats rise to erect bipedal stance for feeding. Specially designed raised cages (RC) were used so that the rats had to rise to erect bipedal stance to eat and drink for 12 weeks. Dual‐energy X‐ray absorptiometry (DEXA) and peripheral quantitative computerized tomography (pQCT) were used to estimate the lean leg mass and bone mineral. Static and dynamic histomorphometry were performed on the triple‐labeled undecalcified sections. We found that making the intact rats rise to erect bipedal stance for feeding increased muscle mass, cortical bone volume, and periosteal bone formation. Orchidectomy increased net losses of bone next to the marrow by increasing bone turnover. Making the ORX rats rise to erect bipedal stance increased muscle mass, partially prevented cancellous bone loss in the PTM, and prevented net cortical bone loss in TX induced by ORX by depressing cancellous and endocortical high bone turnover and stimulating periosteal bone formation. The bone‐anabolic effects were achieved mainly in the first 4 weeks in the PTM and by 8 weeks in the TX. These findings suggested that making the rats rise to erect bipedal stance for feeding helped to increase muscle mass and cortical bone mass in the tibias of intact rats, increase muscle mass, and partially prevented cancellous and net cortical bone loss in ORX rats.

Tetracycline labeling of bone in vivo

SummaryWe have analyzed various aspects of tetracycline labeling technique for the measurement of bone apposition rate in vivo. Our efforts were restricted to those aspects that are frequently questioned when data obtained using this technique are interpreted as representing the rate of bone apposition. Rat bone was labeled in vivo by sequential injections of oxytetracycline at a dose range of 3 to 24 mg/kg body weight and at intervals ranging from 24 to 72 h. The bone apposition rate was calculated by measuring the distance from the first dose of label to the subsequent ones. As these distances are by far too small to be determined accurately by any available micrometer eyepiece, we have used a scanning microscope photometer which allows measurements on slow-forming sites that otherwise would have been considered nongrowing sites. Using these techniques, we have demonstrated that oxytetracycline has no effect on the bone apposition rate when used in the concentrations indicated. In addition, we found that at labeling intervals of 96 h or more, periods of osteoblastic inactivity are likely to be included in measurements at individual sites. The instantaneous apposition rate is thus underestimated at these long time intervals.

Parathyroid hormone stimulates dentin and bone apposition in the thyroparathyroidectomized rat in a dose-dependent fashion

SummaryThe dose-dependency of the effects of parathyroid hormone (PTH) on bone and dentin apposition after both intermittent and continuous administration of the hormone was investigated. The purpose was to compare the sensitivity of these two mineralizing tissues to parathyroid hormone and to provide additional information regarding the direct effect of PTH on mineralized tissue formation. Adult rats were thyroparathyroidectomized and 5 groups of 4 or 5 rats each were given daily subcutaneous injections with different doses of bovine parathyroid hormone. Five more groups of 4 rats each were administered equivalent dosages by means of a continuous infusion pump implanted subcutaneously. An additional group of 4 rats served as controls. All animals were labeled with tetracycline injected on days 7, 9, and 11. The animals were killed on day 12 and blood samples were collected for serum calcium determination. The lower metaphysis of the femur and the left and right mandibles were dissected out, and undecalcified sections of plastic-embedded tissues were prepared. The distances between the three tetracycline bands were measured to determine the amount of bone or dentin formation. Results indicated that both dentin and bone apposition increased with higher dosages of hormone. No overall effect of the method of administration was evident. For both methods, bone apposition showed a more pronounced increase over the control levels than dentin apposition. This suggests that, although both osteoblasts and odontoblasts appear to respond directly to PTH, differences do exist in the magnitude and dose-dependency of the response. No causal relationship was found between increases in serum calcium levels and either bone or dentin apposition at the lower dose levels.

Bone apposition rate as an index of bone metabolism.

The rate of bone apposition was studied by labeling bone with four or five sequential doses of oxytetracycline given at equal intervals of 2 or 4 days in rats and rabbits. The rate was estimated in bone formation sites bearing all the doses. This method has the advantage of assessing the rate of new bone mineralization during the active bone-forming phase only. This index of bone formation was found to be independent of the location of bone samples in the skeleton, and therefore might be under the influence of general body mechanisms only. Preliminary study in the rats indeed yielded results indicating that this bone apposition rate is accelerated by dietary deficiency in calcium.

Bone Formation in a Free, Living Bone Graft Transferred by Microvascular Anastomoses: A Quantitative Microscopic Study Using Fluorochrome Markers

Bone labeling studies with fluorochrome markers (oxytetracycline and DCAF) were carried out in fifteen dogs in which the posterior portion of the ninth rib was transferred, by means of microvascular anastomoses, to a defect fashioned in the mandible. Seven of the dogs received preoperative radiation of the mandible in order to study the performance of the revascularized graft in radiated tissue. In nine animals the bone grafts were labeled in the subperiosteal, the cortical and the endosteal parts, as opposed to the non-labeling of conventional free bone grafts. Determination of the linear bone formation rate in seven dogs (five non-radiated and two radiated), with the use of a scanning microscope photometer, showed no significant difference between the growth rate in the grafts and that in three other skeletal sites studied.

Plasma vitamin D metabolite levels in phosphorus deficient rats during the development of vitamin D deficient rickets

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.

Differences between the effects of phosphate deficiency and vitamin D deficiency on bone metabolism

It has been widely believed that phosphate deficiency causes osteomalacia. Based on this belief, the rickets of familial hypophosphatemia has been attributed to phosphate deficiency associated with the hypophosphatemia. The present studies on rats have, however, demonstrated significant differences between the effects of phosphate deficiency on bone metabolism and the characteristic features of rickets. Weanling rats, maintained on a mildly phosphate deficient diet, had hypercalcemia and hypophosphatemia, and impairment of body growth, bone growth, and bone mineralization. The maximum effect was observed at 5 wk; between 5 and 20 wk the rats improved despite persistent hypophosphatemia. Histologically, at 5 wk the bone showed thick unmineralized osteoid seams covering most bone surfaces, but the epiphyseal cartilage was normal. In addition, the excess osteoid readily incorporated tetracycline indicating normal mineralization and, based on a new sequential pulse labeling technique, the linear bone apposition rate (LBA) was significantly (p < 0.001) increased above control values. This increase was observed within the initial 4 days of phosphate (P) deficiency and persisted up to 15 wk. This effect of P deficiency on LBA was dependent on vitamin D activity. At 4 wk, the mean LBA was 0.106 +/- 0.003 (1 SE) in control rats, 0.149 +/- 0.008 microns/hr in P deficient rats, 0.083 +/- 0.004 microns/hr in vitamin D deficient rats and 0.086 +/- 0.006 microns/hr in rats deficient in both P and vitamin D. We have reported a similar increase in LBA with parathyroid hormone activity. With vitamin D deficiency, phosphate deficient rats showed all the characteristic features of rickets; disorganization of epiphyseal cartilage, excessive unmineralized osteoid, and reduced mineralization based on the incorporation of tetracycline. We conclude that the effects of phosphate deficiency on bone metabolism more closely resembles the effects of PTH activity than the characteristic effects of osteomalacia and rickets.

Skeletal response in rats following the implantation of hypercalcemia-producing Leydig cell tumors☆

The effect of Rice H-500 Leydig cell tumor tissue on bone in rats was assessed by morphometric analysis and tetracycline labeling of the lower femoral metaphyses. The rats in which tumor was implanted showed hypercalcemia, increased osteoclastic bone resorption, inhibition of bone formation with reduction in the bone apposition rate, and a loss in trabecular volume compared with the control rats. There was no evidence of tumor metastasis to bone. The results are consistent with the hypothesis that the Leydig cell tumor secretes a humoral factor capable of causing systemic bone resorption.