Robert R. Hardin’s research while affiliated with The University of Texas Health Science Center at San Antonio and other places

Three studies were carried out. First, the effects of aging on the maturation of the female skeleton were assessed. Second, the hypothesis that has linked ovarian hormone deficiency bone loss to hypercalcemic suppression of the parathyroids leading to a decrease in 1,25-dihydroxyvitamin D synthesis and gut absorption of calcium was examined. Third, the effects of ovariectomy and a combination of ovarian hormone deficiency and low dietary calcium on bone and the calcium-regulating hormones were evaluated. After 6 months, ovariectomy and a low calcium diet independently decreased the density of the ilium, the femur, and the fourth lumbar vertebra as well as the calcium content of the latter two. The effects of the two treatment regimens were additive and more marked in the vertebral bone. Ovariectomy lowered serum calcitonin only in animals fed a normal diet and had no effect on serum PTH and vitamin D metabolites, while a low calcium diet caused a significant increase in serum 1,25-dihydroxyvitamin D. In both dietary regimens ovariectomy resulted in about a 30% decrease in intestinal calcium absorption. A low calcium diet increased morphometric indices of bone formation and bone resorption as did ovariectomy, with resorption exceeding formation. The discussion of our findings led to the conclusion that the aged rat model of ovarian hormone deficiency bone loss qualifies for serious consideration as a practical convenient cost-effective animal model for exploring aspects of the pathogenesis and treatment of postmenopausal bone loss.

Aging caused an increase in serum calcitonin that was markedly suppressed by food restriction. Food restriction had no significant effect on gut calcium absorption, urinary calcium excretion, serum 1,25(OH)2vitamin D, blood ionized calcium, the clearance ofcalcitoninfrom plasma, and its rate of inactivation in vitro by the kidney and liver. In contrast, food restriction modulated an increase with age in calcitonin secreting C-cells, thyroidal calcitonin and thyroidal calcitonin mRNA pools. We conclude that the lowering of age-related increase in serum calcitonin levels by food restriction is likely because of lowered thyroid calcitonin pools rather than changes in the metabolism of calcium, which is the primary stimulus for calcitonin secretion. Our findings also suggest that food restriction decreases agerelated increase in calcitonin levels, at least in part, by modulating the expression of the calcitonin gene.

Studies were carried out to explore the influence of soy protein and food restriction on age-related changes in serum PTH and bone. Three groups of male Fischer 344 rats were studied from 6 weeks of age. Group A rats were fed ad libitum diet A, which has casein as the protein source. Group B rats were fed diet B (with casein as protein source) at 60% of the mean ad libitum food intake. Group C rats were fed ad libitum diet C, which has soy protein as the protein source. The animals were killed at periodic intervals beginning at 6 months of age after an overnight fast. Serum PTH, measured with an intact N-terminal-specific RIA, and immunoreactive calcitonin increased progressively with aging. The increase was markedly suppressed by food restriction, and in the case of PTH by the soy protein diet as well. Serum creatinine started to increase after 18 months of age, and both dietary regimens of groups 2 and 3 retarded the increase. Aging was associated with a fall in serum 25-hydroxyvitamin D, and loss of bone occurred during the terminal part of life in the ad libitum-fed animals. These were prevented by food restriction, while the soy protein diet delayed the onset of bone loss. We conclude from these findings and other data from this study that in the male F344 rats 1) an age-related increase in serum PTH precedes an age-related increase in serum creatinine concentration; 2) an age-related decline in renal function probably contributes to age-related hyperparathyroidism, which, in turn, contributes to senile bone loss; 3) food restriction inhibits age-related hyperparathyroidism and senile bone loss; 4) on the basis of the data from rats fed a soy protein-containing diet, a decline in renal function and progressive hyperparathyroidism are not inevitable consequences of aging in the ad libitum fed rats.

A new, sensitive parathyroid hormone (PTH) radioimmunoassay that appears specific for the intact hormone, and its validation for measuring rat PTH are described. The assay is based on antibody C2-7 from chicken immunized with bovine PTH; it has a detection limit of 6 pg of bPTH per assay tube and measures basal PTH in most rats; it is responsive to provoked changes in endogenous PTH concentration, and the intra-assay and inter-assay coefficients of variation are 6.0% and 7.2%, respectively. Multiple dilutions of rat serum and parathyroid gland extract, result in competitive inhibition curves that are parallel to that of highly purified bPTH. Under our assay conditions the C2-7 antibody cross-reacts well with intact PTH but synthetic fragments of the hormone (1-34bPTH, 1-34hPTH, 28-48hPTH, 44-68hPTH, 53-84hPTH) do not depress tracer (125l-bPTH) binding to the antibody. Studies designed to validate the assay gave predictable results such as enhanced secretion of the hormone in response to EDTA infusion, and failure to detect the hormone in serum following thyroparathyroidectomy. In addition, we made the novel observation that in F344 rats circulating immunoreactive PTH increases progressively with aging.

Studies were carried out on male F344 rats to examine the influence of aging and life-prolonging food restriction on bone and circulating parathyroid hormone levels. In ad libitum fed animals, the weight, density and calcium content of the femur increased with age and achieved their peak levels by 12 months of age. These levels remained stable until about 24 months and by 27 months of age the ad libitum fed animals had lost appreciable amounts of bone. The maturation of the femurs of the animals maintained on 60% of the ad libitum food intake was delayed and their bones were lighter, less dense and contained less calcium than bones from ad libitum fed rats of corresponding ages. But at 6, 12 and 24 months of age, the femur strength to body weight ratios were very highly significantly greater (P less than 0.0001) for the restricted animals compared to the ad libitum fed controls. Circulating immunoreactive parathyroid hormone increased progressively with aging in the animals fed ad libitum and the animals that experienced bone loss at advanced age also had the highest level of the hormone. In contrast, in the food restricted animals aging was not associated with a marked increase in serum parathyroid hormone or with senile bone loss. The data are discussed in relation to the mechanism of the observed changes.

Studies were carried out in rats to examine the role of calcitonin deficiency in the pathogenesis of ovariectomy-induced osteopenia. The parathyroid glands of 80 female Wistar rats were autotransplanted to their thigh muscle and the animals divided into 4 groups. Group 1 rats were sham ovariectomized, and thyroidectomized to make them calcitonin deficient; Group 2 rats were thyroidectomized, and ovariectomized to make them deficient in ovarian hormones as well; Group 3 rats were sham thyroidectomized and sham ovariectomized, and Group 4 rats were sham thyroidectomized and ovariectomized. A fifth group of rats were unoperated upon and served as controls. Thyroidectomized animals were maintained on thyroxine replacement and 11 months after ovariectomy all the animals were bled, killed and their femurs dissected out. In both the thyroid intact and thyroidectomized animals, ovariectomy decreased femur density significantly (P less than 0.01). Similarly, ovariectomy resulted in a decrease in femur calcium (P less than 0.01) in both groups of animals, and in a significant decrease in serum calcitonin (P less than 0.05) in the thyroid intact animals. We conclude from these findings that ovarian hormone deficiency can cause bone loss independently of lowering circulating calcitonin levels.

As a result of reports indicating that dehydroepiandrosterone (DHEA) has bone anabolic properties, studies were carried out to determine whether DHEA will prevent osteopenia due to ovarian hormone deficiency. Female Sprague Dawley rats were randomized into four groups: Group 1 rats were ovariectomized; Group 2 rats were sham-operated upon; and Group 3 rats were ovariectomized and given subcutaneous injections of DHEA five times per week. Groups 1 and 2 rats were injected similar volume of solvent vehicle, and Group 4 rats were killed at the beginning of the study to serve as baseline controls. Six months after ovariectomy, Groups 1, 2 and 3 rats were bled, killed and their femurs dissected out. The following observations were made. The mean food intake of the rats over the experimental period was 20.9 gm per rat per day for the control, ovariectomized and DHEA-treated animals. Compared to age-matched controls, ovariectomy resulted in an increase in body weight, and in a decrease in: plasma calcium (P<0.005), serum calcitonin (P<0.01), femur density (P<0.005), femur calcium (P<0.01) and the ratio of cortical to periosteal area (P<0.03). None of these changes was reversed by DHEA administration. When the changes in cross-sectional areas at the midpoint of the femoral diaphysis were expressed as percentage of the baseline levels, the medullary and cortical areas were increased 32% and 11% respectively in ovariectomized animals, and 14% and 17% respectively in the sham-operated controls. The changes in cross-sectional areas induced by ovariectomy were slightly augmented by DHEA administration: medullary area (32% vs. 35%), and cortical area (11% vs. 15%). Although we have confirmed that ovariectomy induces osteopenia in rats, our findings indicate that dehydroepiandrosterone does not prevent the bone loss.

The effects of aging on the calcemic action of parathyroid hormone were evaluated. In the first and second studies, 1-, 8-, 12-, and 22-month-old male Wistar rats were parathyroidectomized (PTX) or thyroparathyroidectomized (TPTX), injected with parathyroid hormone and bled at 0, 3, 6, 9, and 12 h and plasma calcium determined. In the third study, 2-and 15-month-old TPTX rats were injected with parathyroid hormone and bled at 0, 1, 2, 5, 15, 30 and 120 minutes and plasma immunoreactive parathyroid hormone measured. Since the rats were prestarved, subsequent rise in plasma calcium is due primarily to the effect of parathyroid hormone on bone. The greatest increase in plasma calcium in PTX rats occurred 12 h after the initial injection of parathyroid hormone and was (mean ± S.E.) 2.98±0.48, 1.90±0.24, 0.45±0.19 and 0.2±0.20 mg/100 ml for 1-, 8-, 12-and 22-month-old rats respectively. The peak levels of plasma immunoreactive parathyroid hormone in young and mature rats injected with the hormone were not statistically different. The kidneys of the oldest rats (22-month-old) showed no histological evidence of overt renal disease, and the plasma creatinine and urea of the rats did not increase progressively with age. In TPTX rats, the oldest animals were again much less responsive to the calcemic action of parathyroid hormone than the younger animals. However, the inhibitory effect of aging on the calcemic action of the hormone was more marked in PTX than in TPTX rats.