Infrared analysis of rat bone: age dependency of amorphous and crystalline mineral fractions.

Quantitative infrared spectrophotometric analysis of whole femurs from male rats demonstrates that anorphous calcium phosphate is a major component of bone mineral. The amount of amorphous calcium phosphate in whole bone decreases while the crystalline bone apatite increases during early stages of bone formation. Mature rat bone contains constant levels of both amorphous and crystalline calcium phosphate.

Effect of age and magnesium depletion on bone magnesium pools in rats.

In vivo and in vitro studies were carried out to characterize the exchangeable bone magnesium pool and determine what effect age and magnesium depletion has on bone magnesium. A highly significant correlation was found between the size of the in vitro elutable and in vivo exchangeable bone magnesium (r=0.97). To show that the exchangeable bone magnesium was the surface-limited bone magnesium, elution studies were performed 4 h after the in vivo administration of radiomagnesium. Specific activity in the eluant was 85% of that found in the serum at time of death, suggesting that the elutable and exchangeable bone magnesium pools were largely the same pool. Bone magnesium concentration fell with increasing age. The entire fall in bone magnesium was a result of a decrease in the surface-limited fraction. Since bone crystals have been shown to enlarge with aging with resulting contraction of the surface area, this would be the most apparent explanation for this finding. During magnesium depletion, magnesium concentration in both the exchangeable and nonexchangeable pools decreased. The fractional change in the exchangeable pool was much larger than the change in total or nonexchangeable bone magnesium, suggesting that the surface-limited magnesium pool is available during magnesium depletion. The change in size of the nonexchangeable bone magnesium pool appeared to be more related to the duration of magnesium depletion than the change in serum magnesium levels. The fall in magnesium concentration in this pool is probably a consequence of continuing formation of low magnesium bone during the depletion period.

Bone mineral density in Australia compared with the United States.

Bone density measurements are currently being performed throughout the world in the diagnosis and management of osteoporosis as well as in research into this major health problem. However, it is not clear to what extent bone mineral density (BMD) values determined by dual-photon absorptiometry at one center can be applied to another. This is particularly relevant for the quantitative comparison of results from studies carried out in different laboratories. Furthermore, many centers now acquiring densitometers may not have the resources to determine their own normal range, relying instead on a "normal" range provided by the manufacturer. The question of the comparability of BMD data obtained in different centers was examined by comparing the normal range for the lumbar spine and proximal femur in 203 normal white Australian women and 892 normal white U.S. women, obtained using the same model densitometer. The two populations were compared according to decade. From superimposition of the Australian individual values on the North American normal ranges, only minor differences between the two populations were seen at any of the sites measured at any decade. None of these minor differences were statistically significant. This study shows a close similarity between BMD values in both the proximal femur and lumbar spine in normal white women in Australia and North America, provided the same model densitometer is used. Thus data obtained from different centers in populations with similar ethnic composition may be compared directly. These findings provide for the first time a sound basis for the quantitative comparison of the at times conflicting studies carried out in widely differing settings around the world.

Influence of lactation and pregnancy + lactation on mechanical properties and mineral content of the rat femur.

The quality of bone was assessed from femurs of rats both during lactation and after pregnancy + lactation. Mechanical properties of stiffness, strength, toughness, and ductility were measured, along with standard measurements of dry weight, ash weight, and total bone mineral. No changes occurred during the first week of lactation. During the second and third weeks of lactation all bone parameters except ductility decreased significantly. These data are consistent with bone losing mineral in order to supplement the dietary calcium intake necessary for milk production. In other experiments, femurs were collected from nulliparous rats and from rats that had previously undergone 1-3 pregnancy + lactations. The largest changes in bone mineral and mechanical properties occurred after a single pregnancy + lactation period, although significant further decreases in stiffness and strength occurred after the second pregnancy + lactation. No additional losses occurred following the third pregnancy + lactation. Even 5 months after only one pregnancy + lactation period, the bone quality was still impaired as all bone properties were lower than in nulliparous controls. Because the changes, especially stiffness and strength, were relatively larger than the changes in dry and ash weights of bone, measurements of these mechanical properties provide a more sensitive method to evaluate the quality of bone.

The effect of age and menopause on bone mineral density of the proximal femur.

Although the menopause has been associated with increased bone loss at several skeletal sites, it has not previously been noted in the hip, yet estrogen therapy has been reported to reduce the incidence of hip fractures. We investigated the effect of age and menopause on bone loss in the proximal femur by measuring bone mineral density (BMD) of the femoral neck, Ward's triangle, and trochanter by dual-photon absorptiometry in 263 normal women aged 20-84. Multiple regression analyses revealed a significant decrease in BMD of the femoral neck and Ward's triangle with age in both pre- and postmenopausal women (p less than 0.001). In the trochanter the decrease with age was significant only in postmenopausal women (p less than 0.001). Further analysis revealed that BMD decreased faster at all sites in the early postmenopausal years. During the first 6 years postmenopause, the decrease in BMD of the femoral neck and trochanter was 3-10 times higher than the change in the decade prior to menopause. About 20% of the lifetime femoral neck loss and 30% of the trochanteric loss occurred in the early postmenopausal period. It is concluded that both age and menopause are major determinants of BMD in the proximal femur. These findings could explain why estrogen therapy has been reported to prevent hip fracture. The rapid early postmenopausal loss in BMD of the proximal femur demonstrates the importance of starting estrogen replacement therapy immediately after menopause for maximum effect.

Aging and dietary modulation of rat skeleton and parathyroid hormone.

Studies were carried out on SPF F344 male rats to evaluate the effects of aging and life-prolonging food restriction, without malnutrition, on rat skeleton and circulating PTH. Six-week-old F344 rats were divided into five groups. Group 1 rats were fed ad libitum a diet that contained 21% protein. Group 2 rats were fed 60% of the mean food intake of group 1 rats from 6 weeks of age for the rest of their lives. Group 3 rats were fed 60% of the ad libitum food intake until 6 months of age and then switched to ad libitum feeding. Group 4 rats were fed ad libitum until 6 months of age, and then switched to 60% of the ad libitum food intake. Group 5 rats were fed ad libitum a diet that contained only 12.6% protein so that these animals ingested the same amount of protein per day as the group 2 rats. In group 1 animals, bone length, weight, density, and calcium content increased rapidly with age and plateaued at about 12 months of age. There was no evidence of bone loss in these animals until about 24 months of age, but by 27 months, the animals had lost appreciable amounts of bone. The circulating immunoreactive PTH levels of the animals increased with advancing age, with a marked rise at 27 months. The age-related changes in bone and serum PTH levels of rats in groups 3 and 5 were similar to those of group 1 animals, except that a terminal increase in serum PTH did not occur in group 5 rats. In the groups 2 and 4 animals which were food restricted for the longest period, bone growth and maturation were slowed down, but the animals did not experience senile bone loss or marked terminal increase in circulating PTH. The salutary effects of food restriction were, therefore, not due specifically to the restriction of protein intake or to restricting food intake only during the period of rapid growth.

Hypotensive action of parathyroid hormone in hypoparathyroid and hyperparathyroid rats.

Experimental and clinical data suggest an association between chronic hyperparathyroidism and hypertension, but acute infusion of parathyroid hormone causes vasodilation and hypotension. These observations imply that chronic and acute parathyroid states affect blood pressure through different mechanism(s), either by modification of vascular receptors or by an ionophoretic effect of parathyroid hormone. The effect of parathyroid status induced by dietary calcium manipulations or by surgical ablation of the parathyroid gland on the hypotensive response of parathyroid hormone infusion was studied in rats. At 4 weeks of age 24 male rats were divided into four equal groups. Three groups were sham-operated, and one group was thyroparathyroidectomized. Only the thyroparathyroidectomized group was treated with thyroxine, 10 micrograms/kg/day. The control and thyroparathyroidectomized groups were raised on a 1.4% calcium diet; the other two groups were raised on 0.005% and 2.8% calcium diets. After 8 weeks on the diets, parathyroid hormone was infused through a venous cannula at 5 and 10 micrograms/kg doses and blood pressure was measured through arterial cannulas. The results indicate that hyperparathyroidism and hypocalcemia induced by the low calcium diet attenuated the hypotensive response to parathyroid hormone compared with responses in rats raised on a 1.4% calcium diet. In hypoparathyroid rats (2.8% Ca diet) with hypercalcemia, the hypotensive response was also reduced. However, in hypoparathyroid (thyroparathyroidectomized) rats with hypocalcemia, the hypotensive response was enhanced. The data suggest that chronic parathyroid status, as well as hypercalcemia, alters the hypotensive response to parathyroid hormone infusion, presumably by altering the vascular parathyroid hormone receptors or by some other mechanism.

Skeletal and body-composition effects of anorexia nervosa.

Eleven female patients (aged 18-46 y) with anorexia nervosa were measured by use of dual-photon absorptiometry for 1) bone mineral content (BMC, in g) and bone mineral density (BMD, in g/cm2) of the total skeleton and its regions, 2) BMD of the lumbar spine and the proximal femur, and 3) total body soft-tissue composition. The patients weighed 44.4 kg, approximately 15 kg less than normal peers (n = 22). The fat mass (3.35 kg) and content of soft tissue (7.8%) were four and three times lower (p less than 0.001) respectively, than those in normal women (15.1 kg and 26%, respectively). The total skeleton mineral (1921 g) was approximately 25% less than that of young normal women. The BMC as a fraction of the lean tissue mass was approximately 4.9% in the patients and 5.9% in normal women. Total body and femoral BMD averaged only 10% and 13% lower than those of normal women, respectively; however, spinal BMD was particularly reduced (approximately 25%, p less than 0.001).

Tissue zinc status of genetically diabetic and streptozotocin-induced diabetic mice.

A structural and functional relationship exists between zinc and insulin. In the present study zinc concentrations of various tissues from genetically diabetic and streptozotocin-induced diabetic mice and their appropriate control mice were determined. The zinc concentrations were depressed in serum and femur of C57BL/Ks-db+/db+ mice (db/db) when compared with their nondiabetic heterozygote controls (db/m) and homozygous controls (m/m). No differences were noted in the hepatic or renal Zn concentration of the db/db, db/m, or m/m mice. Zinc supplementation in the drinking water for a 4-wk period had no effect on serum or tissue zinc concentration. Hyperzincuria was noted in the db/db mice. No differences were noted in the Zn concentration of serum or tissue in streptozotocin-induced diabetic mice compared to their controls. These data suggest that zinc deficiency may play a role in the pathogenesis of the insulin resistance present in type II (insulin independent) diabetics.

Distribution of lamellae in human femoral shafts deformed by bending with inferences on mechanical properties.

On the basis of previous investigations indicating that the distribution of osteonic and interstitial lamellae in a normal femur depends on its mechanical properties, a procedure has been devised to provide information on the distribution of lamellae in human femora diaphyses deformed by bending. To achieve this, exactly plane parallel cross sections, 100 micron thick, were prepared from the portion of maximum bending, using an annular blade microtome. An index of the distribution of longitudinal lamellae (whose fiber bundles and crystallites have a longitudinal course and withstand loading by tension) and transverse lamellae (whose fiber bundles and crystallites have a transverse course and withstand loading by compression) was determined using circularly polarized light as illuminating source and a Quantimet 720 image analyzing computer. The results show that, even in a pathologically deformed bone, both the microscopic structure of sections at the level of osteonic and interstitial lamellae, and their macroscopic shape may be governed by the distribution of the forces active in bone.

The effect of lactation on the mineral distribution profile of the rat femur by single photon absorptiometry.

The distribution of bone mineral in excised femurs from lactating and nonlactating control rats was localized and quantitated using single photon absorptiometry. Bone mineral content was measured proximodistally in nine sequential 0.32 cm increments along the femur length and then plotted as a function of the distance from the proximal end of the femur. The plots described a mineral distribution profile and revealed that bone loss following lactation was site specific, being greater in the metaphyseal regions (23-36%) than in the diaphyseal area (14-20%). The change in total bone mineral of femur caused by lactation was estimated by integrating the mineral distribution profile plots. The 28% lower bone mineral determined by this method was in close agreement with the 27% difference determined from femur dry weight measurements. Reproducibility of measurements with 10 repositionings of a single femur was within 4% at all but the 2 most distal sites. Variation in the estimated total bone mineral was 1.3%.

Increased mechanical strength of the vitamin D-replete rat femur by the treatment with a large dose of 24R,25(OH)2D3.

The mechanical properties of the rat femur treated with a large dose of 24R,25(OH)2D3 were examined and their relationship with the mass and mineral contents of the bone were investigated. Male Wistar rats were fed diet containing 0, 0.025, 1.25, 4.0, or 12.5 ppm 24R,25(OH)2D3 for two years starting six weeks after birth. The rats were killed and their right femurs were removed. The adhering soft tissue were stripped off. Radiographs were made of the femur and its bone mineral content was measured by single photon absorptiometry. Then a three-point bending test was done with pressure exerted in the plane of natural extension. After mechanical testing, non-decalcified cross-sections of the femur were prepared at the mid diaphysis as close to the test fracture site as possible. On x-ray images, the cortical thickness was clearly increased in groups treated with larger doses, and the mid-cortical segmental mineral content of the femur increased dose-dependently to about 150%. The mechanical parameters in the treated animals also increased significantly, strength to 120% (p less than 0.01), energy-absorption capacity to 124% (p less than 0.05), and structural stiffness to 183% (p less than 0.01). Segmental bone mineral contents showed the positive correlations with strength and structural stiffness in both the control and the 24R,25(OH)2D3 treated groups as well. We concluded that the increase in the bone density in animals administered high doses of 24R,25(OH)2D3 was accompanied by an increase in mechanical strength of the bone.

Femoral bone density in young male adults with stress fractures.

Femoral bone mineral density (BMD) was measured by dualphoton absorptiometry in 41 young military recruits who had one or several stress fractures, during their physical training program. These fractures involved the following locations: Femur (neck: n = 10, diaphysis: n = 2), calcaneus (n = 10), tibia (n = 8), fibula (n = 3), metatarsus (n = 8). The stress fracture group generally had a lower bone density than that of a control group, consisting of 48 young military recruits matched for age, height and weight. However, the BMD was significantly lower (-10%) in patients with femoral and calcaneal locations, but it did not differ for other locations. To determine the possible effect of this intense physical activity on bone mineral mass, bone mass was measured again in 35 subjects from the control group at the end of their training. The BMD remained stable or increased in 28 subjects, but decreased significantly (greater than 2%) in 7 subjects, demonstrating the individual variability in the adaptation of bone to this stress. Our results suggest that lowered bone mass could be a factor that encourages the development of stress fractures (femoral and calcaneal) in young subjects submitted to intense physical activity to which they are not accustomed.

Accuracy of dual photon absorptiometry in excised femurs.

We investigated the accuracy of assessment of bone mineral content (BMC) by dual photon absorptiometry (DPA). Measurements were compared between BMC and ashed weight using two related scanners. The BMC in different locations of the femur was determined. Twelve cadaver femurs were cleaned of all soft tissue, divided into four parts (head, neck, trochanteric region, and shaft), and measured for BMC in an ethanol/water solution. The bones were then ashed and weighed. Volumetric density was also determined. The correlation coefficient between ash weight and BMC was 0.99 with an s.e.e. of 0.51 g and relative error of 4.8%. Similar correlations were seen within each region. The correlation between the machines was 0.99. Differences in volumetric density were found, with the density of the shaft greater than other regions, and the neck greater than the head or trochanteric regions.

Diagnosis of osteoporosis: usefulness of photon absorptiometry at the radius.

The photon-absorption method (Cameron-Sorenson) is commonly used in the diagnosis of osteoporosis to measure bone mineral in appendicular bones. Although this method gives accurate and reproducible results when applied to the distal radius and midradius, separation between osteoporotic patients and age-matched normals was less than satisfactory because of a large overlap. By contrast, a radiographic index based on the pattern of trabecular bone at the proximal femur (Singh index) gave a better separation between the two populations. The Singh index discriminates better, probably because osteoporotic patients have a greater proportion of loss of trabecular bone of the axial skeleton than of cortical bone of the appendicular skeleton.

Comparison of bone density measurements from different skeletal sites.

Absorptiometric measurements from multiple sites in 212 consecutive patients were examined to determine the interrelationships among bone mineral content values obtained from the lumbar spine, hip, and forearm. Dual photon absorptiometry was used to examine the spine, femoral neck, Ward's triangle, and greater trochanter while the radius and ulna were studied with single photon absorptiometry. All studies were performed on the same day. Concurrent measurements were available for the spine and hip in 197 patients, for the spine and forearm in 151 patients, and at all three sites for 146 patients. Variable degrees of correlation were found among the mineral content values from the six sites, with r values ranging from 0.40 to 0.93. The femoral neck and Ward's triangle showed the highest degree of correlation (r = 0.93); the ulna and lumbar spine demonstrated the poorest correlation (r = 0.40). Relationships between distant anatomic sites exhibited a large amount of variability (large standard error of the estimate) even when highly correlated. Useful predictions of bone mineral content could be obtained only for sites in close proximity. These results suggest that bone mineral content determinations in the upper extremities by single photon absorptiometry may not be useful for predicting mineralization in clinically more significant areas such as the lumbar spine and hip.

Heparin-induced osteoporosis in rats.

In order to study the effect of heparin in inducing osteoporosis, 30 female rats were divided in two groups and treated with daily injections of 2 IU heparin/g body weight for 33 and 65 days and compared with the same number of rats acting as controls. The mineral bone mass in the femora of the animals was measured quantitatively. A significant (p less than 0.001) reduction in bone mineral mass was found in the heparin-treated animals. This effect was present to the same degree after 33 days as after 65 days of treatment. It is concluded that heparin in this dose causes osteoporosis in rats after 33 days and that the described method can be used as an experimental model for further studies on this topic.

The distribution of lithium and its effects on the distribution and excretion of other ions in the rat.

1. In rats, lithium (ca 1 mEquiv/kg body weight) decreased brain sodium and magnesium, bone sodium and calcium and increased muscle calcium, plasma magnesium, urinary calcium and urine volume.2. Lithium was particularly concentrated in bone.

Quantitative computed tomographic evaluation of femoral bone mineral content in renal osteodystrophy compared with radial photon absorptiometry.

The computed tomography (CT) numbers of cortical bone at the level of 20 cm (CT20) and of spongiosa in the lateral condyle at the level of 2 cm (CT02) from the distal end of the femur were obtained by a quantitative CT method and compared with the bone mineral density of mostly cortical bone within the radius (BMD) by photon absorptiometry. The study included 47 patients with chronic renal failure not dialyzed or induced to regular hemodialysis within 4 weeks of the study (group 1), 28 patients on regular hemodialysis for more than one month (group 2), and ten healthy volunteers (group 3). The measures of bone mineral content (BMC), namely CT20, CT02, and BMD, were compared in terms of their abilities to distinguish members in the various groups. For group 1 and group 3, the greatest variation in BMC was in the difference in CT02, which was primarily a measurement of the BMC of spongiosa. For groups 1 and 2, the greatest variation was in the difference in BMD, which was primarily a measurement of the BMC of cortex. The reproducibility of CT02 was estimated as almost equal to the difference in CT02 values at intervals of 10 months' duration of hemodialysis. The results indicated that CT02 was a useful measurement for evaluating the progress in the early stage of the renal osteodystrophy, and it is recommended that the bone mineral measurement with this QCT method should be performed once or twice a year.

Beta 2-microglobulin amyloidosis (AB2M) in patients undergoing long-term hemodialysis. A new type of amyloid.

Amyloidosis has been increasingly recognized in association with renal failure and chronic hemodialysis. This report describes three patients who had long-term hemodialysis (between 7-18 years), in whom deposits developed of a new type of amyloid of beta 2-microglobulin origin. Beta 2-microglobulin amyloid (AB2M) was found in multiple organs, i.e., bone, subendocardium, gastrointestinal blood vessels, tongue, and carpal tunnel connective tissue. AB2M displayed characteristic amyloid features on conventional light and polarized microscopic examination after congo red staining. However immunostaining with anti-amyloid A protein, kappa, and lambda antisera were negative. The studied material reacted positively with beta 2-microglobulin antisera, identifying AB2M in all three cases. Ultrastructural study revealed an unusual curvi-linear fibrillar configuration. AB2M appears to be a new subtype of systemic amyloidosis secondary to renal failure and long-term hemodialysis.