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The objective of the present study was to investigate the effect of changing skeletal Zn load (mobilization/restoring) on bone mineral composition and bone tissue metabolism. For this purpose, 36 65Zn-labelled, young-adult female rats were fed with either a purified diet with sufficient Zn (21 microg/g, control) for 26 days, or deficient Zn (1.4 microg/g) for 12 days followed by 14 days repletion with the control diet. The animals were killed at the onset of the study (reference: n=4), at the end of the Zn deficiency episode (control: n=4; Zn deficiency: n=4), subgroups (n=4) of Zn repleted animals at repletion days 2, 4, 7, 10 and 14, and at day 14 the remaining controls also (n=4). Zn deficiency reduced skeletal Zn concentration from 198 to 155 microg/g of bone dry matter. About half of mobilized skeletal Zn was refilled within 2 days of repletion and was completely restored until the end of the study. Concentrations of bone ash, Ca, P and Mg remained constant (means in bone dry matter: 51% bone ash, 191 mg Ca/g, 95 mg P/g, 4.4 mg Mg/g). Blood plasma concentrations of osteocalcin and daily urinary excretions of pyridinoline PYD and dexoxypyridinoline DPD were unaffected by treatment (mean: 57 ng/ml, 222 nmol/day, 137 nmol/day). Also daily urinary excretions of Ca, P and Mg remained fairly constant (means: 0.26 mg/day, 16 mg/day, 1.5 mg/day). 65Zn autoradiography of femur sections revealed a pronounced Zn exchange in the area of the metaphysis and epiphysis. We conclude that transient mobilization and restoration of skeletal Zn occurs mainly in trabecular bone, and does not involve major changes in bone mass, macro mineral content, or bone tissue turnover in young-adult rats.

Bone loss and long-term persistence of osteoporosis with increased fracture risk are common after liver transplantation. It is unknown whether transplantation-induced disruption of hepatic nerves, serving numerous regulatory metabolic and sensory functions, is herein involved. To test this possibility, we measured bone mineral density (BMD) by peripheral quantitative computed tomography (pQCT) and studied dynamic histomorphometry, radiocalcium kinetics, and biochemical parameters in 7 liver-transplanted and 7 sham-operated inbred rats. Although liver function was normal in TX rats, trabecular BMD of the first lumbar vertebra and total BMD of the femoral diaphysis were decreased by 13% and 6%, respectively, 9 months postsurgery. The breaking force of the femur was significantly lower by 21%. However, bone mass in the femoral and tibial metaphysis was preserved as evidenced by pQCT measurements and histomorphometry. Trabecular width and wall thickness were significantly decreased in vertebral cancellous bone, whereas indices of bone formation and resorption were normal or slightly reduced. Serum minerals, mineral balance, fractional and net absorption of Ca and Mg, serum calciotropic hormones, IGF-I, leptin, specific activity of 45Ca in bone, 45Ca excretion, and biochemical indices of bone formation and bone resorption remained unchanged. We conclude that liver transplantation-related denervation causes cancellous and cortical bone loss in well-innervated bone sites such as the lumbar spine and the long bone diaphysis. Cancellous bone loss in TX rats is due to an impairment of osteoblast team performance and subsequent trabecular thinning. The mechanism uncovered by our study may contribute to long-term bone loss after liver transplantation.

To determine whether an "atherogenic" diet (excess of cholesterol and neutral fat) induces pathological calcification in various organs, including the kidney, and abnormal oxalate metabolism, 24 male Sprague-Dawley rats were fed either normal lab chow (controls, n = 12) or the cholesterol- and fat-rich experimental diet (CH-F, n = 12) for 111 +/- 3 days. CH-F rats developed dyslipidemia [high blood levels of triglycerides, total, low-density lipoprotein (LDL)-, very low-density lipoprotein (VLDL)-, high-density lipoprotein (HDL)-bound cholesterol, total phospholipids], elevated serum total alkaline phosphatase and lactate dehydrogenase (LDH) levels, in the absence of changes in overall renal function, extracellular mineral homeostasis [serum protein-corrected total calcium, magnesium, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D)], plasma glycolate and oxalate levels. There was a redistribution of bone calcium and enhanced exchange of this within the extraosseous space, which was accompanied by significant bone calcium loss, but normal bone histomorphometry. Liver oxalate levels, if expressed per unit of defatted (DF) dry liver, were three times higher than in the controls. Urinary glycolate, oxalate, calcium and total protein excretion levels were elevated, the latter showing an excess of proteins > 100 kD and a deficit of proteins > 30-50 kD. Urinary calcium oxalate supersaturation was increased, and calcium phosphate supersaturation was unchanged. There were dramatically increased (by number, circumference, and area) renal calcium phosphate calcifications in the cortico-medullary region, but calcium oxalate deposits were not detectable. Electron microscopy (EM) and elemental analysis revealed intratubular calcium phosphate, apparently needle-like hydroxyapatite. Immunohistochemistry of renal tissue calcifications revealed co-localization of phospholipids and calcium phosphate. It is concluded that rats fed the CH-F diet exhibited: (1) a spectrum of metabolic abnormalities, the more prominent being dyslipidemia, hyperoxaluria, hypercalciuria, dysproteinuria, loss of bone calcium, and calcium phosphate nephrocalcinosis (NC); and (2) an interaction between calcium phosphate and phospholipids at the kidney level. The biological significance of these findings for the etiology of idiopathic calcium urolithiasis in humans is uncertain, but the presented animal model may be helpful when designing clinical studies.

Primary disturbances in mineral metabolism and deficiencies in insulin and insulin-like growth factor-I (IGF-I) have been implicated in the pathogenesis of diabetic osteopenia. This prompted us to investigate whether normal bone minerals and bone morphology are preserved after pancreas transplantation. To this end, 8 inbred rats (transplants) were compared with 9 sham-operated rats (controls) 20 months after orthotopic pancreas transplantation. While basal levels of insulin remained unaffected by transplantation, an oral glucose load elicited hyperinsulinemia (integrated incremental response: mean +/- SEM, 62+/-8 nmol l(-1) 60 min in transplants vs. 32+/-6 nmol l(-1) 60 min in controls; p<0.01) in the presence of normal glucose levels. Fecal and urinary excretion and fractional intestinal absorption of calcium, magnesium and phosphorus, net calcium absorption and the respective serum mineral levels were unchanged after transplantation, as were those of the calciotropic hormones. Serum osteocalcin and bone alkaline phosphatase remained unaffected, and urinary excretion of pyridinium and deoxypyridinium were unchanged. Fasting plasma IGF-I concentration was significantly decreased in transplants (930+/-42 ng ml(-1)) vs. control rats (1074+/-49 ng ml(-1); p < 0.05). Despite similar physical and chemical properties of bone in both groups, histomorphometry revealed slight osteopenia in transplant rats, as reflected by a 38% reduction in the cancellous bone area of the proximal tibial metaphysis. Plasma IGF-I levels were significantly correlated with bone mineral apposition rate (r=0.70, p<0.02), osteoblast perimeter (r=0.60, p<0.05) and osteoid perimeter (r=0.60, p<0.05). In conclusion, pancreas transplantation preserves physical and chemical properties of bone, but bone metabolism is not completely normal after transplantation, as evidenced by decreased cancellous bone. This might have resulted from the insulin resistance associated with the lowering of the plasma IGF-I level, which was correlated with the mineral apposition rate.

In humans, gastric surgery results in in osteopenia via mechanisms that are insufficiently understood; surgery-induced changes in the hormonal axes involving the stomach, thyroid, and the parathyroids may play a role. To study this in more detail, we evaluated calcium (Ca), magnesium (Mg), and phosphorus (P) metabolism as well as physical, chemical, and histomorphometric bone parameters in rats rendered hypergastrinemic by fundectomy (FX). In independent experiments, the response to an oral Ca challenge was investigated in intact rats versus FX, and in thyroidectomized versus thyroid-intact FX rats. Sixteen weeks following FX, body weight was approximately 80% that of sham-operated controls. In urine, P excretion was elevated fivefold, the pH was significantly decreased, and cAMP excretion was elevated as compared with controls; serum parathyroid hormone (PTH), calcitonin, 25OHD, Ca, Mg, and P were normal; gastrin and 1,25(OH)2D were elevated. On the basis of bone ash mineral content, FX rats developed significant osteopenia, and histomorphometry indicated only slightly elevated bone turnover and mineralization. Following oral Ca, thyroid-intact FX rats developed hypercalcemia, serum gastrin decreased, and calcitonin increased significantly; in thyroidectomized FX rats, calcitonin remained at baseline levels although there was a similar degree of hypercalcemia; PTH decreased during the hypercalcemic period in both groups. Serum gastrin did not correlate with calcitonin or PTH, and in multivariate regression analysis the only predictor of serum 1, 25(OH)2D was urinary phosphorus. It was concluded that in the FX rat (1) osteopenia is not caused by intestinal Ca malabsorption, vitamin D, Ca deficiency, or secondary hyperparathyroidism; (2) osteopenia may be related to PTH-independent urinary hyperexcretion of P, followed by a rise of serum 1,25(OH)2D; (3) the existence of endocrine axes among gastrin, calcitonin, and PTH cannot be substantiated. FX osteopenia appears to be related to gastric acid abolition, and the reactive hypergastrinemia probably stabilizes the mass and turnover of bone.

It is an important question for clinical therapy of osteoporosis with vitamin D metabolites whether these compounds exert their beneficial effects on the skeleton indirectly through an increase in intestinal calcium absorption or whether there is also a major direct component of action on bone. In this study, female 6-month-old Fischer rats were either ovariectomized (OVX) or sham operated. One month before surgery, all rats were placed on a diet containing 0.25% calcium and were kept on this diet throughout the study. Beginning 3 months post-OVX, groups of OVX rats orally received vehicle, a calcium supplement, low dose (0.025 microg/kg x day) or high dose (0.1 microg/kg x day) 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or combinations of low and high dose 1,25-(OH)2D3 with the calcium supplement. By 3 months postsurgery, pretreatment OVX controls had lost 74% and 37% of tibial and vertebral cancellous bone, respectively. Two-way factorial ANOVA showed that a 3-month treatment of osteopenic OVX rats with 1,25-(OH)2D3 dose dependently increased vertebral and tibial cancellous bone mass (P < 0.001 and P = 0.021, respectively) and trabecular width (P < 0.001). Furthermore, 1,25-(OH)2D3 increased serum calcium (P = 0.028) and urinary calcium excretion (P < 0.001) and reduced serum PTH levels (P < 0.001), osteoclast numbers (P < 0.001), and urinary collagen cross-links excretion (P < 0.001). Calcium supplementation alone was without therapeutic effect, and there was no significant two-way interaction between the individual treatment effects of 1,25-(OH)2D3 and calcium on bone mass. These data indicate that the anabolic effects of 1,25-(OH)2D3 in osteopenic OVX rats are mediated through a direct activity on bone.

The aim of this study was to investigate the skeletal effects of cyclosporin A (CsA) in a dose range relevant to clinical medicine in lumbar vertebral cancellous bone of aged male rats and to correlate these effects with possible changes in serum testosterone levels. Thirty-one 18-month-old male Wistar rats were divided into four weight-matched groups and subcutaneously injected with either 0, 1, 3, or 5 mg of CsA/kg of body weight three times per week After 4 weeks of treatment, all rats were killed after in vivo fluorochrome labeling and the first lumbar vertebrae analyzed by quantitative histomorphometry. Serum was analyzed for total calcium, creatinine, alkaline phosphatase, osteocalcin, parathyroid hormone, total testosterone, and CsA levels. CsA administration resulted in a dose-dependent increase in serum osteocalcin levels and in histomorphometric indices of cancellous bone turnover in the axial skeleton. Furthermore, CsA-treated rats showed a deterioration of vertebral cancellous bone structure with increased discontinuity of the trabecular bone network due to trabecular plate perforations. Serum testosterone levels were not significantly changed by CsA treatment and were uncorrelated to all biochemical or histomorphometric indices of bone turnover. We conclude that the 4-week administration of CsA at doses that are close to those used in transplantation patients induced high turnover osteopenia in the axial skeleton of aged, 18-month-old male rats, and that these effects were likely not mediated by changes in serum testosterone levels.

Methylmethacrylate (MMA) embedding of undecalcified bone biopsies is a technique widely used for bone histomorphometry. However, conventional MMA embedding causes almost complete loss of enzyme activity and protein antigenicity in the tissues. Recently, an MMA embedding technique has been reported that preserves enzyme activity and antigenic determinants in bone tissue. We describe here a modification of this embedding method. For our modified MMA embedding process, commercially available methacrylates can be used without purification, and the histologic quality of bone sections is comparable to that of conventionally MMA-embedded bone specimens. The technique reported here can be employed for embedding of larger bone samples and is suitable for histochemical and immunohistological applications as well as for routine bone histomorphometry. By addition of methylbenzoate during infiltration and polymerization of the plastic, the antigenicity of the tissue was improved. As applications of this novel technique, demonstration of alkaline phosphatase and tartrate-resistant acid phosphatase as well as positive labeling of Kupffer cells and osteoclasts with the monoclonal antibody ED1 in sections of liver, tibiae, and vertebrae of 3-month-old rats was demonstrated. The method described here might be useful for the inclusion of histochemical and immunohistological methods into bone histomorphometry.

Total gastrectomy (GX) in humans is frequently followed by osteopenia, but the details are unclear. The present investigations in the rat were aimed at elucidating its pathogenesis. Seventeen weeks after GX, we evaluated Ca, Mg and P metabolism as well as bone parameters, including fluorochrome-based bone histomorphometry. In GX rats, fecal Mg was increased, but intestinal absorption of P, Ca, and Mg was within normal limits, as was the urinary excretion of Ca, Mg, hydroxyproline and the pyridinium cross-links. In contrast, urinary P as well as cyclic AMP were significantly increased. In serum of GX rats, gastrin and 25-hydroxyvitamin D (25-OHD) were decreased, and Ca, Mg, P, parathyroid hormone (PTH), calcitonin, and the bone marker osteocalcin were normal, whereas 1,25-dihydroxyvitamin D [1,25(OH)2D] was significantly increased. GX rats had significantly reduced bone density and mineral content, severe high-turnover osteopenia, characterized by normal width but significantly decreased maturation time of osteoid, increased bone formation rate, and increased numbers of osteoclasts. We concluded that after GX (1) there is high-turnover osteopenia with normal mineralization and other histomorphometric features resembling those seen in states with hyperphosphaturia and subsequent hypervitaminosis D; (2) normal serum PTH levels and several indirect indicators of parathyroid gland function argue against the presence of (secondary) hyperparathyroidism, whereas increased bone mobilization due to elevated 1,25(OH)2D explains the maintenance of homeostasis of serum minerals, especially Ca, at the expense of bone mineral; (3) a complex interplay of mineral-metabolic effectors exists, among which low 25-OHD-PTH-independent renal phosphate losses, and high 1,25(OH)2D are prominent features. The presented animal model is recommended for future research in this area.