The effect of cyclosporin A administration and its withdrawal on bone mineral metabolism in the rat

The in vivo administration of cyclosporin A (CsA) has been associated with significant bone loss and increased bone remodeling. To observe whether these changes are reversible, we have investigated the consequences of the administration and withdrawal of CsA immunotherapy on bone mineral metabolism. Three groups of Sprague-Dawley rats were studied for 28 days. Group A received vehicle, and group B received CsA (15 mg/kg BW) for 28 days, while group C received CsA (15 mg/kg BW) for 14 days and then vehicle from days 15-28 by daily gavage. Serum ionized calcium (Ca2+), PTH, bone gla protein, blood urea nitrogen, creatinine, magnesium, phosphorus, and 1.25-dihydroxyvitamin D were measured weekly. Bone histomorphometry was analyzed on days 14 and 28 in groups A and B and on day 28 in group C. CsA administration resulted in reversible hypomagnesemia and mild transient elevation in circulating 1,25-dihydroxyvitamin D levels with no change in Ca2+, PTH, blood urea nitrogen, or phosphorus. Serum bone gla protein levels were significantly increased (P less than 0.002) during CsA therapy, but tended to return to control values after CsA withdrawal. Enhanced bone remodeling and significant trabecular bone loss accompanied CsA administration. Withdrawal of CsA resulted in all of the histomorphometric parameters, except for bone volume, returning to control values within 2 weeks. Incomplete restoration of bone volume occurred 5 weeks after CsA withdrawal. This limited restoration of bone volume despite CsA withdrawal may have important clinical implications.     

Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats

Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham surgery (control). Groups of ovariectomized (OVX) and control rats were injected daily with low, medium, or high doses of 17 beta-estradiol (10, 25, or 50 micrograms/kg BW, respectively). An additional group of OVX and control rats was injected daily with vehicle alone. All rats were killed 35 days after OVX, and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Trabecular bone volume was markedly reduced in vehicle-treated OVX rats relative to that in control rats (12.1% vs. 26.7%). This bone loss was associated with a 2-fold increase in osteoclast surface and a 4-fold increase in osteoblast surface. The bone formation rate, studied with fluorochrome labeling, was also significantly elevated in vehicle-treated OVX rats (0.111 vs. 0.026 micron3/micron2.day). In contrast, treatment of OVX rats with the three doses of estradiol resulted in normalization of tibial trabecular bone volume and a decline in histomorphometric indices of bone resorption and formation. Our results indicate that estrogen treatment provides complete protection against osteopenia in OVX rats. The protective mechanism involves estrogenic suppression of bone turnover. These findings are consistent with the skeletal effects of estrogen therapy in postmenopausal women.     

17 beta-estradiol prevents osteopenia in the oophorectomized rat treated with cyclosporin-A.

Cyclosporin-A (CsA) administered to the oophorectomized rat exaggerates the high turnover osteopenia associated with oophorectomy alone. This study investigated whether 17 beta-estradiol replacement could influence the development of osteopenia in the oophorectomized rat treated with CsA. Ninety female Sprague-Dawley rats, approximately 300 g in weight, were divided into 6 groups of 15 each and treated according to the following protocol: group A were sham operated (control), group B underwent oophorectomy (Ox), group C underwent Ox and received CsA (15 mg/kg, by daily gavage) for 28 days (Ox and CsA), group D underwent Ox and received CsA and a 0.1 mg 17 beta-estradiol pellet (EP) implanted sc (Ox, CsA, and EP), group E underwent Ox and received EP (Ox and EP), and group F, which was intact and nonoperated, received CsA (CsA). Rats were weighed and bled on days -7, 0, 7, 14, 21, and 28 for measurement of blood ionized calcium, bone Gla protein (BGP), 17 beta-estradiol, and PTH. Bone histomorphometry was determined after double tetracycline labeling. On day 28, serum 17 beta-estradiol was undetectable in groups B (Ox) and C (Ox and CsA), and similar to group A (control) in groups D (Ox, CsA, and EP), E (Ox and EP), and F (CsA). Oophorectomy resulted in a significant gain in weight in groups B (Ox) and C (Ox and CsA), which was prevented by 17 beta-estradiol in groups D (Ox, CsA, and EP) and E (Ox and EP). On day 28, serum BGP levels were higher in groups B (Ox; 73.58 +/- 3.63 ng/ml), C (Ox and CsA; 85.05 +/- 9.88), and F (CsA; 81.35 +/- 3.4) compared to group A (control; 46.07 +/- 5.52; P less than 0.05), while 17 beta-estradiol in groups D (Ox, CsA, and EP; 38.65 +/- 2.85) and E (Ox and EP; 41.89 +/- 1.89) prevented this rise (P = 0.01). BGP levels in group C (Ox and CsA) were higher on days 14 and 21 compared to group B (Ox). Groups D (Ox, CsA, and EP) and E (Ox and EP) had elevated blood ionized calcium levels from day 7 onward. Serum PTH levels were unchanged. Histomorphometric analyses of tibial metaphyses revealed increased parameters of bone formation and resorption, with significant loss of bone volume, in groups B (Ox; 12.03 +/- 1.40%) and C (Ox and CsA; 11.43 +/- 2.20) vs. group A (control; 24.36 +/- 2.37; P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Advanced glycation end-products pentosidine and N epsilon-carboxymethyllysine are elevated in serum of patients with osteoporosis

OBJECTIVE: To investigate serum levels of the advanced glycation end-products (AGEs) pentosidine and N epsilon-carboxymethyllysine (CML) in patients classified into different osteoporosis subgroups according to histomorphometric data. METHOD: Serum samples were obtained from 116 osteoporotic patients (34 men, 82 women) classified by bone histomorphometry into subgroups with high turnover (HTO, n = 32), low turnover (LTO, n = 39), normal turnover (NTO, n = 9) and cellular uncoupled osteoporosis (CUO, n = 36). Pentosidine was measured by high-performance liquid chromatography, and CML by a competitive enzyme-linked immunoassay. RESULTS: The entire osteoporosis group had significantly higher pentosidine and CML serum concentrations than healthy subjects. In contrast to healthy subjects, no correlation between levels of AGEs and age could be found. In subgroups characterized by increased bone resorption (HTO, CUO), serum pentosidine correlated significantly with the histomorphometric marker reflecting osteoclast activity/bone resorption (eroded surface as a percentage of trabecular surface). Moreover, in CUO a strong correlation between pentosidine and the mineral apposition rate was found. Surprisingly, in HTO the levels of CML and percentage of eroded surface were significantly negatively correlated. CONCLUSION: AGE-modified proteins may be a cause of disturbed bone remodelling in osteoporosis. Our findings do not support the alternative hypothesis that increased AGEs in serum indicate only a more intensive releasing of AGEs in circumstances of increased bone resorption.     

Differential effects of androgens and estrogens on bone turnover in normal men

The physiologic role of androgens and estrogens in the maintenance of normal bone turnover in men is a fundamental issue in bone biology. To address this question, we randomized 70 men between the ages of 20 and 44 yr to receive one of three treatment regimens. Group 1 (n = 25) received a GnRH analog (goserelin acetate 3.6 mg by sc injection every 4 wk) alone for 12 wk to suppress endogenous gonadal steroids to prepubertal levels. Group 2 (n = 22) received goserelin plus transdermal testosterone (Androderm 5 mg topically daily) to normalize circulating testosterone and estradiol levels. Group 3 (n = 23) received goserelin plus testosterone plus an aromatase inhibitor (anastrozole 1 mg orally daily) to induce selective estrogen deficiency. The selective effects of androgens and estrogens on skeletal homeostasis were then assessed by measuring changes in biochemical markers of bone turnover and analyzing the between-group differences. Bone resorption markers increased in both the hypogonadal group (group 1) and in the group with selective estrogen deficiency (group 3). Urinary deoxypyridinoline excretion increased more in group 1 than in group 3 (P = 0.023), suggesting a significant effect of androgens on bone resorption, whereas serum N-telopeptide levels increased more in group 3 than in group 2 (P = 0.037), suggesting a significant effect of estrogen on bone resorption. Bone formation markers initially declined in all groups and then increased in groups 1 and 3. The between-group comparisons were consistent for all formation markers. Bone formation markers increased more in group 1 than in group 2 (P = 0.001, 0.037, 0.005 for osteocalcin, carboxy-terminal propeptide of type I procollagen, and amino-terminal propeptide of type I procollagen, respectively). Bone formation markers also increased more in group 1 than in group 3 but these differences were not statistically significant (P = 0.065 0.073, 0.099 for osteocalcin, carboxy-terminal propeptide of type I procollagen, and amino-terminal propeptide of type I procollagen, respectively). Taken together, these data suggest that both androgens and estrogens play independent and fundamental roles in regulating bone resorption in men. These data also suggest that androgens may play an important role in the regulation of bone formation in men.     

Effects of teriparatide, alendronate, or both on bone turnover in osteoporotic men

CONTEXT: We have previously demonstrated that alendronate reduces the ability of teriparatide to increase bone mineral density (BMD) in osteoporotic men. The underlying basis for this observation is poorly understood. OBJECTIVE: The primary aim of this study was to determine whether teriparatide increases osteoblast activity when the ability of teriparatide to increase osteoclast activity is suppressed by alendronate. DESIGN: This was a nonblinded, randomized, controlled trial. SETTING: The study was conducted at the General Clinical Research Center of a teaching hospital. PATIENTS: We studied 63 men, age 46-85, with low spine and/or hip BMD. INTERVENTIONS: Subjects received alendronate 10 mg daily (group 1), teriparatide 37 microg sc daily (group 2), or both (group 3) for 30 months. Teriparatide was begun at month 6. MAIN OUTCOME MEASURES: The primary endpoint was the change in serum N-telopeptide, osteocalcin, and amino-terminal propeptide of type 1 procollagen. RESULTS: In men receiving teriparatide monotherapy (group 2), levels of all bone turnover markers increased markedly during the first 6 months of teriparatide administration and then declined toward baseline during the next 18 months. In men who received combination therapy (group 3), bone turnover marker levels declined in the first 6 months (while receiving alendronate alone) and then returned to baseline levels (N-telopeptide) or above (osteocalcin and amino-terminal propeptide of type 1 procollagen) after teriparatide was added. Changes in each marker were significantly different between groups 1 and 2 (all P values < 0.001), groups 1 and 3 (all P values < 0.001), and groups 2 and 3 (all P values < 0.03). CONCLUSIONS: As with BMD, alendronate impairs the action of teriparatide to increase bone turnover in men.     

Alendronate and estrogen effects in postmenopausal women with low bone mineral density. Alendronate/Estrogen Study Group

The bisphosphonate alendronate and conjugated equine estrogens are both widely used for the treatment of postmenopausal osteoporosis. Acting by different mechanisms, these two agents decrease bone resorption and thereby increase or preserve bone mineral density (BMD). The comparative and combined effects of these medications have not been rigorously studied. This prospective, double blind, placebo-controlled, randomized clinical trial examined the effects of oral alendronate and conjugated estrogen, in combination and separately, on BMD, biochemical markers of bone turnover, safety, and tolerability in 425 hysterectomized postmenopausal women with low bone mass. In addition, bone biopsy with histomorphometry was performed in a subset of subjects. Treatment included placebo, alendronate (10 mg daily), conjugated equine estrogen (CEE; 0.625 mg daily), or alendronate (10 mg daily) plus CEE (0.625 mg daily) for 2 yr. All of the women received a supplement of 500 mg calcium daily. At 2 yr, placebo-treated patients showed a mean 0.6% loss in lumbar spine BMD, compared with mean increases in women receiving alendronate, CEE, and alendronate plus CEE of 6.0% (P < 0.001 vs. placebo), 6.0% (P < 0.001 vs. placebo), and 8.3% (P < 0.001 vs. placebo and CEE; P = 0.022 vs. alendronate), respectively. The corresponding changes in total proximal femur bone mineral density were +4.0%, +3.4%, +4.7%, and +0.3% for the alendronate, estrogen, alendronate plus estrogen, and placebo groups, respectively. Both alendronate and CEE significantly decreased biochemical markers of bone turnover, specifically urinary N-telopeptide of type I collagen and serum bone-specific alkaline phosphatase. The alendronate plus CEE combination produced slightly greater decreases in these markers than either treatment alone, but the mean absolute values remained within the normal premenopausal range. Alendronate, alone or in combination with CEE, was well tolerated. In the subset of patients who underwent bone biopsies, histomorphometry showed normal bone histology with the expected decrease in bone turnover, which was somewhat more pronounced in the combination group. Thus, alendronate and estrogen produced favorable effects on BMD. Combined use of alendronate and estrogen produced somewhat larger increases in BMD than either agent alone and was well tolerated.     

1,25-Dihydroxyvitamin D3 modulates glucocorticoid-induced alteration in serum bone Gla protein and bone histomorphometry

Glucocorticoid excess is associated with alterations in the vitamin D endocrine system. The aim of this study was to assess change in serum bone Gla protein (BGP) after low and high dose cortisone acetate treatment and to assess whether these alterations are altered or attenuated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] administration. Five groups of rats were studied and compared to a control group [cortisone acetate in doses of 0.2, 3.3, and 5.0 mg/100 g BW; 1,25-(OH)2D3 in a dose of 100 ng/100 g BW; and a combination of 1,25-(OH)2D3 (100 ng/100 g BW) plus cortisone acetate (3.3 mg/100 g BW)]. Each animal received daily sc injections for 27 days. BGP decreased significantly by day 7 in the two groups receiving high doses of cortisone acetate compared to control group values (65.20 +/- 4.38 vs. 150.18 +/- 6.13 ng/ml in the intermediate dose group and 91.57 +/- 5.30 vs. 150.18 +/- 6.13 ng/ml in the high dose group; P less than 0.01); this effect persisted until day 28. Histomorphometry revealed decreased formation and resorption in the two high dose cortisone acetate groups, whereas low dose cortisone acetate produced no histological change. The combination therapy lessens any change in BGP until day 28 when BGP was lower than the control value (P less than 0.01); histomorphometry showed that combination therapy prevents the effect of cortisone acetate by increasing bone formation and resorption. The data demonstrate that high doses of cortisone acetate suppress bone formation and that this is reflected in the low serum BGP values. Thus, BGP may be a marker of glucocorticoid-induced bone disease. 1,25-(OH)2D3 protects against glucocorticoid-induced bone disease and the normal BGP level reflects this.     

Anabolic effect of human synthetic parathyroid hormone-(1-34) depends on growth hormone

We tested whether GH is required for the anabolic effect of PTH on bone, using sham-operated (sham) and hypophysectomized (HX) young male rats. HX rats were supplemented daily with 3% sucrose water, T4, and corticosterone. Rats received vehicle or human PTH-(1-34) at 8 micrograms/100 g, sc, once daily, alone or in combination with rat or ovine GH at 0.2 mg/100 g, sc, twice daily or 12 micrograms ovine GH/100 g.day by continuous sc infusion. After 12 days, rats were sedated, and blood, femurs, and tibias were removed. Femur trabecular and cortical bone calcium (Ca), dry weight (DW), and hydroxyproline were measured. PTH increased bone Ca, DW, and hydroxyproline in shams by approximately 30%, but consistently failed to induce an anabolic response in HX rats. GH alone stimulated systemic growth in HX rats and increased their bone Ca and DW by 2-fold. The anabolic effect of PTH was restored in HX rats given both PTH and GH. Total bone mass in these rats was approximately 20% more (P less than 0.05) than the bone mass of rats given GH alone. When food was restricted in shams to limit systemic growth, PTH still induced an increase in bone mass. We conclude that GH or GH-dependent factors, such as insulin-like growth factor-I, which increases in PTH-treated bones in vitro, are required for the anabolic response of bone to PTH in vivo.     

Prospective study of histomorphometry,biochemical bone markers and bone densitometric response to pamidronate in β-thalassaemia presenting with osteopenia-osteoporosis syndrome

This study aimed to evaluate bone remodelling disorders in thalassaemia by using pamidronate (PD) infusion with or without hormone replacement therapy (HRT) as a diagnostic-therapeutic tool. In this prospective study, 24 adult thalassaemia major (TM) and 10 thalassaemia intermedia (TI) patients received either PD and HRT or HRT only (controls) for 3 years. Eugonadal patients with TI had PD only. Bone remodelling was assessed by dual energy X ray absorptiometry (DXA scan), type 1-collagen biochemical bone markers (BBM) and histomorphometry of iliac crest biopsy before and after PD. As a group, thalassaemics had a significant improvement in spinal and femoral bone mineral density Z scores following PD (P < 0·01) compared to the controls. Although BBM were comparable pre-therapy, they were significantly lower in the PD cohort (P < 0·001) compared to the control group. All patients had osteopenia, diminished osteoid formation and bone volume on histomorphometry pre-therapy with high turnover bone disease (HTO) in TM and low-turnover disease (LTO) in TI. In TM, bone volume improved significantly, whereas TI patients showed little or no response to PD. In conclusion, histomorphometry data suggest that TM patients have a distinct pathology of high turnover bone disease compared to TI patients, who have low-turnover disease.     

Gonadotropin-releasing hormone antagonist plus testosterone: a potential male contraceptive

No effective hormonal contraceptive has yet been devised for men. Through their suppressive effect on gonadotropin secretion, GnRH antagonists inhibit both testosterone (T) production and spermatogenesis in animals. Long term administration of an antagonist alone would result in androgen deficiency; this would cause unacceptable physiological and behavioral sequellae in men. Therefore, androgen replacement must be included in any GnRH antagonist regimen used in human male contraception. We tested the hypothesis that the combination of a GnRH antagonist plus T would suppress spermatogenesis in the male primate to azoospermic levels while maintaining normal serum T levels. We examined the effects of the GnRH antagonist Deterelix [N-Ac-DNal(2)1-DpCl-Phe2-DTrp3-DhArg(Et2)6 -DAla10-GnRH], alone and with simultaneous T replacement, on sperm production and serum T levels in adult male monkeys (n = 22). After 12 weeks of daily sc antagonist injection, all animals that received antagonist alone (n = 5) and those that 750 micrograms/kg.day antagonist plus T (n = 5) were azoospermic. After 16 weeks, four of five animals that received 250 micrograms/kg.day antagonist plus T became azoospermic. Control animals (n = 7) received daily injections of vehicle; sperm counts increased somewhat during the study period in that group. Castrate range T levels were achieved in animals receiving antagonist alone. T levels in the groups that received T supplementation and in the control group were in the normal male range throughout the treatment period. Sperm counts returned to the pretreatment range in all animals during the recovery period. We conclude that the combination of a GnRH antagonist plus T can induce azoospermia reversibly in this nonhuman primates species, and that a similar combination may be an effective contraceptive regimen in men. The GnRH antagonist alone may be an effective treatment for androgen-dependent neoplasia.     

Loss of nitrogen from organs in rats induced by exogenous glucagon

Rats weighing 220 g were injected sc with zinc protamin glucagon 20 micrograms once daily (recurrent hyperglucagonemia) and zinc protamin glucagon 60 micrograms three times daily (chronic hyperglucagonemia); the controls received the vehicle three times daily. In the first group blood glucagon rose to above 200 ng/liter for 5 h every day; in the second group it constantly stayed above 600 ng/liter. After both 2 (n = 5) and 14 (n = 5) days treatment the control total blood alpha-amino-nitrogen (AAN) concentration was 4.3 +/- 0.1 mmol/liter, and the urea nitrogen synthesis rate was 4.9 +/- 0.4 mumol/(min.100 g BW) (mean +/- SEM) in controls. In recurrent hyperglucagonemic rats, treated for both 2 (n = 5) and 14 (n = 5) days, total AAN was 3.6 +/- 0.2 mmol/liter (P less than 0.05 vs. control) and urea nitrogen synthesis rate 4.5 +/- 0.8 mumol/(min.100 g BW). In chronic hyperglucagonemic, treated for both 2 (n = 5) and 14 (n = 5) days, total AAN was 2.2 +/- 0.1 mmol/liter (P less than 0.05 vs. control) and UNSR 7.9 +/- 0.8 mumol/(min.100g BW) (P less than 0.05 vs. control). The urea excretion was identical in controls and during recurrent hyperglucagonemia, but it was increased by 50% during chronic hyperglucagonemia. Food intake was the same in all groups. N Balances decreased from 10 mmol/24 h to 5 mmol/24 h (P less than 0.05) by chronic hyperglucagonemia. The total organ N content did not change by recurrent hyperglucagonemia, but in chronic hyperglucagonemia it decreased to 65-85% (P less than 0.01) in carcass, intestines, liver, and kidneys. In conclusion chronic but not recurrent hyperglucagonemia increases the rate of urea synthesis and decreases the blood amino acid concentration. This is suggested to be a reason for the loss of N from organs by chronic hyperglucagonemia.     

Endocrine and pharmacological suppressors of bone turnover protect against osteopenia in ovariectomized rats

This study was designed to test the hypothesis that endocrine and pharmacological suppressors of bone turnover prevent the development of osteopenia during estrogen deficiency. Sham-operated control and ovariectomized (OVX) rats were treated intermittently with vehicle alone, estrogen, or the diphosphonate compounds etidronate disodium (EHDP) and NE-58095 [2-(3-pyridinyl)2-hydroxyethylidene-1,1-bisphosphonate disodium] for 35 or 70 days after surgery. Their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Vehicle-treated OVX rats were characterized by decreased cancellous bone volume and 3- to 4-fold increases in osteoblast surface, osteoclast surface, bone formation rate, and bone resorption rate. Treatment of OVX rats with estrogen and NE-58095 provided complete protection against bone loss and significantly depressed all of the above indices of bone turnover. OVX rats treated with EHDP exhibited at least partial protection against bone loss and decreased bone turnover. EHDP induced a mild mineralization defect, as indicated by a prolonged mineralization lag time at the tibial endocortical surface. The new diphosphonate compound NE-58095 did not impair bone mineralization. Our results indicate that endocrine and pharmacological suppressors of bone turnover prevent the development of osteopenia during the early stages of estrogen deficiency. If confirmed by clinical trials in humans, diphosphonate compounds may prove to be an alternative to estrogen for the prevention of postmenopausal bone loss.     

Parathyroid hormone as a therapy for idiopathic osteoporosis in men: effects on bone mineral density and bone markers

Osteoporosis in men poses a unique therapeutic challenge. Clinical studies have focused largely on the more prevalent problem of post-menopausal osteoporosis, with few gender-specific studies exploring treatment options in men. Idiopathic osteoporosis in middle-aged men presents an additional dilemma, because in the majority of patients it is a low bone turnover state for which there are currently no available anabolic agents. We conducted an 18-month randomized, double blind, placebo-controlled trial of 23 men with idiopathic osteoporosis, 30-68 yr old (mean age +/- SEM, 50 +/- 1.9 yr). All patients received 1,500 mg calcium and 400 IU vitamin D daily. Ten patients were randomized to receive 400 IU PTH-(1-34), and 13 patients received vehicle, administered by daily sc injection. Serum and urinary biochemistries, including markers of bone turnover were measured every 3 months. Bone densitometry of the lumbar spine, hip, and radius was performed every 6 months. PTH-(1-34) was associated with a marked 13.5% increase in bone mass at the lumbar spine, whereas that in the control group did not change (P < 0.001). The mean lumbar spine T-score improved from -3.5 +/- 0.2 to 2.4 +/- 0.4. Femoral neck bone mineral density in the PTH-treated group increased 2.9% (P < 0.05). The 1/3 site of the distal radius showed no change from baseline in the PTH-treated group. There were no significant changes in serum calcium concentration, 24-h urinary calcium excretion, or 1,25-dihydroxyvitamin D in either group. All markers of bone turnover increased in the PTH-treated patients, with the greatest changes in serum osteocalcin and urinary N-telopeptide (230% and 375% above baseline by 12 months, respectively; P < 0.001). Free pyridinoline and markers of bone formation that showed little correlation with each other at baseline, became highly correlated in the PTH-treated group (r = 0.1; P = 0.29 at baseline; to r = 0.7; P < 0.0001 at 18 months), a pattern absent in the control patients. The best predictor of the lumbar spine response to PTH at 18 months was the combination of pyridinoline at baseline and osteocalcin at 3 months (70% of the variance). PTH is a potent stimulator of skeletal dynamics in men with idiopathic, low turnover osteoporosis; is associated with substantial increases in lumbar spine and hip bone density; and may prove to be an efficacious anabolic agent in men with this disorder.     

Inhibition by human interleukin-1 alpha of parathyroid hormone-related peptide effects on renal calcium and phosphorus metabolism in the rat.

Humoral hypercalcemia of malignancy (HHM) is at least partly caused by tumor secretion of PTH-related peptide (PTHrP), but there is growing evidence for cosecretion with PTHrP of other bone-resorbing peptides, such as the cytokine interleukin-1 alpha (IL-1 alpha). Administration of PTHrP in vivo and in vitro generally mimics the actions of PTH itself, with increases in both resorption and formation of bone. However, bone in HHM is characterized by uncoupling of bone turnover, with increased resorption and decreased formation. We performed experiments to determine whether IL-1 alpha might alter the effects of PTHrP and produce uncoupling. Thus, we administered to 100-g male rats by sc osmotic minipumps synthetic PTHrP-(1-34) alone (2 micrograms/100 g/day), recombinant IL-1 alpha alone (1.5 micrograms/100 g/day), both peptides together at the previous doses, or vehicle only. We infused 5 groups of 12 rats each (PTHrP, IL-1 alpha, PTHrP plus IL-1 alpha, ad libitum fed control, and controls pair-fed to the PTHrP plus IL-1 alpha group) for 14 days. At the end of the study, blood and urine were taken for chemical measurements, and tibias and femurs were harvested for histomorphometry and extraction of RNA from periosteal cells. As expected, PTHrP induced hypercalcemia, relative hypophosphatemia, phosphaturia, and reduced bone mass. Osteoblast number was increased, but osteoclast number was not. Indices of bone formation were unchanged or reduced. The dose of IL-1 alpha chosen had no statistically significant effect, except for reduced longitudinal bone growth, but when combined with PTHrP, IL-1 alpha reduced hypercalcemia, hypophosphatemia, and phosphaturia. In contrast to the blood and urine effects, IL-1 alpha did not interact significantly with PTHrP's effect on bone measurements. Northern analysis of periosteal cell mRNA showed that PTHrP reduced expression of osteocalcin, but not glyceraldehyde-3-phosphate dehydrogenase; IL-1 alpha had no additional effect. These data suggest that 1) continuously administered PTHrP alone may induce uncoupled bone turnover with decreased cortical bone formation; 2) IL-1 alpha appears to inhibit strongly the renal effects of PTHrP and weakly (if at all) its actions on bone and, thus, to decrease its hypercalcemic, phosphaturic, and hypophosphatemic actions; and 3) cosecretion of IL-1 alpha, and possibly other peptide cytokines, with PTHrP may modify the clinical expression of HHM.     

Effect of combined risedronate and hormone replacement therapies on bone mineral density in postmenopausal women

Both hormone replacement therapy (HRT) and bisphosphonates are efficacious in the prevention and treatment of postmenopausal osteoporosis. Combined therapy with bisphosphonate and HRT is likely to be used in clinical practice, and limited data are available regarding its efficacy and safety. This was a 1-yr, double blind, placebo-controlled study in which 524 postmenopausal women received daily treatment with conjugated equine estrogens (0.625 mg) alone or in combination with risedronate (5 mg). Women who had not undergone hysterectomy received medroxyprogesterone acetate (up to 5 mg, daily or cyclically) at the discretion of the investigator. The primary efficacy end point was the percent change from baseline in mean lumbar spine bone mineral density (BMD) at 1 yr. Changes in BMD at the proximal femur and forearm, bone turnover markers, and histology and histomorphometry were also assessed. At 12 months, significant (P < 0.05) increases from baseline in lumbar spine BMD were observed in both treatment groups (HRT-only, 4.6%; combined risedronate-HRT, 5.2%); the difference between the two groups was not statistically significant. Both therapies led to significant increases in BMD at 12 months at the femoral neck (1.8% and 2.7%, respectively), femoral trochanter (3.2% and 3.7%), distal radius (1.7% and 1.6%), and midshaft radius (0.4% and 0.7%). The differences between groups were statistically significant (P < 0.05) at the femoral neck and midshaft radius. Both combined risedronate-HRT and HRT-only produced significant decreases in the biochemical markers of bone turnover, with somewhat greater decreases in the combined treatment group. Bone biopsy data showed normal bone structure and normal mineralization with either treatment. Expected decreases in bone turnover were observed and were greater in the combined treatment group (68-79% reduction relative to baseline values, P < 0.005). Overall, combined treatment had a safety profile similar to that of HRT-only, including bone and gastrointestinal safety profiles. In conclusion, the combined treatment with risedronate and HRT had a favorable effect on BMD similar to that of HRT alone at the lumbar spine and slightly, but significantly, greater than that of HRT alone at the femoral neck and midshaft radius. The combined treatment was well tolerated, and there were no adverse effects on the skeleton.     

Skeletal effects of calcitonin in ovariectomized rats

Although calcitonin (CT) has been shown to be effective for the prevention of bone loss in early postmenopausal women, the skeletal effects of the hormone specifically during the early stages of estrogen deficiency have not been characterized histomorphometrically to date. The current study involves use of the ovariectomized (OVX) rat as an animal model for early postmenopausal bone loss to perform such a histomorphometric analysis. One group of OVX rats was injected sc with salmon CT on alternate days for a 6-week period. Additional groups of OVX and sham-operated control rats were treated with vehicle alone. In comparison to control rats, the proximal tibia of vehicle-treated OVX rats were characterized by a 3-fold decrease in cancellous bone volume and significant increases in osteoblast surface (+200%), osteoclast surface (+143%), mineralizing surface (+111%), mineral apposition rate (+36%), bone formation rate (+181%), and longitudinal bone growth (+38%). In contrast, treatment of OVX rats with CT normalized tibial cancellous bone volume and significantly decreased all of the above cellular- and fluorochrome-based indices of bone turnover to near control levels. The results indicate that CT treatment depresses bone turnover and prevents the development of osteopenia in OVX rats. These findings are consistent with the bone protective effect of CT in early postmenopausal women and further support the OVX rat as an animal model for the preclinical evaluation of prophylactic treatments for postmenopausal bone loss.     

The effects of combination of alendronate and human parathyroid hormone(1-34) on bone strength are synergistic in the lumbar vertebra and additive in the femur of C57BL/6J mice

A cyclic PTH regimen is as effective as a daily regimen on bone density gain in humans and in improving bone quality in mice. Our previous murine study evaluated the effects of a cyclic PTH regimen in the absence of a bisphosphonate, whereas our human study addressed the effects of a cyclic PTH regimen in the presence of ongoing alendronate (ALN) treatment. Accordingly, the current study examined the effects of cyclic or daily PTH regimens in combination with ALN on bone quality and bone density in mice. Twenty-week-old, female C57BL/6J mice were treated with the following sc injections (n = 10): 1) vehicle for 5 d/wk (control); 2) ALN (20 microg/kg x d) 3 d/wk (ALN); 3) human PTH(1-34) (40 microg/kg x d) 5 d/wk (daily PTH); 4) daily PTH in addition to ALN (daily PTH plus ALN); 5) PTH 5 d/wk and vehicle 5 d/wk alternating weekly (cyclic PTH); 6) cyclic PTH in addition to ALN (cyclic PTH plus ALN); and 7) PTH and ALN alternating weekly (alt PTH and ALN). Bone mineral density was measured weekly by dual-energy x-ray absorptiometry, and at 7 wk, bone markers, bone structure, and bone strength were evaluated by biochemical assays, peripheral quantitative computed tomography and mechanical testing, respectively. At 7 wk, all treatments significantly increased femoral and vertebral bone mineral density. ALN slightly decreased endosteal circumference, whereas PTH increased periosteal circumference, resulting in significant increases in femoral cortical thickness in all groups. PTH and ALN enhanced bone strength synergistically in the lumbar vertebrae and additively in the femur. Combined therapy, however, had no effects on bone markers. The results show that combinations of ALN and PTH, in both daily and cyclic regimens, produce more beneficial effects than treatment with either agent alone, suggesting that the mechanisms of actions of ALN and PTH on bone quality may be complementary.     

Apparent role of melatonin and prolactin in initiating winter fur growth in mink

A study was conducted to determine the effects of exogenous melatonin and bromocriptine (CB-154), an inhibitor of prolactin synthesis and secretion, on the induction of winter fur growth in mink. Melatonin (10 and 120 mg) was administered to mink (N = 5/group) via silastic implants inserted sc over the scapular area during the last week of June 1985. Treatment of mink (N = 5) with CB-154 alone or in combination with 10 mg melatonin (N = 5) consisted of daily sc injections of 2 mg of the drug in sterile saline from June 25 through July 30. Control animals (N = 5) did not receive injections of vehicle or sham implants. Administration of CB-154 alone or in combination with 10 mg melatonin, as well as 120 mg melatonin alone, initiated growth of the winter fur significantly earlier than that of controls or mink treated with 10 mg melatonin (P less than 0.05). These data suggest that inhibition of prolactin secretion by melatonin is requisite for induction of molt of summer fur and growth of winter fur of mink.     

Bone calcium turnover,formation, and resorption in bromocriptine- and prolactin-treated lactating rats

To evaluate the effect of endogenous prolactin (PRL) on bone metabolism, we studied bone calcium turnover by the ⁴⁵Ca kinetic method and bone formation and resorption by bone histomorphometry and biochemical markers in 13-wk-old lactating Wistar rats. For 1 wk, the animals received daily administration of 0.9% NaCl (control) intraperitoneally, 6 mg of bromocriptine/kg of body wt intraperitoneally, or 6 mg of bromocriptine/kg of body wt plus 2.5 mg of ovine PRL/kg of body wt subcutaneously. Bromocriptine, a dopaminergic inhibitor of endogenous PRL secretion, significantly decreased calcium ion deposit rate and calcium resorption rate in femur, tibia, vertebrae 5 and 6, and sternum by 20–42%. By contrast, calcium resorption rate of the vertebrae and the sternum of the PRL-treated group was higher than that of controls, whereas the tibia and sternum exhibited a greater net loss of calcium. The suppression of bone calcium turnover in the bromocriptine-treated group was further supported by a significant decrease in the urinary deoxypyridinoline, a biochemical index of bone resorption, and the histomorphometric data, which showed changes indicative of suppressed bone resorption and formation. The histomorphometric data from the PRL-treated group were not different from those of the control group with the exception of an increase in the longitudinal growth rate. The results suggested a role of endogenous PRL in the stimulation of bone turnover during lactation.