Usefulness of Serum Pyridinoline Cross-Linked Carboxy-Terminal Telopeptides of Type I Collagen and Osteocalcin in the Study of the Bone Resorption Rate in Oophorectomized Rats

Calero, Jesús Alberto; Piedra, C. de la; Curiel, Manuel Dı́az

doi:10.1159/000057387

Cited by 4 publications

(1 citation statement)

References 18 publications

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“…The intra- and interassay CVs of this method were 5 and 9%, respectively. ICTP was determined through an RIA specialized for rats from the Finnish firm Orion Diagnostic [42]. This method’s sensitivity is 1 ng/ml with intra- and interassay CV, being 5 and 9%, respectively.…”

Section: Methodsmentioning

confidence: 99%

Bone Disease Induced by Phenytoin Therapy: Clinical and Experimental Study

et al. 2009

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Previous studies have made references to prolonged treatment with phenytoin as a possible risk factor in the development of osteoporosis and/or osteomalacia. We studied a group of 30 epileptic patients who were under long-term treatment with phenytoin (DPH) in an ambulatory regimen. We found the prevalence of osteoporosis to be 3.3% and of osteopenia to be 56.6%, affecting predominantly the femur, without any significant decrease in bone mineral density of the lumbar spine. These patients were showing signs of bone turnover uncoupling with increases in bone resorption markers. At this time, they also exhibited slight alterations in their phosphocalcium metabolism with trends to hypocalcemia and secondary hyperparathyroidism that was found not to be caused by a vitamin D deficiency as the serum levels of 25(OH)D and 1,25(OH)₂D were normal. With the aims of corroborating these results and to investigate the physiopathological effects on the bone induced by anticonvulsant drugs we developed a further experimental study in which we administered DPH over a 6-week period with a dose of 5 g/kg/day to male Wistar rats that were in the growth phase. This treatment produced a decrease in overall BMD and bone mineral content in the femur. We did not find osteomalacia in the vertebral biopsy, but the administration of DPH to these animals decreased trabecular volume as well as lessened the thickness of osteoid edges together with an uncoupling in bone turnover. There was also a marked decrease in bone formation and a tendency towards increased bone resorption. We have also found a decrease in resistance to fracture by torsion in the biomechanical assay, which translates into an increase in bone fragility. In these male Wistar rats, the administration of DPH produced a tendency towards increasing the markers of resorption and, though changes in serum levels of calcium and phosphorus were not observed, to provoke an increase in the parathyroid hormone levels; with normal levels of 1,25(OH)₂D which has produced the same inclination in rats as in humans.

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Section: Methodsmentioning

confidence: 99%

Bone Disease Induced by Phenytoin Therapy: Clinical and Experimental Study

et al. 2009

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Development and Application of a Serum C-Telopeptide and Osteocalcin Assay to Measure Bone Turnover in an Ovariectomized Rat Model

et al. 2000

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Biochemical markers applicable to the ovariectomized rat model can provide important tools for studying the bone remodeling process in this animal model of postmenopausal osteoporosis. We describe the development and application of two biochemical markers, a C-telopeptide (of type-I collagen) enzyme-linked immunosorbent assay (ELISA) for measuring bone resorption and an osteocalcin radioimmunoassay (RIA) for measuring bone formation in rat serum. The C-telopeptide ELISA is based on an affinity purified polyclonal antibody generated against human sequence DFSFLPQPPQEKAHDGGR. The antibody epitope involves amino acid sequence, which is similar in rat and human carboxyl terminal peptide of type-I (alpha 1) collagen. Sensitivity of the ELISA was 0.3 ng/ml. The averaged intra- and interassay variation was CV <7%. Averaged dilution and spiked recoveries were 91% and 105%, respectively. The second marker developed is a synthetic peptide-based osteocalcin RIA, which does not require isolation and purification of intact osteocalcin from rat bone. Osteocalcin antiserum used in the RIA was generated in rabbits against a synthetic peptide comprising amino acids 33-49 of the rat osteocalcin sequence. The sensitivity of the RIA was 0.15 ng/ml of peptide. The averaged intra (n = 10) and interassay variations for two controls were CV <9% and 12%, respectively. The averaged dilution and spiked recoveries were 99.6%. In vivo validation of the C-telopeptide ELISA and osteocalcin RIA was performed in an ovariectomized (OVX) rat model. In 12-week-old OVX Sprague Dawley rats, the C-telopeptide and osteocalcin concentrations were approximately 65% and 40%, respectively, higher than the sham group. Estradiol repletion significantly lowered the C-telopeptide and osteocalcin concentration to the levels of the sham group. In addition, changes in serum C-telopeptide concentration correlated negatively with trabecular BMD measured by pQCT (r = -0.51, P < 0.001). In conclusion, the C-telopeptide ELISA and osteocalcin RIA exhibited required sensitivity, accuracy, and adequate discriminatory power to be used for measuring bone resorption and bone formation in the ovariectomized rat model.

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Development of a synthetic peptide-based tartrate-resistant acid phosphatase radioimmunoassay for the measurement of bone resorption in rat serum

Srivastava

Libanati

Lane

et al. 2002

Journal of Bone and Mineral Metabolism

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Selective markers of bone turnover provide a convenient and reproducible alternative to the complex and expensive histochemical techniques used commonly to study the effect of pharmacological agents and the pathogenesis of bone disease in the ovariectomized (OVX) rat model. One marker, which has been specifically linked to terminally differentiated osteoclasts and, thus, provides useful insight at cellular levels, is type-5 tartrate-resistant acid phosphatase (TRACP). We describe the development of a TRACP radioimmunoassay (RIA), which requires synthetic peptide for antibody development. To develop the RIA, polyclonal antibodies were generated in goats against a synthetic peptide, DPSVRHQRKCY, corresponding to amino acid residues 267-275 of the rat type-5 TRACP sequence. In the RIA, 50 microl of rat serum, 100 microl of goat anti-TRACP antibodies, and 100 microl of tracer were incubated overnight. The antibody-bound fraction was separated, counted, and unknown values were calculated by comparison with the peptide calibrator. Rat serum shows parallelism with the synthetic peptide calibrator used in the RIA. The sensitivity of the RIA was 24.7 microg/l, and the measuring range was 19-2476 microg/l. The average intra-assay coefficients of variation for (CV) two controls were less than 7%. The average dilution and spike recoveries were 107% and 87%, respectively. We applied our peptide-based RIA to study bone resorption in an OVX rat model. TRACP concentrations in serum in 12-week-old OVX Sprague Dawley rats were 14%-22% (P < 0.05) higher than those in the sham-operated rats, and TRACP concentrations in OVX rats treated with estradiol were 24%-32% lower (P < 0.01) than those in the vehicle-treated OVX group. Similarly, as compared with those in OVX rats, TRACP concentrations decreased to those of sham levels in OVX rats receiving 10 microg/kg per day of alendronate for 10 days. In addition, the TRACP levels determined by RIA showed a significant correlation with serum C-telopeptide (type-I collagen) concentrations (r = 0.56; P < 0.001) measured by an enzyme-linked immunosorbent assay (ELISA) developed earlier for the rat model. In conclusion, we have developed a TRACP RIA that could be used to monitor the rate of bone resorption in the rat model.

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Usefulness of Serum Pyridinoline Cross-Linked Carboxy-Terminal Telopeptides of Type I Collagen and Osteocalcin in the Study of the Bone Resorption Rate in Oophorectomized Rats

Cited by 4 publications

References 18 publications

Bone Disease Induced by Phenytoin Therapy: Clinical and Experimental Study

Bone Disease Induced by Phenytoin Therapy: Clinical and Experimental Study

Development and Application of a Serum C-Telopeptide and Osteocalcin Assay to Measure Bone Turnover in an Ovariectomized Rat Model

Development of a synthetic peptide-based tartrate-resistant acid phosphatase radioimmunoassay for the measurement of bone resorption in rat serum

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