Development and application of a serum C-telopeptide and osteocalcin assayto measure bone turnover in an ovariectomized rat model

Biochemical markers applicable to the ovariectomized rat model can provide important tools for studying the bone remodeling process in this animal mod el of postmenopausal osteoporosis. We describe the development and applicat ion of two biochemical markers, a C-telopeptide (of type-I collagen) enzyme -linked immunosorbent assay (ELISA) for measuring bone resorption and an os teocalcin radioimmunoassay (RIA) for measuring bone formation in rat serum. The C-telopeptide ELISA is based on an affinity purified polyclonal antibo dy generated against human sequence DFSFLPQPPQEKAHDGGR. The antibody epitop e 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%. Averag ed dilution and spiked recoveries were 91% and 105%, respectively. The seco nd marker developed is a synthetic peptide-based osteocalcin RIA, which doe s not require isolation and purification of intact osteocalcin from rat bon e. Osteocalcin antiserum used in the RIA was generated in rabbits against a synthetic peptide comprising amino acids 33-49 of the rat osteocalcin sequ ence. The sensitivity of the RIA was 0.15 ng/ml of peptide. The averaged in tra (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 performe d in an ovariectomized (OVX) rat model. In 12-week-old OVX Sprague Dawley r ats, the C-telopeptide and osteocalcin concentrations were approximately 65 % and 40%, respectively, higher than the sham group. Estradiol repletion si gnificantly lowered the C-telopeptide and osteocalcin concentration to the levels of the sham group. In addition, changes in serum C-telopeptide conce ntration 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 powe r to be used for measuring bone resorption and bone formation in the ovarie ctomized rat model.