Development of a highly sensitive, high-throughput, mass spectrometry-based assay for rat procollagen type-I N-terminal propeptide (PINP) to measure bone formation activity.

Type-I procollagen aminoterminal propeptide (PINP) is a useful biomarker for bone formation activity that is used to monitor treatment of bone disorders including osteoporosis. Studies with human patients under long-term therapy for osteoporosis by daily injection of parathyroid hormone (PTH) demonstrated that the circulating level of PINP at 3 months of treatment, measured by radioimmunoassay, was a good predictor for bone mineral density (BMD) at 18 months. It is important to have PINP assays for other species to elucidate processes of bone formation and for the development of new therapeutic options that can enhance bone formation activity. Currently, only a human PINP radioimmunoassay is commercially available for clinical use, which may not be cross reactive with PINP from other species. For example, rat PINP has little amino acid sequence homology to human PINP. Therefore, we developed a new, highly sensitive, high-throughput mass spectrometry-based assay for PINP from rat plasma or serum that does not rely on antibody reagents. Circulating levels of PINP showed age-dependent changes in rats. Prednisolone treatment, which is known to retard bone formation activity, led to a significant decrease in PINP, whereas PTH treatment dose-dependently increased PINP. The throughput of the assay parallels that of most antibody-based assays so that it can handle a large number of samples that are generated from preclinical animal studies. PINP in rats may serve as a biomarker for bone formation activity, and this assay could be instrumental in studying bone physiology in rat experimental models.

Comparison of results from the semiautomated serum bone alkaline phosphatase isoenzyme assay with the periosteal alkaline phosphatase assay for use in rat models.

BACKGROUND: Assessment of bone formation activity is an important component of pharmacologic efficacy and toxicity evaluations for compounds in development for osteoporosis therapies. Antemortem biomarkers of bone formation and remodeling in rodents are uncommon. While the periosteal alkaline phosphatase (ALP) assay is a postmortem and laborious means of testing bone-building activity, the semiautomated ALP isoenzyme assay is an antemortem assay that is performed on an automated chemistry analyzer after 2 simple dilutions of the initial serum sample and a short incubation. OBJECTIVES: The goal of our investigation was to determine if the serum bone ALP (BALP) data obtained from the semiautomated ALP isoenzyme assay had a similar pattern of response when compared with the periosteal ALP (PALP) assay for use in pharmacologic screening in rats. METHODS: Serum and bone tissue samples were obtained from orchidectomized Wistar rats, a model of clinically induced osteoporosis. Subsequent bone formation was initiated via treatment with one of several compounds. In study 1, orchidectomized male rats were given either vehicle, dihydrotestosterone or a testosterone derivative subcutaneously every 4 days for 28 days. In study 2, orchidectomized male rats were given either vehicle or compounds A, B, or C by oral gavage daily for 15 days. Blood and tibias were collected at necropsy. Serum was analyzed for BALP activity using a semiautomated ALP assay. Tibias from the same rats were analyzed for PALP activity. RESULTS: Serum BALP activity paralleled PALP activity within each group when compared with the controls. CONCLUSION: Our data indicate that the semiautomated serum BALP isoenzyme assay may be used as a biomarker of bone-building potential in rat models of osteoporosis. This assay affords many advantages to investigators of musculoskeletal diseases, including the potential to measure multiple data points in a single study.