Determination of Hydroxyproline in Bone Collagen: Potential Application as a Biomarker for Bone Diseases

Abstract

Hydroxyproline (Hyp), a non-proteinogenic amino acid is a component of the organic material in bone. It has been used for 14C-dating of bone and the measurement of Hyp could be used as a biomarker in bone metabolism. Hydroxyproline is a component of collagen, the main structural protein in bone. The analyses of 14C in collagen and Hyp in human bones may provide timing information about bone processes and diseases, such as osteoarthritis and osteoporosis. The analysis of Hyp in bones (e.g., the determination of Hyp content) primarily relies on a spectrometric technique, liquid chromatography-mass spectrometry (LC-MS), and the determination of 14C content requires accelerator mass spectrometry (AMS). Moreover, to obtain these materials from bone requires the successful extraction of collagen and thr separation of Hyp from the collagen. This study aims at comparing methods for extracting collagen from bone, which do not destroy the Hyp. These methods include the use of either NaOH, KOH or HCl in one stage of the extraction process and separating sufficient Hyp for 14C analysis. This will provide information to determine whether Hyp can be used as a biomarker for bone diseases like osteoarthritis and osteoporosis. A preliminary 14C AMS analysis on collagen extracted by the NaOH method was carried out on human bones previously analyzed for forensic purposes. This demonstrated the ability of this technique to provide recent (post 1950) timing information. The collagen extractions by three different methods were first conducted on modern chicken bone, and the results showed that KOH method is the best bone collagen extraction method, yielding a largest quantity of Hyp. The KOH method was then employed to extract collagen from cow bone as a test of a more human-like (mammalian) material. As this was successful, collagen was extracted from diseased human bone fragments, obtained from the Ottawa Hospital. The data revealed that Hyp was successfully obtained from these bones. The study demonstrates that the extraction as well as the separation methods (preparative HPLC) can provide sufficient Hyp from bones for 14C AMS analysis. This will lead to future studies of Hyp in bone turnover, which may lead to its use as a novel biomarker for bone diseases such as osteoarthritis and osteoporosis.