A collagen extraction and deuterium oxide stable isotope tracer method for the quantification of bone collagen synthesis rates in vivo

Civil, R, Brook, MS, Elliott-Sale, KJ, Santos, L, Varley, I, Lensu, S, Kainulainen, H, Koch, LG, Britton, SL, Wilkinson, DJ, Smith, K, Sale, C ORCID: https://orcid.org/0000-0002-5816-4169 and Atherton, PJ (2021) A collagen extraction and deuterium oxide stable isotope tracer method for the quantification of bone collagen synthesis rates in vivo. Physiological Reports, 9 (10). e14799-e14799. ISSN 2051-817X

Preview

Published Version Available under License Creative Commons Attribution. Download (637kB) | Preview

Abstract

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D2O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D2O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106–0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040–0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166–0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022–0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover.