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 ORCID: https://orcid.org/0000-0003-1122-5099, Santos, L ORCID: https://orcid.org/0000-0002-1915-6780, Varley, I ORCID: https://orcid.org/0000-0002-3607-8921, 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. ISSN 2051-817X

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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 (D2 O) 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 D2 O 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.

Item Type:	Journal article
Publication Title:	Physiological Reports
Creators:	Civil, R., Brook, M.S., Elliott‐Sale, K.J., Santos, L., Varley, I., Lensu, S., Kainulainen, H., Koch, L.G., Britton, S.L., Wilkinson, D.J., Smith, K., Sale, C. and Atherton, P.J.
Publisher:	Wiley
Date:	27 May 2021
Volume:	9
Number:	10
ISSN:	2051-817X
Identifiers:	Number Type 10.14814/phy2.14799 DOI 1442689 Other
Rights:	© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Divisions:	Schools > School of Science and Technology
Record created by:	Laura Ward
Date Added:	02 Jun 2021 14:53
Last Modified:	02 Jun 2021 14:53
URI:	https://irep.ntu.ac.uk/id/eprint/42966

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