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. 2021 May;9(10):e14799.
doi: 10.14814/phy2.14799.

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

Rita Civil  1 , Matthew S Brook  2 , Kirsty J Elliott-Sale  1 , Lívia Santos  1 , Ian Varley  1 , Sanna Lensu  3 , Heikki Kainulainen  3 , Lauren G Koch  4 , Steven L Britton  5   6 , Daniel J Wilkinson  2 , Kenneth Smith  2 , Craig Sale  1 , Philip J Atherton  2
Affiliations

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

Rita Civil et al. Physiol Rep. 2021 May.

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.

Keywords: GC-pyrolysis-IRMS; bone turnover; collagen synthesis; deuterium oxide; stable isotopes.

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Figures

FIGURE 1
FIGURE 1
Change in bound deuterium enrichment (Δδ2H) across the tibia proximal (T‐PRO), mid‐shaft (T‐MID), distal (T‐DIS) and femur (FEM). GC‐pyrolysis‐IRMS limit of detection (LOD) shown as 10 δ2H and GC‐MS LOD shown as 4700 δ2H. Sampling areas of tibia and femur shown in highlighted in grey.
FIGURE 2
FIGURE 2
Collagen fractional synthetic rate (FSR) for the femur (FEM) and the mid‐shaft of the tibia (T‐MID). (a) Data represented as box plots, + represents mean. (b) Individual values. * Wilcoxon matched pairs test p < 0.001
FIGURE 3
FIGURE 3
Collagen fractional synthetic rate (FSR) across the proximal (T‐PRO), mid‐shaft (T‐MID) and distal (T‐DIS) sites of the tibia. (a) Data represented as box plots, + represents the mean. (b) Individual values. * Post hoc Dunn's multiple comparisons test p < 0.001
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