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.

Highlights

  • Understanding bone remodelling in ageing and disease, and developing strategies to maintain bone tissue are vital

  • The average body water enrichment in rats was 0.685 ± 0.089 APE; whilst the average change in the deuterium labelling, expressed as delta per mil deuterium (δ2H), was Femur diaphyses (FEM) 352 ± 38 δ2H, tibia proximal (T-­PRO) 548 ± 45 δ2H, tibia mid-s­ haft diaphyses (T-­MID) 170 ± 21 δ2H and T-­DIS 83 ± 10 δ2H (Figure 1), with the higher the value reflecting the greater incorporation of labelled alanine

  • The calculated average collagen fractional synthetic rates (FSRs) for FEM (0.131 ± 0.078%/day; 95% confidence intervals (95% CI) [0.106–0­ .156]) were significantly greater than the FSR at T-­MID (0.055 ± 0.049%/day; 95% CI [0.040–­0.070]; p < 0.001, Figure 2)

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Summary

| INTRODUCTION

Understanding bone remodelling in ageing and disease (e.g., osteoporosis), and developing strategies to maintain bone tissue are vital. Amino acid isotope tracers, such as carbon (13C), deuterium (2H) or nitrogen (15 N), have been used to measure in vivo synthesis of human musculoskeletal tissues, including bone (Babraj et al, 2005; Scrimgeour et al, 1993; Smeets et al, 2019) These amino acid tracers are, hindered by the heterogeneity of amino acid body pools (Brook et al, 2017; Wilkinson, 2016), and require preparation of high-­cost infusions and venous/arterial cannulation (Wilkinson et al, 2014). Combining sensitive GC-­pyrolysis-­IRMS techniques, this method enables the measurement of slow turnover proteins such as collagen, with the potential to determine short term changes in bone collagen synthesis

| METHODS
| RESULTS
| DISCUSSION

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