Musculoskeletal Physiology Research Group, Sport Health and Performance Enhancement Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, UK.
Clinical, Metabolic and Molecular Physiology, University of Nottingham, Royal Derby Hospital, Derby, UK.
Physiol Rep. 2021 May;9(10):e14799. doi: 10.14814/phy2.14799.
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 (D 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 D 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.
开发安全实用的策略来预防骨组织弱化至关重要，但由于缺乏对骨胶原转换短期（数天-数周）生理学的了解，这一尝试受到了阻碍。为了解决这个问题，我们开发了一种使用氘水（DO）示踪剂掺入技术结合气相色谱-热裂解-同位素比质谱（GC-pyrolysis-IRMS）来定量体内骨胶原合成的方法。46 只来自选择性繁殖模型的雄性和雌性大鼠摄入 DO 3 周。所有大鼠死后均从股骨骨干（FEM）、胫骨近端（T-PRO）、远端（T-DIS）骺-干骺端和胫骨中段骨干（T-MID）获得。脱矿后，分离胶原蛋白蛋白并水解，通过 GC-pyrolysis-IRMS 将氘掺入蛋白结合丙氨酸来确定胶原蛋白的分数合成率（FSR）。FEM 的胶原蛋白 FSR（0.131±0.078%/天；95%CI[0.106-0.156]）大于 T-MID 的 FSR（0.055±0.049%/天；95%CI[0.040-0.070]；p<0.001）。T-PRO 部位的 FSR 最高（0.203±0.123%/天；95%CI[0.166-0.241]），T-DIS 部位最低（0.027±0.015%/天；95%CI[0.022-0.031]）。三个胫骨部位的 FSR 不同（p<0.001）。在此，我们开发了一种灵敏的方法来定量体内骨胶原合成，并确定了特定部位的合成率，这可能适用于人类骨胶原转换的研究。
