背景:骨是一种代谢活跃的组织,含有作为内分泌靶标和效应物的不同细胞类型。Further,骨骼是钙的动态仓库,磷和其他必需矿物质。响应于机械/内分泌刺激,组织基质经受恒定的周转。骨转换需要高能量水平,使脂肪酸成为骨细胞的重要来源。然而,目前对骨细胞代谢的认识较差。这部分是由于骨基质的复杂性和从骨样品中提取小分子的困难。这项研究旨在评估从蛋白质主导的基质中螯合代谢物的效果,以提高代谢组学数据的质量和数量,以发现病理条件下的小分子模式。
方法:从接受髋关节置换手术的65至85岁(高龄)患者中收集人骨样本。分离的皮质和小梁骨粉用脱钙处理,酶(胶原酶I和蛋白酶K)和基于溶剂的代谢物提取方案。用高分辨率质谱(HRMS)分析提取的混合物。使用XCMS和MetaboAnalystR包进行数据分析。
结果:在添加溶剂之前,骨样品的快速酶处理导致代谢物提取的产率明显更高。胶原酶I和蛋白酶K的快速消化在皮质和骨小梁样品中显示出更高的有效性,与显著更高的率(2.2倍)的胶原酶I进一步分析显示显著富集的途径,如从头脂肪酸生物合成,鞘糖脂代谢和脂肪酸氧化-过氧化物酶体。
结论:这项工作提出了一种用于HRMS代谢组学的骨样品制备的新方法。骨基质构象在分子水平上的破坏有助于分子释放到提取溶剂中,因此,可以导致更高质量的结果和可信的生物标志物发现。我们的结果显示老化的骨样品中的β-氧化改变。未来涵盖更多患者的工作值得确定有效的治疗方法以实现健康衰老。
Bone is a metabolically active tissue containing different cell types acting as endocrine targets and effectors. Further, bone is a dynamic depot for calcium, phosphorous and other essential minerals. The tissue matrix is subjected to a constant turnover in response to mechanical/endocrine stimuli. Bone turnover demands high energy levels, making fatty acids a crucial source for the bone cells. However, the current understanding of bone cell metabolism is poor. This is partly due to bone matrix complexity and difficulty in small molecules extraction from bone samples. This study aimed to evaluate the effect of metabolite sequestering from a protein-dominated matrix to increase the quality and amount of metabolomics data in discovering small molecule patterns in pathological conditions.
Human bone samples were collected from 65 to 85 years old (the elderly age span) patients who underwent hip replacement surgery. Separated cortical and trabecular bone powders were treated with decalcifying, enzymatic (collagenase I and proteinase K) and solvent-based metabolite extraction protocols. The extracted mixtures were analyzed with the high-resolution mass spectrometry (HRMS). Data analysis was performed with XCMS and MetaboAnalystR packages.
Fast enzymatic treatment of bone samples before solvent addition led to a significantly higher yield of metabolite extraction. Collagenase I and proteinase K rapid digestion showed more effectiveness in cortical and trabecular bone samples, with a significantly higher rate (2.2 folds) for collagenase I. Further analysis showed significant enrichment in pathways like de novo fatty acid biosynthesis, glycosphingolipid metabolism and fatty acid oxidation-peroxisome.
This work presents a novel approach for bone sample preparation for HRMS metabolomics. The disruption of bone matrix conformation at the molecular level helps the molecular release into the extracting solvent and, therefore, can lead to higher quality results and trustable biomarker discovery. Our results showed β-oxidation alteration in the aged bone sample. Future work covering more patients is worthy to identify the effective therapeutics to achieve healthy aging.