目的:代谢组学作为一种解决生物学问题的方法,其应用呈指数级增长。因此,这是采用最佳做法的关键时刻。众所周知,破坏的组织氧供应迅速改变细胞能量电荷。然而,解剖后延迟的小鼠组织冷冻改变广泛代谢组的速度和程度没有很好的描述。此外,组织基因型如何调节这种代谢组学漂移以及追踪的13C同位素体分布可能发生变化的程度尚未得到解决.
方法:通过结合液相色谱(LC)和气相色谱(GC)-质谱(MS),我们测量了255只小鼠肝脏代谢物的水平在30秒后如何变化,1分钟,3分钟,和10分钟的冻结延迟。然后,我们进行了概念测试延迟冻结实验,评估小鼠心脏和骨骼肌的广泛代谢组学漂移,野生型(WT)和线粒体丙酮酸载体(MPC)敲除小鼠肝脏之间的差异代谢组学变化,以及从13C标记的葡萄糖到小鼠肝脏的13C同位素丰度和富集的变化。
结果:我们的数据表明,解剖后延迟的小鼠组织冷冻导致广泛代谢组的快速缺氧驱动重塑,诱导假阴性和假阳性基因型之间的差异,以及13C同位素分布的重组。值得注意的是,我们表明嘌呤核苷酸降解产物的增加是肝脏和心脏冷冻延迟的一个特别高的动态范围标记。
结论:我们的发现提供了以前没有的,系统地说明了广泛的,在组织冷冻延迟的早期几分钟内发生的多结构域代谢组变化。他们还提供了一部小说,小鼠肝脏离体的详细资源,缺氧代谢组学重塑。
Metabolomics as an approach to solve biological problems is exponentially increasing in use. Thus, this a pivotal time for the adoption of best practices. It is well known that disrupted tissue oxygen supply rapidly alters cellular energy charge. However, the speed and extent to which delayed mouse tissue freezing after dissection alters the broad metabolome is not well described. Furthermore, how tissue genotype may modulate such metabolomic drift and the degree to which traced 13C-isotopologue distributions may change have not been addressed.
By combined liquid chromatography (LC)- and gas chromatography (GC)-mass spectrometry (MS), we measured how levels of 255 mouse liver metabolites changed following 30-second, 1-minute, 3-minute, and 10-minute freezing delays. We then performed test-of-concept delay-to-freeze experiments evaluating broad metabolomic drift in mouse heart and skeletal muscle, differential metabolomic change between wildtype (WT) and mitochondrial pyruvate carrier (MPC) knockout mouse livers, and shifts in 13C-isotopologue abundances and enrichments traced from 13C-labled glucose into mouse liver.
Our data demonstrate that delayed mouse tissue freezing after dissection leads to rapid hypoxia-driven remodeling of the broad metabolome, induction of both false-negative and false-positive between-genotype differences, and restructuring of 13C-isotopologue distributions. Notably, we show that increased purine nucleotide degradation products are an especially high dynamic range marker of delayed liver and heart freezing.
Our findings provide a previously absent, systematic illustration of the extensive, multi-domain metabolomic changes occurring within the early minutes of delayed tissue freezing. They also provide a novel, detailed resource of mouse liver ex vivo, hypoxic metabolomic remodeling.