Mouse tissue harvest-induced hypoxia rapidly alters the in vivo metabolome, between-genotype metabolite level differences, and 13C-tracing enrichments

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Hypoxia; Metabolomics; Mitochondrial pyruvate carrier; Purine degradation; Quality control; Stable isotope tracing.