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PO1-6-7

Determination of serum metabolites in mouse based on stable isotope-resolved metabolomics

[Speaker] Xuemei Qin:1
[Co-author] Ting Linghu:1, Junsheng Tian:1, Xiang Zhang:2, Gunahua Du:3
1:Modern Research Center for Traditional Chinese Medicine, Shanxi university, China, 2:Department of Chemistry, University of Louisville, USA, 3:Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, China

Background: Metabolites in a metabolome are derived from enzymatic reactions and form a network where the outputs of preceding enzymatic reactions provide inputs to others. In metabolite profiling metabolomics, the measured concentration of a metabolite is its summed abundance if this metabolite is synthesized in more than one pathway. The relative contribution of those pathways to the synthesis of that metabolite remains ambiguous. Stable isotope-resolved metabolomics (SIRM) uses heavy isotope tracers (e.g., 13C, 18O, 15N) to identify and discern pathways involved in biochemical processes, by measuring the incorporation of heavy atoms into the metabolites produced downstream of the tracer(s).
Methods: We developed a device for injection of stable isotope labeled tracer, e.g. 13C-glucose, to mouse caudal vein. We then applied the developed device to a SIRM study. Each of 36 mouse were injected with 3 mL of 0.05, 0.1, 0.15, 0.2 mmol/kg/min 13C-glucose for 5, 6, 7 h, respectively. After sacrificing mouse, about 1.5 mL blood were collected from each mouse. Metabolites were extracted from mouse blood using 50% acetonitrile. The extracted metabolites were then analyzed by LC-MS.
Results: The developed device was composed of a mouse fixator, microinjection needle and syringe pump. While mouse is confined in the device, it can breathe freely and did not have observable injure even after 7 h of continuous injection. Sample pretreatment methods as well as LC-MS operation and instrument parameters were optimized for high sensitive detection of the metabolites involved in glycolysis and TCA cycle. The optimal analytical method was established by comparing the number and the degree of heavy isotope incorporation in each metabolite. Overall, 13C incorporation in metabolites reached a plateau in 6 h injection of 0.1 mmol/kg/min 13C-glucose.
Conclusions: A mouse caudal vein injection device was built, and the method of stable isotope-resolved metabolomics was established using model mouse. This method developed in this study can be used for any mechanistic research on disease development and drug treatment.
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