Brain Adenylosuccinate Is Dramatically Increased Under Global Ischemia Without Evidence for Purine Nucleotide Cycle Activation

Abstract

It is well documented that adenosine and adenine nucleotides, such as ATP, ADP, and AMP, undergo significant alterations within seconds upon brain ischemia. For their accurate quantification, in situ deactivation of enzymes involved in their metabolism is required to prevent postmortem alterations. Thus, techniques such as high energy head-focused microwave irradiation (MW) or freeze-blowing are often used prior to metabolome analysis. However, alterations of another important purine nucleotide, adenylosuccinate (AdSucc), under brain ischemia have not been previously addressed. AdSucc is an intermediate in purine nucleotide de novo synthesis. Over 50 years ago, it was also proposed to have a role in brain energy metabolism through the purine nucleotide cycle (PNC) similar to that in muscle, with, to the best of our knowledge, no follow-up studies. In the present study, we applied MW and LC–MS analysis for mouse brain AdSucc quantification in situ at baseline and upon 30 s, 2 min, and 10 min of global ischemia. Our data indicate that in situ enzyme deactivation is required for brain AdSucc quantification. We report, for the first time, that brain AdSucc is dramatically increased 19-fold at 30 s ischemia and 77-fold at 2 min, from 0.007 ± 0.001 to 0.136 ± 0.026 and 0.555 ± 0.036 nmol/mg of brain wet weight (ww), respectively, without further increase at 10 min, positioning it as one of the major brain metabolites under ischemia (~0.56 mM). Quantification of PNC and tricarboxylic acid cycle (TCA) metabolites did not support the role of AdSucc induction in the activation of these pathways under ischemia. Importantly, a significant AdSucc increase up to ~0.56 mM did not affect its precursor aspartate (Asp), which remained at ~1 mM (0.923 ± 0.036 nmol/mg ww) during ischemia, indicating that AdSucc is not produced by the condensation reaction between Asp and IMP in the PNC catalyzed by adenylosuccinate synthase (ADSS). Further studies are required to elucidate the mechanisms for AdSucc increase and its role under brain ischemia.