Microwave irradiation decreases <scp>ATP</scp>, increases free [Mg²⁺], and alters <i>in vivo</i> intracellular reactions in rat brain

<jats:title>Abstract</jats:title><jats:p>Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the <jats:italic>in vivo</jats:italic> state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free <jats:styled-content style="fixed-case">NAD</jats:styled-content>(<jats:styled-content style="fixed-case">P</jats:styled-content>)<jats:sup>+</jats:sup>/<jats:styled-content style="fixed-case">NAD</jats:styled-content>(<jats:styled-content style="fixed-case">P</jats:styled-content>)<jats:styled-content style="fixed-case">H</jats:styled-content> ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of <jats:styled-content style="fixed-case">ATP</jats:styled-content> were about 30% lower, whereas those of <jats:styled-content style="fixed-case">ADP</jats:styled-content>,<jats:styled-content style="fixed-case"> AMP</jats:styled-content>, and <jats:styled-content style="fixed-case">GDP</jats:styled-content> were higher in the microwave‐irradiated compared with the freeze‐blown brains. In addition, the hydrolysis of approximately 1 μmol/g of <jats:styled-content style="fixed-case">ATP</jats:styled-content>, a major <jats:italic>in vivo</jats:italic> Mg<jats:sup>2+</jats:sup>‐binding site, was related to approximately five‐fold increase in free [Mg<jats:sup>2+</jats:sup>] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ∆G' of <jats:styled-content style="fixed-case">ATP</jats:styled-content> hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and <jats:styled-content style="fixed-case">TCA</jats:styled-content> cycle metabolites, the phosphorylation potential, and the ∆G' of <jats:styled-content style="fixed-case">ATP</jats:styled-content> hydrolysis do not represent the <jats:italic>in vivo</jats:italic> state when using microwave‐fixed brain tissue.</jats:p>