Importance of spontaneous alpha-ketoacid decarboxylation in experiments involving peroxide

The potential role of spontaneous alpha-ketoacid decarboxylation as a source of interference in experiments involving peroxide was investigated. The assay of pyruvate dehydrogenase activity in isolated renal mitochondria was employed as an example. Spontaneous peroxide-induced pyruvate decarboxylation competed significantly with enzymatic decarboxylation at peroxide concentrations greater than 50 microM. Corrected values for enzymatic decarboxylation could be obtained by subtracting spontaneous decarboxylation rates from rates obtained in the presence of mitochondria. At higher peroxide concentrations (greater than 200 microM), reaction product accumulates (acetoacetate) to levels which may have regulatory effects on mitochondrial metabolism. The divalent cations, Ca2+ and Mg2+, both accelerate spontaneous peroxide-induced pyruvate decarboxylation while other components of the assay medium had an inhibitory effect on the reaction. The results are discussed in relation to the currently accepted reaction mechanism. Investigators who perform experiments involving reactive oxygen species should be familiar with this often overlooked reaction.