Comparison of FTIR and NADH-Dependent Oxygen Uptake Analyses As Metabolomic Evaluation Methods.

The Nicotinamide Adenine Dinucleotide (NADH)-dependent oxygen uptake assay has been proven to be an effective metabolomic method for evaluating respiration in isolated mitochondria or specific complex inhibition. This is a labor intensive method however, with high variability. Fourier transform infrared spectroscopy (FTIR) can also be used to indicate respiration inhibition, inhibitor mode of action, and dose response in mitochondria and is much less labor intensive. In a previous study conducted by Heckman et al. (2002), site of action was determined by using oxygen electrode assay methods to measure inhibition for each protein of oxidative phosphorylation individually. With FTIR, more rapid elucidation of site of action can be determined with spectra similarities of unknown treated mitochondria with that of mitochondria treated with inhibitors that have previously reported sites of action. The objective of these experiments was to evaluate and compare the effect of eight chemicals on the respiration of mitochondria isolated from wheat (Triticum aestivum L.) seedlings. Three main groups were shown in the dendrogram performed from cluster analysis. All chemicals were separated into two groups, and the third group contained untreated mitochondria and the solvent blank. The third group demonstrates that there was no effect on the mitochondria due to the solvent that was used as a vehicle for the chemicals. The grouping for each chemical agrees with their previously reported mode of actions, and by comparison the mode of action of trinexapac-ethyl was confirmed. FTIR spectroscopy will be a very powerful and efficient method to use in future mitochondrial respiration studies.