Metabolites are resolved by reverse phase ion pairing HPLC and detected and quantified using electrochemical analysis (GSH and GSSG) and fluorescence (NADH and NADPH) and UV/VIS (NAD+, NADP+, CoASH, and acetyl-CoA) spectroscopy as we have described previously (see below). The identities of relevant compounds are confirmed by GC-MS and routine spiking of experimental samples with known quantities of standards to ensure accurate peak assignment. A major advantage conferred by these methods is the ability to analyze all redox couples of interest using biological samples containing less than 0.25 mg of protein and two extraction protocols. The types of samples amenable to analyses include isolated organelles, protein homogenates, and flash frozen tissue or cells from invertebrates and vertebrates. Samples are prepared for analysis of GSH, GSSG, NADP+, and NAD+ by extraction with 5% meta-phosphoric acid. Importantly, extraction with 5% meta-phosphoric acid yields protein pellets that can be further probed by non-reducing gel electrophoresis followed by Western blot analysis for the relative level of oxidized to reduced thioredoxin. A separate extraction with 125 mM KOH enables analysis of NADPH and NADH as well as AMP, ADP, and ATP. The limits of detection for the various compounds are:
These values are given for heart tissue, however metabolite concentrations vary less than 1-fold between tissues and would therefore have similar detection limits.
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