Tissue lysates obtained through cell lysis buffer processing (described above) were batch processed for citrate synthase activity as previously described by our laboratory (Kephart et al., 2015 (link)). This metric was used as a surrogate for mitochondrial content per the findings of Larsen et al. (2012) (link) suggesting citrate synthase activity highly correlates with transmission electron micrograph (TEM) images of mitochondrial content (r = 0.84, p < 0.001). The assay principle is based upon the reduction of 5,50-dithiobis(2- nitrobenzoic acid) (DTNB) at 412 nm (extinction coefficient 13.6 mmol/L/cm) coupled to the reduction of acetyl-CoA by the citrate synthase reaction in the presence of oxaloacetate. Briefly, five μg of skeletal muscle protein was added to a mixture composed of 0.125 mol/L Tris–HCl (pH 8.0), 0.03 mmol/L acetyl-CoA, and 0.1 mmol/L DTNB. The reaction was initiated by the addition of 5 μL of 50 mmol/L oxaloacetate and the absorbance change was recorded for 1 min. The average CV values for all duplicates was 4.6%.
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