Fig 1. Effect of sample processing on GLDH quantification.

GLDH is located within the mitochondrial matrix and it has been hypothesized that failure to remove intact mitochondria during blood processing for serum/plasma can alter GLDH quantifications. Therefore, we processed blood samples collected from mice in multiple ways to determine if mitochondrial removal impacted measurements (A). All blood samples initially underwent a standard low speed spin to remove red blood cells and debris and to isolate plasma. An aliquot from each of these samples underwent an additional high-speed spin that was forceful enough to pellet any intact mitochondria that may have been present in the standard plasma to obtain post-mitochondrial supernatant (16,000xg plasma). GLDH was then quantified in the fresh samples with the expectation that if the assay reagents can penetrate the mitochondrial membrane, removal of intact mitocondria would result in a reduction in GLDH levels. However, if the reagents cannot penetrate the undamaged membrane, the presence of intact mitochondria in plasma will not be consequential until these mitochondria are lysed (B). We performed three freeze/thaw cycles on our samples which would presumably lyse any mitochondria that remained in the plasma samples. This lysis should liberate GLDH from damaged mitochondria and result in a rise in GLDH levels if the assay reagents cannot detect GLDH in intact mitochondria.