Mitochondrial diseases, caused by defects in the electron transport chain (ETC), are progressive, often fatal, diseases that can affect any age. Mitochondrial diseases can be difficult to recognize because of their protean manifestations. It is estimated that around 90 genes, both nuclear and mitochondrial, are necessary to encode for all the subunits in complexes I-V of the ETC. A myriad more are necessary for their correct assembly and function. Consequently, it is not practical to use genetic testing for confirmation of suspected mitochondrial disease. Functional assays of ETC activity, however, can be quite useful in narrowing in on a defect as isolated to a specific complex or affecting the mitochondria as a whole. Muscle biopsy is the gold standard. Unfortunately, this is an invasive procedure and if handled improperly can lead to both false-positive or false-negative results. Consequently, there is a need for the development of additional testing modalities. The purpose of this abstract is to describe our progress developing a new diagnostic test that seeks to use platelets as a source of mitochondria for study. Each platelet contains one to three mitochondria that provide the energy for platelet function. Previous studies have shown that mitochondrial DNA mutations or ETC defects can be detected in mitochondria from platelets when they were not detectable in white blood cells. This disparity may be due to a difference in the selective pressure on these two cell lines because of there different energetic requirements. Based on this theory, we have developed a functional assay of the ETC in platelet mitochondria. We use well-known, polarographic techniques that allow examination of each ETC complex in a single assay by use of a series of specific substrates and inhibitors. Here, however, these techniques are applied in a newly available respirometer that, because it is highly controlled and extremely sensitive, allows polarographic assessment in systems where it was not previously possible. We have been successful in developing assay conditions for platelets that preserve activity in each of the ETC complexes from normal volunteers. There seems to be excellent reproducibility of results with this assay. Additionally, we have optimized the isolation of platelets to minimize the volume of blood required. We are now in the process of collecting data on additional normal volunteers as well as patients with known mitochondrial defects to assess the ultimate utility of this assay. It is our hope that this assay will allow detection of ETC defects in patients and in some cases obviate the need for muscle biopsy.
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