The Activity Of Mitochondrial Oxidative Phosphorylation Enzyme Complexes Of Mitochondrial Cytopathies And Its Significance

Mitochondrial cytopathies is a group of multisystem diseases; mainly affect skeletal muscle, cardiac muscle and central nervous system. So they are also named as mitochondiral encephalomyopathies clinically. Abnormal mitochondrial function is involved in aging and neurodegenerative diseases such as Alzheimer's disease and Parkinson disease. Mitochondrial DNA mutation is one of the causes of these diseases. But most mitochondrial structural and functional proteins are coded by nuclear genome; their genotypes are not strictly corresponding with phenotypes. So, clinical manifestation, radiology characteristics, muscular pathology, mtDNA mutation analysis and measurement of OXPHOS activities are integrated to make a reasonable diagnosis. Theoretical, any mutation affect mitochondrial functions through OXPHOS complexes. So activities of mitochondrial OXPHOS have not only important diagnosis value but can be benefit of deep research of pathogenesis, selection and detection of clinical medication. There are several methods to measure OXPHOS activities, which need complex reagents and reaction conditions. The problem of interlab variation in methods for mitochondrial disease diagnosis has emerged. Especially the use of frozen muscle tissues of mitochondrial cytopathies patients has no perfect methods and credible results. This experiment presents spectrophotometric techniques to study mitochondrial OXPHOS activities, especially considered are techniques that can be successfully both in two-step centrifugation and low-speed centrifugation frozen muscular tissue mitochondrial preparations. We analyze the relationship of genotypes, phenotypes and OXPHOS activity.Object: First, spectrophotometric techniques were used to study mitochondrialOXPHOS activities, especially techniques that can be successfully both in two-step centrifugation and low-speed centrifugation frozen muscular tissue mitochondrial preparations. Mitochondrial preparations of normal fresh and frozen muscular tissue were used as comparison. Second, OXPHOS activities were measured in different subtypes of mitochondrial cytopathies, and analyze the relationship of genotypes, phenotypes and OXPHOS activity. Last, mitochondrial cytopathies were subdivided according their OXPHOS activities deficiency types.Method: All 36 cases are from instituete of neuromuscular disorders of Qilu hospital, Shandong University. Based on clinical manifestation, radiology characteristics and muscular pathology, MELAS(12 patients), CPEO(5 patients), KSS(3 patients), mitochondrial myopathy(3 patients), Leigh syndrome(2 patients) and undefined type mitochondrial cytopathy(2 patients) were identified. Another 9 patients affected by non-mitochondrial cytopathy muscular disorders and 10 normal surgical examples were included in our study as comparison. Different exacting mitochondrial preparation methods were used according the quantity of muscle. Sectrophotometric techniques were presents to study mitochondrial OXPHOS complexes activities, and compared with self citrate synthase activities.Result: Mitochondrial OXPHOS complexes activities have statistic significance between fresh and frozen muscle tissues of normal surgical examples. Different exacting methods also affect the activities. Complex I and III have statistic difference in MELAS patients. Complex I , Illand IV have statistic difference in CPEO patients. Complex V activity decrease in a Leigh syndrome patient, and have good relation with a new nonsense point mutation in ATPase6 gene. Complex I activity decrease is the most significant in all 27 patients, especially in MELAS patients.Conclusion: Although the sectrophotometric methods of OXPHOS measurement are perfect in theory, a lot of miscellaneous and uneasy-conserved reagents are needed, and different lab conditions also affect the result. So controlling the complication of reagents and lab conditions plays a important role in this study. We suggest that the best material is two-step or low-speed centrifugationmitochondrial preparations from fresh muscle tissues. Although different subtypes of mitochondrial cytopathies have variable degree change of OXPHOS activities, it is important to combine the clinical manifestation, radiology characteristics, genetics result and muscular pathology when analyze the patients. Measurement of OXPHOS is useful of discussing pathogenesis of known-mutations and confirming the new mutations, beneficial to lead researches to reduce the range of detecting genes and find the new mutations.