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Hoppel 2016a Abstract MitoFit Science Camp 2016

From Bioblast
Skeletal muscle mitochondria in diagnostic studies.


Hoppel CL (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

Mitochondrial functional testing revolves around the key role played by ATP in energy metabolism and the approach to entering this system. The mitochondrial process involves substrate translocation, metabolism with production of reducing equivalents, NADH+, FADH+, oxidation through a series of oxido-reductases (complexes) ultimately to form water, pumping protons from the matrix side to the intermembrane space, the selective impermeability of the inner membrane to protons, and the flow of protons through Complex V leading to the production of ATP from ADP, Pi, and protons. Integration of this mitochondrial function can be measured as oxidative phosphorylation in freshly isolated mitochondria and in other preparations with intact, fresh mitochondria.

We isolate mitochondria from biopsies of skeletal muscle (vastus lateralis) and rarely liver from patients suspected of a mitochondrial disease. The activity of the electron transfer-pathway is determined in the fresh muscle and in the isolated mitochondria. In addition using spectroscopy and the freshly isolated mitochondria, the rate of ATP production is directly determined using an amino acid, glutamate & malate, and a fatty acid, oleoylcarnitine, as substrates and separately the activity of the adenine nucleotide translocase. We also directly measure Complex V (ATPase), and creatine kinase in frozen-thawed isolated mitochondria. Proteomic analysis is done using Blue Native Gel Electrophoresis to quantify supercomplexes and unincorporated complexes. Finally, we do quantitative phospholipid analysis with characterization of cardiolipin molecular species on the isolated mitochondria.

Integrated mitochondrial function is measured as oxidative phosphorylation using 19 different substrates to probe various metabolic pathways and distinct entry points of reducing equivalents into the oxidative apparatus [1]. For example, alpha-glycerol phosphate as a substrate for its dehydrogenase directly reduces coenzyme Q in a unique pathway separate from Complex I, II, or the ETF-Q oxido-reductase. Six different fatty acid substrates are used to assess the varied and exclusive metabolic pathways in fatty acid oxidation. Mitochondrial membrane integrity is directly measured using NADH oxidation. The use of oxidative phosphorylation, skeletal muscle and isolated mitochondrial ET-pathway activities provide critical information in situations where ET-pathway defects are accompanied with mitochondrial proliferation, i.e., a five-fold increase in mitochondrial content can move skeletal muscle ET-pathway activity into the low normal range, whereas the defect is clear with isolated mitochondria. i.e., per mg mitochondrial protein the defect is 20% of control, but with 5 times as much mitochondria the 20% appears at the low end of the reference interval.

Integrated mitochondrial function testing in biopsy material provides an excellent approach to the diagnosis of mitochondrial diseases in challenging clinical presentations. Similar type of measurements can be done in cultured skin fibroblasts and cells harvested from blood.

O2k-Network Lab: US OH Cleveland Hoppel CL

Labels: MiParea: mt-Membrane, mt-Awareness 

Stress:Cryopreservation, Mitochondrial disease  Organism: Human  Tissue;cell: Skeletal muscle, Liver  Preparation: Isolated mitochondria  Enzyme: Complex V;ATP synthase, Supercomplex  Regulation: ATP production, Substrate 

Pathway: F, N, Other combinations 

Event: C1  MitoFit Science Camp 2016 


Center Mitochondr Diseases, Depts Pharmacology & Medicine, Case Western Reserve School Med, Cleveland, OH, USA. - [email protected]


  1. Puchowicz MA, Varnes ME, Cohen BH, Friedman NR, Kerr DS, Hoppel CL (2004) Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria-case studies. Mitochondrion 4:377-85. »Bioblast Link