Difference between revisions of "Ye 2013 Anal Biochem"

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(Created page with "{{Publication |title=Ye F, Hoppel CL (2013) Measuring oxidative phosphorylation in human skin fibroblasts. Anal Biochem [Epub ahead of print]. |info=[http://www.ncbi.nlm.nih.gov...")
 
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{{Publication
|title=Ye F, Hoppel CL (2013) Measuring oxidative phosphorylation in human skin fibroblasts. Anal Biochem [Epub ahead of print].
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|title=Ye F, Hoppel CL (2013) Measuring oxidative phosphorylation in human skin fibroblasts. Anal Biochem 437: 52-58
 
|info=[http://www.ncbi.nlm.nih.gov/pubmed/23462540 PMID: 23462540]
 
|info=[http://www.ncbi.nlm.nih.gov/pubmed/23462540 PMID: 23462540]
 
|authors=Ye F, Hoppel CL
 
|authors=Ye F, Hoppel CL
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|journal=Anal Biochem
 
|journal=Anal Biochem
 
|abstract=An approach has been developed to quantitate oxidative phosphorylation in harvested human skin fibroblasts that have been permeabilized with digitonin. In protocol 1, state 3 rates are measured with Complex I and II substrates, followed by uncoupled maximal oxidative capacity measured in the presence of these combined substrates, as well as through Complex IV. In protocol 2, state 3 rates are measured using palmitoylcarnitine to monitor fatty acid oxidation, and duroquinol to assess the flux through Complex III; uncoupled duroquinol oxidation measures maximal oxidative capacity through Complex III. The activity of citrate synthase is determined in every experiment as a marker of the amount of mitochondria per chamber. Data are expressed on the basis of cell count (per million fibroblasts), of protein, or of citrate synthase activity. Cell growth conditions are optimized and it is necessary to keep cultured cells from reaching confluency. Cultures in passages 3 to 10 show reproducible oxidative phosphorylation data. Based on the data from the 15 normal human skin fibroblast lines, we are evaluating the use of this approach to diagnose systemic mitochondrial disease and avoid issues associated with open skeletal muscle biopsy.
 
|abstract=An approach has been developed to quantitate oxidative phosphorylation in harvested human skin fibroblasts that have been permeabilized with digitonin. In protocol 1, state 3 rates are measured with Complex I and II substrates, followed by uncoupled maximal oxidative capacity measured in the presence of these combined substrates, as well as through Complex IV. In protocol 2, state 3 rates are measured using palmitoylcarnitine to monitor fatty acid oxidation, and duroquinol to assess the flux through Complex III; uncoupled duroquinol oxidation measures maximal oxidative capacity through Complex III. The activity of citrate synthase is determined in every experiment as a marker of the amount of mitochondria per chamber. Data are expressed on the basis of cell count (per million fibroblasts), of protein, or of citrate synthase activity. Cell growth conditions are optimized and it is necessary to keep cultured cells from reaching confluency. Cultures in passages 3 to 10 show reproducible oxidative phosphorylation data. Based on the data from the 15 normal human skin fibroblast lines, we are evaluating the use of this approach to diagnose systemic mitochondrial disease and avoid issues associated with open skeletal muscle biopsy.
|keywords=Diagnosis, Systemic mitochondrial disease  
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|keywords=Diagnosis, Systemic mitochondrial disease
 
|mipnetlab=US OH Cleveland Hoppel CL
 
|mipnetlab=US OH Cleveland Hoppel CL
 
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Revision as of 13:31, 31 May 2013

Publications in the MiPMap
Ye F, Hoppel CL (2013) Measuring oxidative phosphorylation in human skin fibroblasts. Anal Biochem 437: 52-58

» PMID: 23462540

Ye F, Hoppel CL (2013) Anal Biochem

Abstract: An approach has been developed to quantitate oxidative phosphorylation in harvested human skin fibroblasts that have been permeabilized with digitonin. In protocol 1, state 3 rates are measured with Complex I and II substrates, followed by uncoupled maximal oxidative capacity measured in the presence of these combined substrates, as well as through Complex IV. In protocol 2, state 3 rates are measured using palmitoylcarnitine to monitor fatty acid oxidation, and duroquinol to assess the flux through Complex III; uncoupled duroquinol oxidation measures maximal oxidative capacity through Complex III. The activity of citrate synthase is determined in every experiment as a marker of the amount of mitochondria per chamber. Data are expressed on the basis of cell count (per million fibroblasts), of protein, or of citrate synthase activity. Cell growth conditions are optimized and it is necessary to keep cultured cells from reaching confluency. Cultures in passages 3 to 10 show reproducible oxidative phosphorylation data. Based on the data from the 15 normal human skin fibroblast lines, we are evaluating the use of this approach to diagnose systemic mitochondrial disease and avoid issues associated with open skeletal muscle biopsy.

Keywords: Diagnosis, Systemic mitochondrial disease

O2k-Network Lab: US OH Cleveland Hoppel CL


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Organism: Human  Tissue;cell: Skeletal muscle  Preparation: Permeabilized cells  Enzyme: Complex I, Complex II; Succinate Dehydrogenase"Complex II; Succinate Dehydrogenase" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property., Complex III, Complex IV; Cytochrome c Oxidase"Complex IV; Cytochrome c Oxidase" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property.  Regulation: Fatty Acid"Fatty Acid" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.  Coupling state: OXPHOS 

HRR: Oxygraph-2k