Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells

The assessment of mitochondrial respiratory chain (RC) enzymatic activities is essential for investigating mitochondrial function in several situations, including mitochondrial disorders, diabetes, cancer, aging and neurodegeneration, as well as for many toxicological assays. Muscle is the most commonly analyzed tissue because of its high metabolic rates and accessibility, although other tissues and cultured cell lines can be used. We describe a step-by-step protocol for a simple and reliable assessment of the RC enzymatic function (complexes I–IV) for minute quantities of muscle, cultured cells and isolated mitochondria from a variety of species and tissues, by using a single-wavelength spectrophotometer. An efficient tissue disruption and the choice for each assay of specific buffers, substrates, adjuvants and detergents in a narrow concentration range allow maximal sensitivity, specificity and linearity of the kinetics. This protocol can be completed in 3 h.

[1]  B. Ames,et al.  Part X • Chapter 27 – Oxidants and aging , 2000 .

[2]  Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans. , 2007, Methods in molecular biology.

[3]  Robert W. Taylor,et al.  Biochemical assays of respiratory chain complex activity. , 2007, Methods in cell biology.

[4]  T. Peng,et al.  Mitochondrial dysfunction in Parkinson's disease. , 1999, Biochemical Society symposium.

[5]  D. Wallace,et al.  Evaluation of procedures for assaying oxidative phosphorylation enzyme activities in mitochondrial myopathy muscle biopsies. , 1990, Biochimica et biophysica acta.

[6]  B. Barrell,et al.  Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding , 1994, Cell.

[7]  M. Jendrach,et al.  Mitochondrial dysfunction: An early event in Alzheimer pathology accumulates with age in AD transgenic mice , 2009, Neurobiology of Aging.

[8]  F. Fontanesi,et al.  Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Polarography and Spectrophotometric Enzyme Assays , 2009, Current protocols in human genetics.

[9]  C. Hoppel,et al.  Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity. , 2007, Mitochondrion.

[10]  G. Attardi,et al.  Polarographic assays of respiratory chain complex activity. , 2007, Methods in cell biology.

[11]  R. Margreiter,et al.  Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells , 2008, Nature Protocols.

[12]  W. Sperl,et al.  The problem of interlab variation in methods for mitochondrial disease diagnosis: enzymatic measurement of respiratory chain complexes. , 2004, Mitochondrion.

[13]  T. Bourgeron,et al.  The measurement of the rotenone-sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. , 1990, Biochemical and biophysical research communications.

[14]  R. Rodenburg,et al.  Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology. , 2006, Clinical chemistry.

[15]  Lichuan Yang,et al.  Assay of mitochondrial ATP synthesis in animal cells and tissues. , 2007, Methods in cell biology.

[16]  C. Hoppel,et al.  Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations. , 1985, Archives of biochemistry and biophysics.

[17]  V. Pertegato,et al.  Optimization of respiratory chain enzymatic assays in muscle for the diagnosis of mitochondrial disorders. , 2011, Mitochondrion.

[18]  Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations. , 2002 .

[19]  M. Roden,et al.  The role of mitochondria in insulin resistance and type 2 diabetes mellitus , 2012, Nature Reviews Endocrinology.

[20]  Charis Eng,et al.  A role for mitochondrial enzymes in inherited neoplasia and beyond , 2003, Nature Reviews Cancer.

[21]  N. Bresolin,et al.  Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting, and mental impairment , 1986, Neurology.

[22]  S. Dimauro,et al.  Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations. , 2002, The Biochemical journal.

[23]  Robert S. Balaban,et al.  Mitochondria, Oxidants, and Aging , 2005, Cell.

[24]  S. Dimauro,et al.  Mitochondrial respiratory-chain diseases. , 2003, The New England journal of medicine.

[25]  L. Scorrano,et al.  Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts , 2007, Nature Protocols.

[26]  Keshav K. Singh,et al.  Genetic insights into OXPHOS defect and its role in cancer. , 2011, Biochimica et biophysica acta.

[27]  A. Baracca,et al.  Evaluating Mitochondrial Membrane Potential in Cells , 2007, Bioscience reports.

[28]  D. Wallace,et al.  Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. , 1996, Methods in enzymology.

[29]  Ann Saada,et al.  Biochemical assays for mitochondrial activity: assays of TCA cycle enzymes and PDHc. , 2007, Methods in cell biology.

[30]  Jörg Haberstroh,et al.  Time-Dependent and Tissue-Specific Accumulation of mtDNA and Respiratory Chain Defects in Chronic Doxorubicin Cardiomyopathy , 2003, Circulation.

[31]  R. Rodenburg,et al.  Mitochondrial ATP synthase: architecture, function and pathology , 2011, Journal of Inherited Metabolic Disease.

[32]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[33]  J. Veerkamp,et al.  Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure. , 1985, Clinica chimica acta; international journal of clinical chemistry.

[34]  R. Rodenburg,et al.  Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. , 2007, Clinical chemistry.

[35]  Ò. Miró,et al.  Mitochondrial Dna Depletion and Respiratory Chain Enzyme Deficiencies are Present in Peripheral Blood Mononuclear Cells of HIV-Infected Patients with Haart-Related Lipodystrophy , 2002, Antiviral therapy.

[36]  J. Mazat,et al.  Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis. , 2009, Mitochondrion.

[37]  M. Beal,et al.  Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases , 2006, Nature.