Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
DiMauro, S. & Schon, E.A. Mitochondrial respiratory-chain diseases. N. Engl. J. Med. 348, 2656–2668 (2003).
Balaban, R.S., Nemoto, S. & Finkel, T. Mitochondria, oxidants, and aging. Cell 120, 483–495 (2005).
Szendroedi, J., Phielix, E. & Roden, M. The role of mitochondria in insulin resistance and type 2 diabetes mellitus. Nat. Rev. Endocrinol. 8, 92–103 (2011).
Chandra, D. & Singh, K.K. Genetic insights into OXPHOS defect and its role in cancer. Biochim. Biophys. Acta 1807, 620–625 (2011).
Eng, C., Kiuru, M., Fernandez, M.J. & Aaltonen, L.A. A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat. Rev. Cancer 3, 193–202 (2003).
Miro, O. 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. Antivir. Ther. 8, 333–338 (2003).
Lebrecht, D., Setzer, B., Ketelsen, U.P., Haberstroh, J. & Walker, U.A. Time-dependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy. Circulation 108, 2423–2429 (2003).
Lin, M.T. & Beal, M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 (2006).
Winklhofer, K.F. & Haass, C. Mitochondrial dysfunction in Parkinson's disease. Biochim. Biophys. Acta 1802, 29–44 (2010).
Hauptmann, S. et al. Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice. Neurobiol. Aging 30, 1574–1586 (2009).
Reisch, A.S. & Elpeleg, O. Biochemical assays for mitochondrial activity: assays of TCA cycle enzymes and PDHc. Methods Cell Biol. 80, 199–222 (2007).
Villani, G. & Attardi, G. Polarographic assays of respiratory chain complex activity. Methods Cell Biol. 80, 121–133 (2007).
Kuznetsov, A.V. et al. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat. Protoc. 3, 965–976 (2008).
Vives-Bauza, C., Yang, L. & Manfredi, G. Assay of mitochondrial ATP synthesis in animal cells and tissues. Methods Cell Biol. 80, 155–171 (2007).
Janssen, A.J. et al. Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology. Clin. Chem. 52, 860–871 (2006).
Solaini, G., Sgarbi, G., Lenaz, G. & Baracca, A. Evaluating mitochondrial membrane potential in cells. Biosci. Rep. 27, 11–21 (2007).
Spinazzi, M. et al. Optimization of respiratory chain enzymatic assays in muscle for the diagnosis of mitochondrial disorders. Mitochondrion 11, 893–904 (2011).
Medja, F. et al. Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis. Mitochondrion 9, 331–339 (2009).
Gellerich, F.N., Mayr, J.A., Reuter, S., Sperl, W. & Zierz, S. The problem of interlab variation in methods for mitochondrial disease diagnosis: enzymatic measurement of respiratory chain complexes. Mitochondrion 4, 427–439 (2004).
Trounce, I.A., Kim, Y.L., Jun, A.S. & Wallace, D.C. Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol. 264, 484–509 (1996).
Salviati, L. et al. Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations. Biochem. J. 363, 321–327 (2002).
Angelini, C. et al. Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting, and mental impairment. Neurology 36, 1048–1052 (1986).
Zheng, X.X., Shoffner, J.M., Voljavec, A.S. & Wallace, D.C. Evaluation of procedures for assaying oxidative phosphorylation enzyme activities in mitochondrial myopathy muscle biopsies. Biochim. Biophys. Acta 1019, 1–10 (1990).
Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, 287–295 (2007).
Palmer, J.W., Tandler, B. & Hoppel, C.L. Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations. Arch. Biochem. Biophys. 236, 691–702 (1985).
Jonckheere, A.I., Smeitink, J.A. & Rodenburg, R.J. Mitochondrial ATP synthase: architecture, function and pathology. J. Inherit. Metab Dis. (2011).
Barrientos, A., Fontanesi, F. & Diaz, F. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays. Curr. Protoc. Hum. Genet. 63, 19.3.1–1 (2009).
Grad, L.I., Sayles, L.C. & Lemire, B.D. Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans. Methods Mol. Biol. 372, 51–66 (2007).
Rowley, N. et al. Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding. Cell 77, 249–259 (1994).
Janssen, A.J. et al. Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. Clin. Chem. 53, 729–734 (2007).
Moghaddas, S., Distler, A.M., Hoppel, C.L. & Lesnefsky, E.J. Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity. Mitochondrion 8, 155–163 (2008).
Fischer, J.C. et al. Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure. Clin. Chim. Acta 145, 89–99 (1985).
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).
Chretien, D., Bourgeron, T., Rotig, A., Munnich, A. & Rustin, P. The measurement of the rotenone-sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem. Biophys. Res. Commun. 173, 26–33 (1990).
Kirby, D.M., Thorburn, D.R., Turnbull, D.M. & Taylor, R.W. Biochemical assays of respiratory chain complex activity. Methods Cell Biol. 80, 93–119 (2007).
Acknowledgements
This work has been supported by a donation from Stevanato Group to M.S., in memory of its founder G. Stevanato; from Telethon Italy grant no. GGP09207; and from a grant from Fondazione Cariparo. This research is part of a project of the Telethon-funded Italian Collaborative Network on Mitochondrial Disorders (GUP09004). The funding source had no role in the conduction of the study. We are grateful to L. Santinello for her assistance as librarian.
AUTHOR CONTRIBUTIONS
M.S. and A.C. designed, and performed experiments, analyzed data and wrote the paper; V.P. performed experiments. L.S. and C.A. analyzed data and critically revised the paper.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Video 1
Muscle homogenization. (SWF 7352 kb)
Table 1
Respiratory chain enzyme activities in illustrative examples of control preparations in muscle and cultured fibroblasts. (PDF 51 kb)
Figure 1
Respiratory chain enzymatic activities in cultured fibroblasts from patients with different mitochondrial disorders. Patients with mutations in two different CIV assembly factors, SURF1 (white bar) and SCO2 (dark grey bar), have isolated CIV deficiency. The patient with mutations in COQ2, a gene required for CoQ10 biosynthesis, displays selective complex II+III deficiency (light grey bar). The patient with the C5545T mtDNA mutation in tRNAtrp, which impairs mitochondrial protein synthesis, has a generalized defect of complexes containing mtDNA encoded subunits (CI, II+III, III, IV; black bar). (PDF 7 kb)
Rights and permissions
About this article
Cite this article
Spinazzi, M., Casarin, A., Pertegato, V. et al. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc 7, 1235–1246 (2012). https://doi.org/10.1038/nprot.2012.058
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2012.058
This article is cited by
-
Role of muscle FOXO gene in exercise against the skeletal muscle and cardiac age-related defects and mortality caused by high-salt intake in Drosophila
Genes & Nutrition (2023)
-
MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma
Journal of Experimental & Clinical Cancer Research (2023)
-
Optimization of differential filtration-based mitochondrial isolation for mitochondrial transplant to cerebral organoids
Stem Cell Research & Therapy (2023)
-
Lipid-droplet associated mitochondria promote fatty-acid oxidation through a distinct bioenergetic pattern in male Wistar rats
Nature Communications (2023)
-
Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics
Scientific Reports (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.