Measuring Rates of ATP Synthesis

  • Matthew J. BirdEmail author
  • Silvia Radenkovic
  • Pieter Vermeersch
  • David Cassiman
Part of the Methods in Molecular Biology book series (MIMB, volume 1862)


Here, we offer you a high-throughput assay to measure the ATP synthesis capacity in cells or isolated mitochondria. More specifically, the assay is linked to the mitochondrial’ electron transport chain components of your interest being either through complex I (with or without a linkage to pyruvate dehydrogenase activity), through complex II, or through the electron transport flavoprotein and complex I (β-oxidation of fatty acids).

Key words

ATP synthesis Oxidative phosphorylation β-oxidation Mitochondria Bioenergetics Oxidative phosphorylation (OXPHOS) 



DC and PV are senior clinical investigators of the Research Foundation-Flanders (FWO-Belgium).

Competing Interests

The authors declare no competing interests.


  1. 1.
    Boyer PD (1997) The ATP synthase–a splendid molecular machine. Annu Rev Biochem 66:717–749CrossRefGoogle Scholar
  2. 2.
    Ataullakhanov FI, Vitvitsky VM (2002) What determines the intracellular ATP concentration. Biosci Rep 22:501–511CrossRefGoogle Scholar
  3. 3.
    Qian T, Cai Z, Yang MS (2004) Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography-electrospray ionization mass spectrometry. Anal Biochem 325:77–84CrossRefGoogle Scholar
  4. 4.
    Wanders RJ, Ruiter JP, Wijburg FA (1993) Studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts: application to mitochondrial encephalomyopathies. Biochim Biophys Acta 1181:219–222CrossRefGoogle Scholar
  5. 5.
    Dröse S (2013) Differential effects of complex II on mitochondrial ROS production and their relation to cardioprotective pre- and postconditioning. Biochim Biophys Acta 1827:578–587CrossRefGoogle Scholar
  6. 6.
    Stepanova A, Shurubor Y, Valsecchi F, Manfredi G, Galkin A (2016) Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart. Biochim Biophys Acta 1857:1561–1568CrossRefGoogle Scholar
  7. 7.
    Patel KP, O'Brien TW, Subramony SH, Shuster J, Stacpoole PW (2012) The spectrum of pyruvate dehydrogenase complex deficiency: clinical, biochemical and genetic features in 371 patients. Mol Genet Metab 105:34–43CrossRefGoogle Scholar
  8. 8.
    Clayton DA, Shadel GS (2014) Isolation of mitochondria from cells and tissues. Cold Spring Harb Protoc 2014(10):pdb.top074542CrossRefGoogle Scholar
  9. 9.
    Banfalvi G (2016) Permeability of Biological Membranes. Springer International Publishing 73-127. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Matthew J. Bird
    • 1
    • 2
    Email author
  • Silvia Radenkovic
    • 2
    • 3
  • Pieter Vermeersch
    • 1
  • David Cassiman
    • 2
    • 4
  1. 1.Laboratory MedicineUniversity Hospitals LeuvenLeuvenBelgium
  2. 2.Hepatology Laboratory, Department of Chronic Diseases, Metabolism and AgeingKU LeuvenLeuvenBelgium
  3. 3.Metabolomics Expertise CenterVIB-KU Leuven Center for Cancer BiologyLeuvenBelgium
  4. 4.Metabolic CenterUniversity of LeuvenLeuvenBelgium

Personalised recommendations