Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios

Salin, K., Villasevil, E. M., Auer, S. K., Anderson, G. J., Selman, C. , Metcalfe, N. B. and Chinopoulos, C. (2016) Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios. Physiological Reports, 4(20), e13007. (doi:10.14814/phy2.13007) (PMID:27798358) (PMCID:PMC5099967)

Salin, K., Villasevil, E. M., Auer, S. K., Anderson, G. J., Selman, C. , Metcalfe, N. B. and Chinopoulos, C. (2016) Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios. Physiological Reports, 4(20), e13007. (doi:10.14814/phy2.13007) (PMID:27798358) (PMCID:PMC5099967)

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Abstract

The use of tissue homogenate has greatly aided the study of the functioning of mitochondria. However, the amount of ATP produced per oxygen molecule consumed, that is, the effective P/O ratio, has never been measured directly in tissue homogenate. Here we combine and refine existing methods previously used in permeabilized cells and isolated mitochondria to simultaneously measure mitochondrial ATP production (JATP) and oxygen consumption (JO2) in tissue homogenate. A major improvement over existing methods is in the control of ATPases that otherwise interfere with the ATP assay: our modified technique facilitates simultaneous measurement of the rates of “uncorrected” ATP synthesis and of ATP hydrolysis, thus minimizing the amount of tissue and time needed. Finally, we develop a novel method of calculating effective P/O ratios which corrects measurements of JATP and JO2 for rates of nonmitochondrial ATP hydrolysis and respiration, respectively. Measurements of JATP and JO2 in liver homogenates from brown trout (Salmo trutta) were highly reproducible, although activity declined once homogenates were 2 h old. We compared mitochondrial properties from fed and food‐deprived animals to demonstrate that the method can detect mitochondrial flexibility in P/O ratios in response to nutritional state. This method simplifies studies examining the mitochondrial bioenergetics of tissue homogenates, obviating the need for differential centrifugation or chemical permeabilization and avoiding the use of nonmitochondrial ATPase inhibitors. We conclude that our approach for characterizing effective P/O ratio opens up new possibilities in the study of mitochondrial function in very small samples, where the use of other methods is limited.

Item Type:Articles
Additional Information:We acknowledge financial support from a European Research Council Advanced Grant (number 322784 to NBM).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Anderson, Mr Graeme and Auer, Dr Sonya and Selman, Professor Colin and Martin, Miss Eugenia and Metcalfe, Professor Neil and Salin, Dr Karine
Authors: Salin, K., Villasevil, E. M., Auer, S. K., Anderson, G. J., Selman, C., Metcalfe, N. B., and Chinopoulos, C.
College/School:College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
Journal Name:Physiological Reports
Publisher:American Physiological Society
ISSN:2051-817X
Copyright Holders:Copyright © 2016 The Authors
First Published:First published in Physiological Reports 4(20): e13007
Publisher Policy:Reproduced under a Creative Commons License

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