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However, the amount of <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content> 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 <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content> production (<jats:styled-content style=\"fixed-case\"><jats:italic>J<\/jats:italic>ATP<\/jats:styled-content>) and oxygen consumption (<jats:italic>J<\/jats:italic>O<jats:sub>2<\/jats:sub>) in tissue homogenate. A major improvement over existing methods is in the control of <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content>ases that otherwise interfere with the <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content> assay: our modified technique facilitates simultaneous measurement of the rates of \u201cuncorrected\u201d <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content> synthesis and of <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content> 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 <jats:styled-content style=\"fixed-case\"><jats:italic>J<\/jats:italic>ATP<\/jats:styled-content> and <jats:italic>J<\/jats:italic>O<jats:sub>2<\/jats:sub> for rates of nonmitochondrial <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content> hydrolysis and respiration, respectively. Measurements of <jats:styled-content style=\"fixed-case\"><jats:italic>J<\/jats:italic>ATP<\/jats:styled-content> and <jats:italic>J<\/jats:italic>O<jats:sub>2<\/jats:sub> in liver homogenates from brown trout (<jats:italic>Salmo trutta<\/jats:italic>) were highly reproducible, although activity declined once homogenates were 2\u00a0h old. We compared mitochondrial properties from fed and food\u2010deprived 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 <jats:styled-content style=\"fixed-case\">ATP<\/jats:styled-content>ase 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.<\/jats:p>","DOI":"10.14814\/phy2.13007","type":"journal-article","created":{"date-parts":[[2016,10,25]],"date-time":"2016-10-25T01:19:11Z","timestamp":1477358351000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":32,"title":["Simultaneous measurement of mitochondrial respiration and <scp>ATP<\/scp> production in tissue homogenates and calculation of effective P\/O ratios"],"prefix":"10.14814","volume":"4","author":[{"given":"Karine","family":"Salin","sequence":"first","affiliation":[{"name":"Institute of Biodiversity Animal Health and Comparative Medicine University of Glasgow Glasgow UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eugenia 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