Assessing mitochondrial dysfunction requires defining the specific function to be evaluated. Mitochondria primarily generate ATP through oxidative phosphorylation, but other functions like reactive oxygen species production, apoptosis, and calcium regulation are also important. Dysfunction can be assessed in isolated mitochondria, cells, or in vivo, with varying degrees of experimental control and physiological relevance. Methods to measure mitochondrial function include flux measurements, which provide more insight into ATP production than measurements of intermediates and potentials. For isolated mitochondria, the best assay is respiratory control, measuring respiration rate in response to ADP. For intact cells, the best assay is cell respiratory control, measuring ATP production, proton leak, coupling efficiency, and other parameters. Measurements of membrane potential provide additional information. The proton circuit is central to mitochondrial function, involving electron transport complexes and proton movement across the inner membrane. The proton circuit is quantified by measuring potential and flux, with the pmf being the sum of membrane potential and pH gradient. The pmf is crucial for ATP synthesis and is affected by proton leak, ATP turnover, and other processes. Modular kinetic analysis allows the study of individual modules of the proton circuit, such as substrate oxidation, ATP turnover, and proton leak, and their interactions. This approach helps identify the primary causes of dysfunction. The respiratory control ratio (RCR) is a key measure of mitochondrial function, reflecting the ability to generate ATP in response to ADP. The RCR is influenced by various factors, including substrate oxidation, ATP turnover, and proton leak. Absolute respiration rates provide insights into the mechanism of dysfunction, while the P/O ratio is a less reliable indicator of mitochondrial dysfunction. Measuring pmf and respiration rates together provides more comprehensive information. Modular kinetic analysis is a powerful tool for understanding mitochondrial dysfunction, allowing the identification of primary and secondary causes. The choice of assay depends on the context, with isolated mitochondria, intact cells, and in vivo models each offering different advantages. The best approach is to use a combination of methods to ensure accurate and informative results.Assessing mitochondrial dysfunction requires defining the specific function to be evaluated. Mitochondria primarily generate ATP through oxidative phosphorylation, but other functions like reactive oxygen species production, apoptosis, and calcium regulation are also important. Dysfunction can be assessed in isolated mitochondria, cells, or in vivo, with varying degrees of experimental control and physiological relevance. Methods to measure mitochondrial function include flux measurements, which provide more insight into ATP production than measurements of intermediates and potentials. For isolated mitochondria, the best assay is respiratory control, measuring respiration rate in response to ADP. For intact cells, the best assay is cell respiratory control, measuring ATP production, proton leak, coupling efficiency, and other parameters. Measurements of membrane potential provide additional information. The proton circuit is central to mitochondrial function, involving electron transport complexes and proton movement across the inner membrane. The proton circuit is quantified by measuring potential and flux, with the pmf being the sum of membrane potential and pH gradient. The pmf is crucial for ATP synthesis and is affected by proton leak, ATP turnover, and other processes. Modular kinetic analysis allows the study of individual modules of the proton circuit, such as substrate oxidation, ATP turnover, and proton leak, and their interactions. This approach helps identify the primary causes of dysfunction. The respiratory control ratio (RCR) is a key measure of mitochondrial function, reflecting the ability to generate ATP in response to ADP. The RCR is influenced by various factors, including substrate oxidation, ATP turnover, and proton leak. Absolute respiration rates provide insights into the mechanism of dysfunction, while the P/O ratio is a less reliable indicator of mitochondrial dysfunction. Measuring pmf and respiration rates together provides more comprehensive information. Modular kinetic analysis is a powerful tool for understanding mitochondrial dysfunction, allowing the identification of primary and secondary causes. The choice of assay depends on the context, with isolated mitochondria, intact cells, and in vivo models each offering different advantages. The best approach is to use a combination of methods to ensure accurate and informative results.