title: Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers.
abstract: Oxidative phosphorylation (OXPHOS) efficiency, defined as the ATP-to-O ratio, is a critical feature of mitochondrial function that has been implicated in health, aging, and disease. To date, however, the methods to measure ATP/O have primarily relied on indirect approaches or entail parallel rather than simultaneous determination of ATP synthesis and O2 consumption rates. The purpose of this project was to develop and validate an approach to determine the ATP/O ratio in permeabilized fiber bundles (PmFBs) from simultaneous measures of ATP synthesis (JATP) and O2 consumption (JO2 ) rates in real time using a custom-designed apparatus. JO2 was measured via a polarigraphic oxygen sensor and JATP via fluorescence using an enzyme-linked assay system (hexokinase II, glucose-6-phosphate dehydrogenase) linked to NADPH production. Within the dynamic linear range of the assay system, ADP-stimulated increases in steady-state JATP mirrored increases in steady-state JO2 (r(2) = 0.91, P < 0.0001, n = 57 data points). ATP/O ratio was less than one under low rates of respiration (15 μM ADP) but increased to more than two at moderate (200 μM ADP) and maximal (2,000 μM ADP) rates of respiration with an interassay coefficient of variation of 24.03, 16.72, and 11.99%, respectively. Absolute and relative (to mechanistic) ATP/O ratios were lower in PmFBs (2.09 ± 0.251, 84%) compared with isolated mitochondria (2.44 ± 0.124, 98%). ATP/O ratios in PmFBs were not affected by the activity of adenylate kinase or creatine kinase. These findings validate an enzyme-linked respiratory clamp system for measuring OXPHOS efficiency in PmFBs and provide evidence that OXPHOS efficiency increases as energy demand increases.
authors.affiliation: Department of Kinesiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina Daniel.lark@vanderbilt.edu.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Lark
authors.affiliation: Department of Kinesiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Torres
authors.affiliation: Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: Chien-Te
authors.initials: CT
authors.lastname: Lin
authors.affiliation: Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: Terence E
authors.initials: TE
authors.lastname: Ryan
authors.affiliation: Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: Ethan J
authors.initials: EJ
authors.lastname: Anderson
authors.affiliation: Department of Kinesiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: P Darrell
authors.initials: PD
authors.lastname: Neufer
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism; D000818:Animals; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000502:physiology*; D009249:NADP / Q000378:metabolism; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption / Q000502:physiology
chemical_list: D009249:NADP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase; D000263:Adenylate Kinase
keywords: adenosine 5′-triphosphate synthesis; adenosine 5′-triphosphate-to-oxygen ratio; oxidative efficiency; permeabilized myofiber