title: Optical spectroscopy in turbid media using an integrating sphere: mitochondrial chromophore analysis during metabolic transitions.
abstract: Recent evidence suggests that the activity of mitochondrial oxidative phosphorylation complexes (MOPCs) is modulated at multiple sites. Here, a method of optically monitoring electron distribution within and between MOPCs is described using a center-mounted sample in an integrating sphere (to minimize scattering effects) with a rapid-scanning spectrometer. The redox-sensitive MOPC absorbances (∼465-630 nm) were modeled using linear least squares analysis with individual chromophore spectra. Classical mitochondrial activity transitions (e.g., ADP-induced increase in oxygen consumption) were used to characterize this approach. Most notable in these studies was the observation that intermediates of the catalytic cycle of cytochrome oxidase are dynamically modulated with metabolic state. The MOPC redox state, along with measurements of oxygen consumption and mitochondrial membrane potential, was used to evaluate the conductances of different sections of the electron transport chain. This analysis then was applied to mitochondria isolated from rabbit hearts subjected to ischemia/reperfusion (I/R). Surprisingly, I/R resulted in an inhibition of all measured MOPC conductances, suggesting a coordinated down-regulation of mitochondrial activity with this well-established cardiac perturbation.
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. chessdj@mail.nih.gov
authors.forename: David J
authors.initials: DJ
authors.lastname: Chess
authors.forename: Eric
authors.initials: E
authors.lastname: Billings
authors.forename: Raúl
authors.initials: R
authors.lastname: Covian
authors.forename: Brian
authors.initials: B
authors.lastname: Glancy
authors.forename: Stephanie
authors.initials: S
authors.lastname: French
authors.forename: Joni
authors.initials: J
authors.lastname: Taylor
authors.forename: Heather
authors.initials: H
authors.lastname: de Bari
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
authors.forename: Robert S
authors.initials: RS
authors.lastname: Balaban
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D002244:Carbon / Q000378:metabolism; D003470:Culture Media; D004734:Energy Metabolism; D006321:Heart / Q000502:physiology; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D055095:Optics and Photonics / Q000379:methods*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010477:Perfusion; D011817:Rabbits; D015427:Reperfusion Injury; D015203:Reproducibility of Results; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D003470:Culture Media; D002244:Carbon; D000255:Adenosine Triphosphate
keywords: Electron transport chain; Ischemia/reperfusion; Mitochondria; Spectroscopy