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J. Biol. Chem., Vol. 280, Issue 26, 25119-25126, July 1, 2005

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Conformational Dependence of Intracellular NADH on Metabolic State Revealed by Associated Fluorescence Anisotropy^*

Harshad D. Vishwasrao, Ahmed A. Heikal¶, Karl A. Kasischke||, and Watt W. Webb||**

From the Howard Hughes Medical Institute, Center for Neurobiology & Behavior, Columbia University, New York, New York 10032, the ^¶Department of Bioengineering, Pennsylvania State University, University Park, Pennsylvania 16802, and the ^||School of Applied & Engineering Physics, Cornell University, Ithaca, New York 14853

Global analysis of fluorescence and associated anisotropy decays of intrinsic tissue fluorescence offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH in neural tissue. Using this technique, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration. The concentration of free NADH, and that of an enzyme bound form with a relatively low lifetime, increases preferentially over that of other enzyme bound NADH species. Concomitantly, the intracellular viscosity is reduced, likely due to the osmotic swelling of mitochondria. These conformation and environmental changes effectively decrease the tissue fluorescence average lifetime, causing the usual total fluorescence increase measurements to significantly underestimate the calculated concentration increase. This new discrimination of changes in NADH concentration, conformation, and environment provides the foundation for quantitative functional imaging of neural energy metabolism.

Received for publication, March 7, 2005 , and in revised form, April 26, 2005.

^* This work was performed in the Developmental Resource for Biophysical Imaging Opto-Electronics, which was supported by Grant 9-P41-EB001976 from the National Institute of Biomedical Imaging & Bioengineering, National Institutes of Health (NIH). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a Paper of the Week.

Supported in part by NIH, National Institute of General Medical Sciences Research Training Grant GM08267.

^** To whom correspondence should be addressed: School of Applied and Engineering Physics, Cornell University, 223 Clark Hall, Ithaca, NY 14853. Tel.: 607-255-3331; Fax: 607-255-7658; E-mail: www2{at}cornell.edu.

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