Ratiometric methodology for NAD(P)H measurement in the perfused rat heart using surface fluorescence

D. A. Scott, L. W. Grotyohann, J. Y. Cheung and R. C. Scaduto Jr Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033. The surface fluorescence of the isolated perfused rat heart has been evaluated for the purpose of NAD(P)H quantitation. With the use of excitation at 340, 380, 415, and 430 nm with emission detection at 500 +/- 20 nm, the intensities at 340 and 380 nm excitation were found to be linearly related during NAD(P)H oxidation/reduction induced by changes in substrate availability. Changes in cardiac NAD(P)H caused similar changes at 340 and 380 nm excitation, but those at 340 nm were of greater magnitude. Isolated cardiac mitochondria exhibited essentially identical optical properties during changes in NAD(P)H content induced by changes in substrate availability and by NAD(H) oxidation/reduction caused by coupled phosphorylation of ADP. The changes in redox status of both isolated mitochondria and the intact perfused heart can be expressed by a 340/380 excitation fluorescence ratio because of these relationships. This value assumed a minimum and maximum value under conditions of complete oxidation and reduction, respectively. Use of this ratio in the perfused heart avoids the artifacts caused by cardiac motion and tissue stretch. Removal of motion artifacts with an excitation ratio could only be accomplished if the measurements at 340 and 380 nm were estimated at the same point in the cardiac cycle. A method of cardiac waveform reconstruction and signal averaging is described to obtain these data from sequential measurements. With these techniques, the reduction of cardiac NAD(P)H can be expressed as a percentage of the range obtained between minimum and maximum reduction. The described technique is of general utility in the assessment of cardiac bioenergetics.