Determination of pyridine nucleotide fluorescence from the perfused heart using an internal standard

Mitochondrial reduced nicotinamide adenine dinucleotide (NADH) is a key intermediate in energy metabolism in the heart, which can be qualitatively monitored using nondestructive surface fluorescence techniques. However, this optical technique is subject to artifacts from alterations in tissue absorbance, motion of the heart, and variations in excitation intensity. In this study rapid-scan fluorescence emission spectroscopy was used in conjunction with an internal fluorescence standard to compensate for these optical artifacts. The fluorescence spectra obtained from heart had a maximum at 460 nm and a shoulder at 415 nm. Dilution of heart homogenates resulted in a fluorescent spectrum characteristic of suspensions of mitochondria, indicating that absorption of fluorescence by tissue components produces an inner filter effect. This internal filter was characterized, and isobestic points with regard to O2 were found at 425 and 450 nm. Alterations in the inner filter effect due to changes in tissue oxygenation were eliminated by monitoring the NADH at 425 nm. Motion artifacts and excitation source fluctuations were corrected by loading heart cells with an internal fluorescent standard, 5(6)-carboxy-2',7'-dichlorofluorescein (ClCF). Motion of the heart and changes in excitation intensity altered the fluorescence detected from both NADH and ClCF. The use of the NADH-to-ClCF ratio detected at isobestic wavelengths (425 nm NADH and 520 nm ClCF) gives a relative measure of NADH fluorescence, which adequately compensates for both internal absorbance and motion artifacts.