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

Area of Science:

• Biophysics
• Biochemistry
• Cardiovascular Physiology

Background:

• Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is crucial for cellular energy metabolism.
• Quantifying NAD(P)H in the intact heart is challenging due to motion and physiological changes.
• Surface fluorescence offers a non-invasive method for assessing NAD(P)H levels.

Purpose of the Study:

• To develop and validate a fluorescence-based method for NAD(P)H quantitation in the isolated perfused rat heart.
• To establish a reliable indicator of cardiac redox status.
• To overcome artifacts associated with cardiac motion and tissue stretch.

Main Methods:

• Utilized surface fluorescence with excitation at 340, 380, 415, and 430 nm and emission detection at 500 nm.
• Investigated the linear relationship between fluorescence intensities at 340 and 380 nm during NAD(P)H redox changes.
• Employed cardiac waveform reconstruction and signal averaging to synchronize measurements and remove motion artifacts.

Main Results:

• Fluorescence intensities at 340 and 380 nm excitation showed a linear relationship during NAD(P)H oxidation/reduction.
• A 340/380 nm excitation fluorescence ratio was established to represent cardiac redox status.
• This ratio effectively corrected for artifacts caused by cardiac motion and tissue stretch when measurements were synchronized.

Conclusions:

• The 340/380 nm excitation fluorescence ratio provides a robust measure of cardiac NAD(P)H redox state in the perfused rat heart.
• The developed method, incorporating signal averaging, accurately quantifies NAD(P)H reduction as a percentage of the full range.
• This technique offers general utility for assessing cardiac bioenergetics and metabolic function.