Noninvasive Approach For Quantitative Analysis Of Energy Metabolism And Mitochondrial Anomalies In Living Cells

Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) as key metabolic cofactors in energy metabolism in eukaryotic cells. As a result, there has been recent resurgence in using these intrinsic cofactors as natural probes for diagnostic purposes in cancer, diabetes, apoptosis, and neurodegenerative diseases. Here, we used autofluorescence dynamics assay to quantify the concentration and enzyme binding of intrinsic NADH and FAD in living cells. In these studies, cancer (Hs578T) and normal (Hs578Bst) breast cells are used as model systems to examine the sensitivity of the proposed assay to cell pathology. Our non-invasive, quantitative assay includes multiphoton microscopy and spectroscopy of NADH and FAD. Two-photon fluorescence lifetime imaging of cellular autofluorescence, in a calibrated microscope, is used to construct NADH and FAD concentration images in live cells. In addition, time-resolved associated anisotropy of cellular autofluorescence provides direct quantification of the molar fractions of free and enzyme-bound cofactors in both normal and transformed cells. Targeted inhibition of complex IV of the electron transport chain in Hs578Bst, using KCN, confirm the sensitivity of cellular autofluorescence to changes in the respiratory state activities. Comparative studies of the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. This fluorescence dynamics assay on natural coenzymes (i.e., without the need for exogenous fluorescence dyes) is applicable to other metabolic and signaling pathways in live cells, which contrasts with conventional biochemical techniques that require cell destruction.