Mitochondrial function is essential to the life of all eukaryotic cells and human health by supporting the energy demands for all cellular processes [1
]. In addition, mitochondria play a key mechanistic role in programmed cell death (apoptosis), free radical generation and oxidative stress (a hallmark of aging), and biomolecular sensing of glucose, oxygen and nitric oxide. As a result, it is not surprising that mitochondrial dysfunction is widely linked to a range of diseases and health problems such as cancer, Alzheimer’s disease and other neurodegenerative diseases [2
]. Mitochondria have also been a target for developing therapeutic drugs, due to their role in cell survival and health related conditions [6
]. For example, some anticancer agents are designed to stimulate proapoptotic mitochondrial events in tumor cells [7
]. Mitochondrial anomalies dysregulate energy production, which supports many pathways of intermediary metabolism, and, therefore, have also attracted attention as another target for drug discovery and clinical intervention [10
]. Mitochondrial dysfunction and oxidative stress have been associated with the majority of neurodegenerative diseases. As a result, the reduction of mitochondrial oxidative stress is another target for new therapeutic drugs aimed at either preventing or slowing down the progression of neurodegenerative disorders [11
]. Uncoupling of the mitochondrial electron transport chain (ETC) has also been a pharmacological target for treating obesity [12
]. 2,4 dinitrophenol has been used to induce some weight loss by enhancing energy expenditure via mitochondrial uncoupling. Efficient biomarkers and noninvasive techniques that would allow for real time monitoring of mitochondrial function would, therefore, provide a powerful tool towards effective diagnosis of health problems and an opportunity for early therapeutic intervention.
Decades after the pioneering work by Chance and colleagues [13
], there is a resurgent interest in using intracellular coenzymes, such as reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) – see for chemical structure, as intrinsic biomarkers for metabolic activities and mitochondrial anomalies. For example, NADH and its oxidized form (NAD+
) are involved in mitochondrial function, energy metabolism, calcium homeostasis, gene expression, oxidative stress, aging and apoptosis. Chance and Williams, in a series of seminal reports, demonstrated that the respiratory chain activities of isolated in vitro
mitochondria correlate with the redox coenzymes in the ETC [17
]. Mutated mitochondrial DNA in cancer, for example, results in an alteration of the conformations (and function) of NADH dehydrogenase (complex I) and cytochrome C (complex III) in the ETC, which leads to the generation of free radicals and apoptosis [22
]. The reduced NADH phosphate (NAD[P]H) is involved in the reductive biosynthesis of fatty acids and steroids, antioxidation and oxidative stress, and the oxidized form, NADP+
, participates in calcium homeostasis [23
]. Some evidence suggests that intracellular NADH concentration is greater (up to tenfold, by some estimates) than that of NAD(P)H [24
]. The sensitivity of the autofluorescence of these pyridine nucleotides to cell pathology remains inconsistent and may depend on the experimental techniques, conditions and cell lines [26
]. Reduced FAD (FADH2
) and FAD are another redox pair associated with respiration in all eukaryotic cells. Some data suggest that flavoproteins, such as lipoamide dehydrogenase (LipDH) and electron transfer flavoprotein, contribute significantly to the cellular autofluorescence [28
]. FAD is largely associated with mitochondria and the oxidative phosphorylation pathway [30
]. NAD(P)H, FADH2
and their oxidized counterparts are critical for a broad array of oxidation–reduction (redox) reactions in living cells [23
]. In particular, the redox ratio (NAD+
:NADH) allows for real-time monitoring of the metabolic state of a cell during pathophysiological changes.
The chemical structure of NAD(P)H, FADH2 and their oxidized forms
Based on their functional role in cell biology, intracellular NADH and FAD have a diagnostic potential as natural biomarkers for cellular redox reactions, energy metabolism and mitochondrial anomalies under different pathophysiological conditions [34
]. Importantly, these coenzymes are naturally fluorescent and, therefore, genuine, noninvasive imaging of metabolic activities can be carried out in living cells and tissues. The autofluorescence properties of NADH and FAD eliminate potential toxicity, nonspecific binding and interference with biomolecular functions that are associated with the use of exogenous dyes. In addition, the autofluorescence of these coenzymes can be excited using distinct illumination wavelengths, ranging from UV to infrared regions, for complementary imaging using one photon (1P) - and two photon (2P)-fluorescence microscopy, respectively. Finally, the NADH and FAD autofluorescence is sensitive to protein binding and local environment. When combined, these properties are essential for a useful biomarker such as NADH and FAD.
The objective of this article is to highlight recent development of these intracellular co enzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. The structure and spectroscopic properties of these coenzymes is reviewed as a guide for experimental design and data interpretation. Recent studies on NADH and flavin are highlighted within the context of their biological function in metabolic activities and mitochondrial function. In addition, fluorescence based methods for monitoring cellular coenzymes are compared with conventional biochemical assays that require cell lysates. Finally, the potential and challenges associated with using these coenzymes as biomarkers is discussed and compared with conventional, exogenous markers for mitochondria. This article is not intended to be comprehensive. Rather, it is written to serve as biological, biomedical and technical resource for new researchers who may be interested in exploiting these natural biomarkers for diagnostic and mechanistic studies.