The discovery of the optical properties of reduced nicotinamide adenine dinucleotide (NADH; earlier names, DPNH, diphosphopyridine nucleotide, or PN, pyridine nucleotide), has led to very intensive research since the early 1950s. The reduced form of this molecule, NADH (Fig. 3.3a, b) [15], absorbs light at 320–380 nm (Fig. 3.3c) and emits fluorescent light in the 420- to 480-nm range (Fig. 3.3d) [16].

As the oxidized form, NAD⁺, does not absorb light in this range, it was possible to evaluate the redox state of the mitochondria by monitoring the UV absorbance (this section) or blue fluorescence of NADH (Sect. 3.3).

In 1951, Theorell and Bonnichsen found a shift in the absorption spectrum of DPNH upon addition of alcohol dehydrogenase (ADH) [17]. In the same year, Theorell and Chance described a new spectrophotometric technique for measuring the formation and disappearance of the compound of ADH and DPNH (Fig. 3.4) [18]. This work was based on previous publications that also appeared in 1951 [19, 20]. Moreover, in 1951, Chance and Legallias described a rapid and sensitive spectrophotometer for the measurement of reaction kinetics [21]. A year later (1952), Chance and Castor showed the applicability of this new technique to the measurements of pyridine nucleotide enzymes of muscle homogenate or intact cells [22]. In this study they cast doubt about Warburg’s assumption that the Warburg effect was caused by suppressed mitochondrial function. Immediately afterward (1952), Chance used the same device to monitor respiratory pigments, including NADH, in metabolizing cells [23]. Chance and Neilands (1952) found that the interaction between NADH and lactic dehydrogenase (LDH) of the heart led to a shift in the absorption band [24]. In 1952 and 1953, Chance and Castor showed the dynamics of respiratory pigment behavior (including NADH) in ascites tumor cells [25, 26]. In 1954, Chance and Williams briefly described new sensitive differential spectrophotometric methods applied to the study of reduced NADH in isolated rat liver mitochondria [27]. The same approach was used by Connelly and Chance in monitoring NADH in stimulated frog nerve and muscle preparations [28]. The oxidation of NADH in the muscle was similar to its oxidation in isolated mitochondria upon addition of ADP. In a comprehensive paper on “Enzyme mechanisms in living cells,” Chance described in detail the measurements of the respiratory enzymes, including NADH [29].

A major milestone in NADH monitoring was the technique presented in 1954 by Chance [30] using a double-beam spectrophotometer to determine the appropriate wavelengths in measurements of respiratory enzymes.

The detailed descriptions of the respiratory chain and oxidative phosphorylation in the mitochondria, published in 1955 by Chance and Williams, established our basic knowledge of mitochondrial function [1, 3, 4, 31, 32]. In the latter five papers, Chance and Williams defined, for the first time, the metabolic states of isolated mitochondria in vitro, depending on the substrate, oxygen, and ADP levels (Fig. 3.1). In addition, they correlated those metabolic states to the oxidation–reduction levels of the respiratory enzymes. The physiological significance of those metabolic states was discussed in 1956 by Chance and Williams [33], and later by Chance and other collaborators [34–38]. In 1959, Chance et al. described a highly sensitive recording micro-spectrophotometer that enabled the monitoring of NADH absorbance in a single cell [39].

The fact that NADH was monitored by the difference in the absorption spectrum of its reduced form limited the use of that technique to the study of mitochondria in vitro, and in very thin tissue samples (e.g., muscle) or in cell suspension. To provide a method more specific than absorption spectroscopy, fluorescence spectrophotometry in the near-ultraviolet range (UV-A) was applied for NADH measurement. The initial model of fluorescence recorder was described by Theorell and Nygaard in 1954 (Fig. 3.5) [40, 41] and Theorell, Nygaard, and Bonnichsen [42]. Using a modified device, Boyer and Theorell (1956) showed that the fluorescence of DPNH was shifted and the intensity was increased upon combination of DPNH and liver alcohol dehydrogenase-ADH [43]. The first detailed study using fluorescence spectrophotometry of NADH in intact Baker’s yeast cells and algae cells was published in 1957 by Duysens and Amesz [44] (Fig. 3.6).

In the next 5 years (1958–1962), the monitoring of NADH fluorescence was significantly expanded, led by Chance and collaborators. In a first preliminary study, Chance et al. [45] performed simultaneous fluorometric and spectrophotometric measurements of the reaction kinetics of bound pyridine nucleotides (PN) in the mitochondria. In the same year (1958) Chance and Baltscheffsky presented preliminary results of measuring the fluorescence of intramitochondrial PN (Fig. 3.8b) [46]. In this study, they proved the connection between the mitochondrial metabolic state and the redox state of NADH as measured by spectral fluorometry in mitochondria isolated from rat liver as seen in Fig. 3.8b [3].

The correlation between the enzymatic assay of PN and sensitive spectrophotometry was investigated by Klingeberger et al. using rat liver, heart, kidney, and brain [47].

In 1959, Chance and collaborators were able to expand the use of NADH fluorometry to various experimental models, from isolated mitochondria to intact tissue. Intramitochondrial pyridine nucleotides were analyzed in connection to the ADP-ATP cycle [48]. To monitor NADH localization in intact cells, Chance and Legallais developed a unique differential micro-fluorometer with a very high spatial resolution [49]. This approach was used, in various cells, to identify the intracellular localization of NADH fluorescence signals [50–52]. The next step was to apply the fluorometric technique to the higher organization level of animal tissues. Together with Jobsis, Chance measured in vitro changes in muscle NADH fluorescence following stimulation [53]. In another paper, published by Chance and Theorell [54], the authors came to the very significant conclusion that “The oxidation and reduction state of mitochondrial pyridine nucleotide without a measurable change of cytoplasmic fluorescence suggest that compartmentalization of mitochondrial and cytoplasmic pyridine nucleotide occurs in vivo, at least in the grasshopper spermatid.” In another article, Chance and Hollunger elaborated on the energy-linked reduction of the mitochondrial pyridine nucleotides [55].

Intensive use of the in vivo NADH monitoring approach started in 1962. The “classical” paper on in vivo monitoring of NADH was published in 1962 by Chance et al. [8]. They were able to simultaneously monitor the brain and kidney of anesthetized rats using two micro-fluorometers. In 1962, Chance and collaborators elaborated on this kind of in vivo monitoring and used it in other rat organs [56–58].

To verify the source of NADH fluorescence signals monitored in vivo, it was necessary to freeze the monitored tissue and to biochemically measure its pyridine nucleotide content. Initially, Avi-dor et al. [82] compared the fluorescence signal in isolated mitochondria to the concentration of pyridine nucleotides. Chance et al. (Fig. 3.14a) compared the fluorescent and analytical measurements of DPNH and TPNH using in vivo liver [83]. It became clear that ischemia, limiting the availability of oxygen, led to a large increase in the fluorescent signal. At the same time, DPNH showed a marked increase whereas TPNH remained unchanged. These results suggested that the source of the in vivo fluorescent signal is mainly from NADH.

In 1966, Chance summarized the studies comparing the fluorescence signals with the biochemical analysis of pyridine nucleotides [72]. It was found (Fig. 3.14b) that DPNH was the main source of fluorescence change in the in vitro beating heart. This observation held true under anoxia and under treatment with amytal. Chance et al. showed a clear correlation between the fluorescence signal measured in the heart and the biochemical analysis of NADH in the tissue.

To test conditions where NADH becomes oxidized, the hyperbaric oxygenation effect was tested in the rat liver in vivo [84]. NADH was found to be the main source for the increase of in vivo signal under amytal treatment. Under hyperbaric hyperoxic conditions, the contribution of TPNH (NADPH) was larger. In the same study, Chance et al. showed that in the brain cortex, under anoxic or hyperbaric conditions, NADH was the main source of the fluorescence signal (Fig. 3.15a, b) [72].

In 1971, Jobsis et al. [85] compared the corrected NADH fluorescence signal (after subtracting the reflectance signal) to its concentration measured in a brain tissue sample after induction of convulsions or anoxia (Fig. 3.16a). A very clear correlation was found, but it was concluded that “a quantitative interpretation of the changes of fluorescence in terms of nanomoles of NADH and NADPH is not truly warranted at this point.” Other investigators performed similar comparisons between the fluorescence signals and the analytical measurements. Shimazaki et al. showed a good correlation between the fluorescence of NADH and its concentration in control cornea as well as in cyanide-treated cornea [86].

NADH fluorescence and enzymatically determined NADH levels were similarly compared in the brain subjected to ischemia [87]. Toth et al. found a clear correlation between the two parameters under various metabolic perturbations of the spinotrapezius muscle (Fig. 3.16b) [88]. In the in vivo muscle, cyanide treatment and ischemia caused an increase in both parameters whereas the glycolysis inhibitor IAA induced a decrease in both. The same effects have been recently found by Toth et al., testing cat sartorius muscle in vivo [89]. A significant correlation was found between the fluorescence signal change in ischemia and the amount of NADH determined enzymatically (Fig. 3.16c). In 2005, Uppal et al. developed a technique to estimate the concentration of NADH in human blood samples [90]. They compared the fluorescence signals to the biochemical analysis results.

The intracellular localization of the signals measured from NADH had been discussed even before NADH fluorescence was measured.

It is important to understand that the excitation and emission wavelengths of NADH (350 and 460 nm) are well separated from other endogenous chromophores, as described by Anderson-Engels and Wilson [91], and that the 460-nm fluorescent emission originates primarily from NADH bound to mitochondria.

Klingenberg et al. [47], who used mitochondria from various organs, found that the sum of [DPN] and [DPNH] was of the same order of magnitude in the liver, heart, kidney, and brain. Using mitochondria of rat liver and kidney, Avi-Dor et al. [82] came to the following conclusions:

The average fluorescence yield of reduced pyridine nucleotide in mitochondria is six to eight times the yield of DPNH and TPNH in aqueous solution.

The yield of mitochondrial DPNH is substantially higher than the yield of mitochondrial TPNH.

The significance of these conclusions is that the in vivo fluorometric technique provides information about the redox state of NADH in the mitochondria with a negligible contribution of the cytoplasm. Estabrook arrived at the same conclusion using rat liver mitochondria by comparing the fluorometric technique with the spectrophotometric approach [92]. The same conclusion was drawn by Chance et al. [8, 65] for rat brain and kidney in vivo, and by Jöbsis et al. [93] for cat brain induced to epileptic activity. Jöbsis and Duffield [94], using the fluorometric technique in intact toad sartorius muscle, concluded “that cytoplasmic NADH does not interfere materially with these measurements.” A similar conclusion was reached by Chapman [95], positing that “mitochondrial NADH is the sole significant origin of labile fluorescence under the condition used.” The same was reported later by Jöbsis and Stainsby [93] for the mammalian skeletal muscle. O’Connor [96], who used in vivo monitoring of cat brain in combination with biochemical assays, stated the following: “The cortical fluorescence recorded with in vivo fluorometric techniques originated from mitochondrial NADH.” The same results and conclusions were obtained by Nuutinen [97] using isolated perfused rat heart. Accordingly, “The NADH + NADPH fluorescence of the intact tissue originates largely from the mitochondria.” In our own studies summarized in a review paper [81], we also confirmed the notion that most of the labile NADH signal in the brain originated from the mitochondria. Our conclusion was based on studies of the rat brain.

In preliminary experiments, we used a time-sharing fluorometer to monitor both NADH and flavoproteins (Fp) fluorescence from a small brain area free of large blood vessels [98–100]. As seen in Fig. 3.17 (right), exposure of the brain to anoxia led to NADH reduction (increased fluorescence signal) simultaneously with the reduction of Fp (a decrease in fluorescence signal). These similarities in the kinetics of response to anoxia suggest that the signal originates from the same cellular compartment, that is, the mitochondria. Such similarities were also found in a decapitation model (data not shown). Additional indirect evidence comes from other decapitation model studies performed by us [100, 101]. We found, in the awake rat, that the NADH fluorescence increase starts within 1 s and reaches its maximal level within less than 1 min (Fig. 3.17, right). The same timing pattern has been described previously [102] when effects of decapitation were studied in an anesthetized mouse brain. It has been reported that after decapitation, a large enhancement of glycolysis occurs, but this may have only started several minutes after the decapitation [103]. Lowry’s findings also suggest that if NADH increases dramatically several minutes after the decapitation, it may derive from the cytosolic source. In no decapitated animal (more than 200 animals) did we ever find such a secondary increase in NADH fluorescence after the initial maximal increase. The initial increase is correlated to the decrease in pO₂ as measured by a multiparametric technique (see Fig. 3.17, left) [104]. Eng et al. [64], studying single rat cardiac myocytes, asserted that “These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originated from mitochondrial NADH.” Coremans et al. [105] confirmed that “The results show that the NADH fluorescence/UV reflectance ratio can be used to monitor the mitochondrial redox state of the surface of intact blood perfused myocardium.”