Although NADH was monitored in isolated cells from human tissues [2], the first attempt to apply NADH fluorometry to human tissues in vivo was made in 1971 by Jobsis et al. [3].

The fluorometric technique was used in patients (Fig. 9.1) to evaluate the relative rate of oxygen consumption and the bioenergetics activity of the intact cerebral cortex of patients. Using NADH fluorescence micro-fluorometry (Fig. 9.1a, b), they monitored the exposed brain of neurosurgical patients undergoing treatments for focal cerebral seizures. They correlated the electrocorticographic data to the NADH redox state under direct cortical stimulation of the monitored area. The recorded changes (Fig. 9.1c), namely, the oxidation of NADH (decrease of fluorescence), were very similar to those obtained in analogous procedures in the cat brain [4].

A few years later, the collaboration between Austin and Chance [5, 6], led to the recording of NADH in the brain of patients subjected to micro-anastomosis of the superficial temporal artery (STA) to the middle cerebral artery (MCA). They found an improvement of cerebral oxidative metabolism after the anastomosis, which was correlated to the elevated blood flow and increased tissue pO₂. Using a different type of fluorometer, Van Buren et al. showed a decrease in NADH (oxidation) resulting from cortical stimulation in epileptic patients [7]. Cortical electrical activity was compared with NADH fluorescence in patients undergoing operation following focal epilepsy. NADH was oxidized by 3–15 % in response to cortical stimulation only in 60 % of the patients. The topography of spread of NADH changes after cortical stimulation was usually asymmetrical in the human epileptigenic cortex.

Barlow et al. [8] used the fluorometric technique to measure NADH and flavoprotein fluorescence. In patients they monitored the fluorescence of flavoprotein in the human brain after anastomosis of the superficial temporal artery to the middle cerebral artery. They “demonstrated increased rate of cortical oxidative metabolism after the surgical procedures.”

During the same year Fein and Jobsis studied the changes in brain energetics in patients undergoing STA-MCA micro-anastomosis [9]. They tested six patients and concluded that “the mechanism of energy production may be more sensitive to ischemic anoxia than the mechanism of energy utilization.” Later on Fein and Olinger monitored patients after transient ischemic attacks (TIAs) [10, 11]. The brain of these patients, who had undergone an extracranial–intracranial bypass, was stimulated, and changes in NADH were recorded. They concluded that “persistent reversible abnormalities of cortical mitochondrial metabolism exist in a significant number of patients with history of TIAs who are suitable candidates for bypass surgery. Chance, in a review article in 1983 [12], compares three types of brain imaging techniques; optical—the surface fluorescence, NMR and radio isotopes, emphasizing the advantages and disadvantages of each one.”

The laser-based fluorimeter developed by Renault et al. [13] was used to monitor NADH redox state in the endomyocardium of patients during left heart catheterization [14]. They injected contrast medium into the right coronary artery and recorded a large transient increase in NADH fluorescence. It was hard to interpret the response because a control injection of saline solution was not reported. Later on the same group monitored the human heart during induced variations in left ventricular end-diastolic pressure (LVEDP) and during coronary arteriography. NADH laser fluorometry therefore seems to be promising as a means of exploring myocardial metabolism during cardiac catheterization [15]. During coronary angiography, injection of the contrast medium induces a rise in NADH in patients with a significant degree of coronary stenosis.

Figure 9.2 shows the effect of catheterization on NADH fluorescence measured in patients.

In another study, NADH was monitored in patients with McArdle’s disease (muscular phosphorylase deficiency), supporting the hypothesis that in this disease reduction of flux through the Embden–Meyerhof pathway causes decrease in the mitochondrial ability to produce NADH during ischemia [16]. Later on, NADH was monitored in the knee extensor muscle in 30 healthy males. NADH increased maximally within the first seconds of contraction [17].

Under amyotrophic sclerosis there is impairment of mitochondrial function as presented by decrease in the activity of NADH:CoQ oxidoreductase compared to the control group. In this study NADH was not monitored in vivo, but rather an isolated muscle fiber was used [18].

The same group of investigators monitored muscle fiber in vitro isolated from patients suffering from sporadic amyotrophic lateral sclerosis (SALS). They monitored flavoprotein/NAD(P)H ratio using autofluorescence imaging and found heterogeneous distribution of mitochondrial respiratory chain defect in 11 of 17 patients with SALS [19].

Intracellular fluorophores were monitored in isolated muscle cells, having the advantage of avoiding the problems of phototoxicity and photo-bleaching [20].

It is important to note that other studies in which spectroscopy was used to detect cancer cells or tumors were published. In most of them, monitoring was not directly related to the changes to NADH redox state itself, but rather the wide spectrum was measured, including the area of NADH emission spectrum [21–29].

In the past 10 to 20 years the low-temperature scanner for measurement of NADH and Fp fluorescence was applied to the identification of cancer and its malignancy [30–35].

Mayevsky and his group attempted to apply the NADH monitoring system to the evaluation of the human kidney during a transplantation procedure. We measured the transplanted kidney cortex immediately after the surgical procedures in the operation room [36, 37].

As of today, in vivo monitoring of the liver in the human has not been performed. Nevertheless, two papers described the changes in liver redox state after transplantation. A correlation between flavoproteins/pyridine nucleotides in human liver frozen tissue and arterial blood acetoacetate/β-hydroxybutyrate ratio was found. They concluded that the hepatic mitochondrial redox state of pyridine nucleotide can be evaluated by arterial blood [38]. Intramitochondrial oxidoreduction state was evaluated in frozen samples of liver grafts obtained from living donors. There was a delay in oxidation of the intramitochondrial oxidoreduction state as compared to blood oxygenation, probably caused by the cold preservation condition. Those findings indicate the importance of microcirculation in the normalization of the oxidoreduction state [39].

Although the current review is concerned with in vivo monitoring of NADH in human organs, we are including a short review of monitoring in cells and tissues. Spectroscopy of cells and tissues in vitro is used to differentiate between normal and cancer cells of human organs.

One of the problems of determining melanoma in situ is the lack of suitable techniques. This group has developed a fluorescence technique for qualitative and quantitative determination of nuclear cataract in human lenses. This technique is based on the fluorescence of a natural chromophore in the tissue that is excited at 366 nm, excites maximally at 475 nm (probably NADH), and is the first to be changed following metabolic changes [40]. Lohmann et al. [41] reported the monitoring of lung tissue. Fluorescence tomography (with a peak at 460 nm) together with other established methods might be used for determination of the histology of a biopsy sample. Schneckenburger et al. [42] found that skin fibroblasts from patients with mitochondrial disease showed high levels of fluorescence. This technique was used also on patients with moles of melanoma, as there is an increase in metabolic rate in cancerous cells that is observed mainly at the borders between healthy tissue and cancerous tissue. The fluorescence in the melanoma tissue was sixfold higher than that of the normal tissue.

Autofluorescence was used in colonic tissue to distinguish between neoplastic (abnormal growing) and hyperplasic (normal growing) tissue. This technique is significantly ideal for tissue diagnosis because it does not require administration of exogenous fluorescent dyes to the patients [43].

Konig et al. [44] claimed that early diagnosis of a diseased tissue, especially in the field of cancer, is not yet available. Characterization of the tissue metabolic state can successfully distinguish between healthy and malignant tissue. This study monitored bladder mucosa carcinoma and prostatic tissue. Laser-induced fluorescence is a technique that could perform “optical biopsy” in gastrointestinal mucosa. Monitoring of esophageal specimens showed good accuracy in distinguishing between normal and malignant tissue [45].

Because cancer cells usually show higher glycolytic activity, this activity affects their NADH/NAD⁺ level. When frozen slices of bronchial tissue were monitored, NADH fluorescence was higher in normal tissue than in malignant tissue [46].

Sterenborg et al. [47] found that the detection of skin cancer using the fluorescence of natural chromophores, especially NADH, was possible. Pfeifer et al. [48] used the fluorescence method to distinguish between healthy, cancerous, and necrotic tissue in lung tumor diagnostics. It was demonstrated that NADH fluorescence can be used for differentiation between tumorous and necrotic tissue and that flavin measurements are suitable for differentiation between healthy and nonhealthy tissue.

A compact probe of the laser spectrometer was used in various models: in hippocampal slices, to differentiate between necrotic and nonnecrotic lung tissue; for monitoring cell growth; and for bladder carcinoma diagnosis (this last showed lower fluorescence in malignant cells than normal cells) [49].

During the past 20 years we have developed three types of multiparametric monitoring systems that were applied to patients at various clinical locations. The concept of our development was to provide the clinicians with new physiological information that will improve their ability to diagnose the stage of pathological conditions in real time.