Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies

Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathological states, the real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. The goals of the present review are the following: 1) to provide an historical overview of NADH fluorescence monitoring and its physiological significance; 2) to present the solid scientific ground underlying NADH fluorescence measurements based on published materials; 3) to provide the reader with basic information on the methodologies used in the past and the current state of the art fluorometers; and 4) to clarify the various factors affecting monitored signals, including artifacts. The large numbers of publications by different groups testify to the valuable information gathered in various experimental conditions. The monitoring of NADH levels in the tissue provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. Although NADH signals are not calibrated in absolute units, their trend monitoring is important for the interpretation of physiological or pathological situations. To understand tissue function better, the multiparametric approach has been developed where NADH serves as the key parameter. The development of new light sources in UV and visible spectra has led to the development of small compact units applicable in clinical conditions for better diagnosis of patients. real-time tissue viability; tissue spectroscopy; patient monitoring     

NADH levels and solventogenesis in Clostridium acetobutylicum: New insights through culture fluorescence

Summary The metabolic relationship between the solventogenic state in Clostridium acetobutylicum and intracellular NADH levels was investigated using culture fluorescence as a technique for continuous monitoring of in vivo NADH levels. Continuous culture experiments showed that a transition from acidogenic to solventogenic state was accompanied by a decrease in culture fluorescence, which was interpreted as a decrease in NADH level. It appears that NADH/NAD⁺ turnover rates may be more significant than NADH levels in determining the metabolic state of the cell. This result provides important new information on regulation of the intracellular reduction state in Clostridium acetobutylicum. Culture fluorescence is shown to be a useful technique for non-invasive on-line monitoring of the metabolic state in continuous acetone-butanol fermentations.     

Oxidation-reduction states of NADH in vivo: from animals to clinical use

Mitochondrial dysfunction is part of many pathological states in patients, such as sepsis or stroke. Presently, the monitoring of mitochondrial function in patients is extremely rare, even though NADH redox state is routinely measured in experimental animals. In this article, we describe the scientific backgrounds and practical use of mitochondrial NADH fluorescence measurement that was applied to patients in the past few years. In addition to NADH, we optically measured the microcirculatory blood flow and volume, as well as HbO(2) oxygenation, from the same tissue area. The four detected parameters provide real time data on tissue viability, which is critical for patients monitoring.     

High-resolution visualization of oxygen distribution in the liver in vivo

Microcirculatory failure after stress events results in mismatch in oxygen supply and demand. Determination of tissue oxygen distribution in vivo may help elucidate mechanisms of injury, but present methods have limited resolution. Male Sprague-Dawley rats were anesthetized, prepared for intravital microscopy, and received intravenously the oxygen-sensitive fluorescent dye Tris(1,10-phenanthroline)ruthenium(II) chloride hydrate [Ru(phen)3(2+)]. An impaired hepatic oxygen distribution was induced by either phenylephrine or hemorrhage. Intensity of Ru(phen)3(2+) fluorescence was compared with NADH autofluorescence indicating changes in the mitochondrial redox potential. Ethanol was injected to affect the NADH-to-NAD+ ratio without altering the P(O2). Infusion of Ru(phen)3(2+) resulted in a heterogeneous fluorescence under baseline conditions reflecting the physiological acinar P(O2) distribution. A decrease in oxygen supply due to phenylephrine or hemorrhage was paralleled by an increase in Ru(phen)3(2+) and NADH fluorescence reflecting an impaired mitochondrial redox state. Ethanol did not alter Ru(phen)3(2+) fluorescence but increased NADH fluorescence indicating independence of P(O2) and redox state imaging. Intravenous administration of Ru(phen)3(2+) for intravital videomicroscopy represents a new method to visualize the hepatic tissue P(O2). Combined with NADH autofluorescence, it provides additional information regarding the tissue redox state.     

(Semi-)quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart.

In vivo analysis of the metabolic state of tissue by means of reduced nicotinamide adenine dinucleotide (NADH) fluorimetry is disturbed by tissue movements and by hemodynamic and oximetric effects. These factors cause changes in the absorption of ultraviolet (UV) excitation light by the tissue. Many different methods have been used in the literature to compensate measured NADH fluorescence intensities for these effects. In this paper we show on theoretical grounds that the ratio of NADH fluorescence intensity and UV diffuse reflectance intensity provides a (semi-)quantitative measure of tissue NADH concentrations. This result is corroborated by experiments with tissue phantoms in which absorption and back-scattering properties were varied. Furthermore, we have verified the validity of this compensation method in isolated Langendorff-perfused rat heart preparations. In this preparation oximetric effects (of blood and tissue) are the major determinants of the metabolism-dependent UV diffuse reflectance change. Hemodynamic effects accompanying compensatory vasodilation are negligible. Movement artifacts were eliminated by simultaneously recording fluorescence and reflectance images, using a CCD camera with a biprism configuration. 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.     

Mitochondrial NADH fluorescence is enhanced by complex I binding

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Mitochondrial function and energy metabolism in cancer cells: Past overview and future perspectives

The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.     

Fluorimetric characterisation of metabolic activity of ex vivo perfused pig hearts.

Autofluorescence of tissues and organs is an indicator of the physiological state of cells. The aim of the study was to investigate whether fluorimetric determination of the redox state of the ex vivo perfused pig heart can provide fast online detection of progressive changes in heart muscle tissue. Measurements on six organs perfused in a four-chamber working heart model were performed using a spectroscopic method exploiting the specific and different fluorescence lifetimes of intrinsic fluorophores such as NADH and flavins and providing a means of internal signal referencing. It was shown that the redox potential of heart muscle tissue can be assessed by fluorescence measurement. In the steady-state phase of the beating heart, spectroscopic measurements revealed a change in redox state from an initial constant level to a continuous decrease, accompanied by a decrease in heart performance and indications of changes in electrolyte equilibrium (K(+) concentration). At the same time, troponin I levels in the perfusate increased. The results indicate that fluorimetric determination of heart muscle metabolic activity yields reliable information about the functional status of the ex vivo heart and may be advantageous for the optimisation of ex vivo organ models.     

酿酒酵母胞质内NADH的代谢调控

酿酒酵母(Saccharomyces cerevisiae)的生长过程有大量的胞内NADH产生。有氧途径中,胞外的NADH脱氢酶、三磷酸甘油穿梭酶系是线粒体内NADH氧化的最主要机制。该文主要讨论以下三个方面的内容:不同生理环境下促成线粒体胞内NADH氧化的各主要机制的作用;借助电子传递链开启NADH从胞质脱氢酶到线粒体的通道,各代谢动力学的有序进行;各种酶形成超分子复合物,尤其是起关键调控作用的酶形成具相似生理功能的高整合性功能酶。     

Direct biologically based biosensing of dynamic physiological function

Dynamic regulation of biological systems requires real-time assessment of relevant physiological needs. Biosensors, which transduce biological actions or reactions into signals amenable to processing, are well suited for such monitoring. Typically, in vivo biosensors approximate physiological function via the measurement of surrogate signals. The alternative approach presented here would be to use biologically based biosensors for the direct measurement of physiological activity via functional integration of relevant governing inputs. We show that an implanted excitable-tissue biosensor (excitable cardiac tissue) can be used as a real-time, integrated bioprocessor to analyze the complex inputs regulating a dynamic physiological variable (heart rate). This approach offers the potential for long-term biologically tuned quantification of endogenous physiological function.     

High spatial resolution readout of 3-D metabolic organ structure: an automated, low-temperature redox ratio-scanning instrument

Intraorgan compartmentation of metabolic processes plays an important role in the understanding of the physiological function of the integrated organ under normal as well as under pathological conditions. We describe here a technique by which 3-D information on tissue redox state may be obtained by means of automated scanning of surface fluorescence. The instrument allows for serial scanning of frozen tissue. A typical scan of a tissue volume of 3 X 3 X 2 mm at a linear resolution of 50 micron and a spatial resolution of ca. 3 X 10(-7) ml includes 144,000 single-point measurements of pyridine nucleotide and flavoprotein fluorescence within the tissue block. The scanning process is fully computerized and programs have been developed which allow 2- or 3-D reconstruction of the data in terms of "redox ratio models," exemplified here by a 3-D model of a spreading depression wave in the cerebral cortex of a gerbil.     

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.     

The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions

The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, reports on the amount of free NADH in mitochondria are sparse and contradictory. We first determined the emission spectrum of NADH bound to proteins using isothermal titration calorimetry combined with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins, and the amount is low in state 3 (substrate + ADP present) and high in state 2 (only substrate present) and state 4 (substrate + ATP). By contrast, the amount of free NADH is low but relatively constant, even increasing a little in state 3. Using modeling, we show that these results can be explained by a 2.5- to 3-fold weaker average binding of NADH to mitochondrial protein in state 3 compared with state 4. This indicates that there is a specific mechanism for free NADH homeostasis and that the concentration of free NADH in the mitochondrial matrix per se does not play a regulatory role in mitochondrial metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism.     

Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae

Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpdlp, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.     

Genetically encoded fluorescent sensors for intracellular NADH detection

We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.     

一体化的光声-荧光显微镜用于活体肿瘤成像

报道了一种利用单一波长激发的同时产生光声和荧光信号的显微成像系统,本成像系统具有超高的成像分辨率(<6μm)。借助外源的造影剂在近红外的吸收特性,利用光声-荧光显微成像系统对活体肿瘤进行光声/荧光成像。实验结果表明,光声-荧光显微镜在早期肿瘤的成像和检测等方面具有潜在的应用价值。因此,通过研究和选择适当的双模态造影剂,该系统在不同病理模型中可以提供更准确的组织信息及生理参数。     

Ca²⁺ signals of astrocytes are modulated by the NAD⁺/NADH redox state

Astrocytes are important glial cells in the brain providing metabolic support to neurons as well as contributing to brain signaling. These different functional levels have to be highly coordinated to allow for proper cell and brain function. In this study, we show that in astrocytes the NAD(+) /NADH redox state modulates dopamine-induced Ca(2+) signals thereby connecting metabolism and Ca(2+) signaling. Application of dopamine induced a dose-dependent increase in Ca(2+) signal frequency in these cells, which was dependent on D(1) -receptor signaling, glycolytic activity, an increase in cytosolic NADH and inositol 1,4,5-triphosphate receptor operated intracellular Ca(2+) stores. Application of dopamine at a low concentration (1 μM) did not induce an increase in Ca(2+) signal frequency by itself. However, simultaneously increasing cytosolic NADH content either by direct application of NADH or by application of lactate resulted in a pronounced increase in Ca(2+) signal frequency. This increase could be blocked by co-application of pyruvate, suggesting that indeed the NAD(+) /NADH redox state is regulating Ca(2+) signals. We conclude that at the NAD(+) /NADH redox state metabolic and signaling information is integrated in astrocytes, thereby most likely contributing to precisely coordinate these different tasks of astrocytes.     

Use of fluorometry for monitoring and control of a bioreactor

A new fluorescent bioreactor monitoring probe-multiple excitation fluorometric system (MEFS)-has been developed. This probe was compared to the commercially available BioChem Technology FluroMeasure system (NADH probe). In this task the fluorescence behavior of three model fermentation systems, ethanol fermentation by Candida utilis, phenol fermentation by Pseudomonas putida, and glucose fermentation by Saccharomyces cerevisiae, were examined. The results indicated that the fluorescence intensity and behavior of various cellular fluorophors vary significantly between the different fermentation systems. Monitoring a fermentation process using only NAD(P)H fluorescence provided limited information. The NAD(P)H fluorescence was found not to be the best fluorescence signal for monitoring cell concentrations. The best way of monitoring a bioreactor by fluorometry may be to monitor several fluorophors in the whole culture broth simultaneously and to relate these fluorescence signals to various biological parameters.