Use of NADH fluorescence to determine mitochondrial function in vivo

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. In this paper we are summarizing the following items: (1) presenting the solid scientific ground underlying nicotine amide adenine dinucleotide (NADH) NADH fluorescence measurements based on published materials. (2) Presenting NADH fluorescence monitoring and its physiological significance. (3) Providing the reader with basic information on the methodologies of the fluorometers reflectometers. (4) Clarifying various factors affecting the monitored signals, including artifacts. (5) Presenting the potential use of monitoring mitochondrial function in vivo for the evaluation of drug development. 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 better understand the tissue function, the multiparametric approach has been developed where NADH serves as the key parameter to be monitored.     

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.     

On-line fluorescence measurements in assessing culture metabolic activities

Summary NADH fluorescence aided by a stoichiometric metabolic pathway model and culture dynamics was used to elucidate the unobservable intracellular physiological state in two metabolically different phases during culture of Clostridium acetobutylicum. The validity of the theoretical model was examined over a range of culture pH regimes and initial sugar concentrations. The H₂/CO₂ gas concentration ratio was found to be an important process parameter. NADH fluorescence detection was compared with simultaneous enzymatic measurements. The specific fluorescence (fluorescence per biomass, F/X) provided a distinction between oxidative and reductive culture metabolism independent of the pH or substrate concentration changes. A good indicator of the type of culture activity proved to be the dF/dt parameter. The net fluorescence measurements correlated with butanol accumulation under all growth conditions suggesting the possible use of the fluorescence probe as a butanol probe in this fermentation.     

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.     

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.     

Properties of a Large Complex with NADH Dehydrogenase Activity from Barley Thylakoids

When assays for NAD(P)H-ferricyanide oxidoreductases were performed,activities specific for NADH (0.23 unit (mg protein)^–1)and NADPH (0.68 unit (mg protein)^–1) were detected inchloroplasts isolated from leaves of barley (Hordeum vulgareL.). Activities of chloroplast NADH- and NADPH-ferricyanideoxidoreductase were 5-fold and 25-fold higher, respectively,than the maximum activity that could be attributed to mitochondrialcontamination. Moreover, most of the chloroplast NADH-ferricyanideoxidoreductase (60 to 80%) was solubilized by deoxycholate (DOC)from thylakoids as a single, high-molecular-mass complex thatwas distinguishable from the mitochondrial complex by its lowerelectrophoretic mobility in 3% polyacrylamide, as revealed byreduction of nitro blue tetrazolium (NBT) in the presence ofNADH or NADPH on gels after electrophoresis. The stroma yieldeda single band of a dehydrogenase (66 kDa) that used NADH asits electron donor. Several NADPH-dependent activities weredetected after electrophoresis of the stromal fraction. Moreover,chloroplast-specific activities could be distinguished frommitochondrial activities on the basis of the specificity ofthe donor and the acceptor of electrons, the dependence of theactivities on pH, and the sensitivity to various inhibitors.K_m values for NADH (26 µM) and NADPH (75 µM) werein the same range as those of mitochondrial activities. Mostof the NADPH-dependent activity probably corresponds to thechloroplast ferredoxin-NADP⁺ oxidoreductase. The possibilityis discussed that thylakoid NADH dehydrogenase(s) might be theproduct of chloroplast ndh genes and that this activity is involvedin chlororespiration. (Received April 25, 1994; Accepted December 5, 1994)     

Nitric oxide regulation of mitochondrial oxygen consumption II: Molecular mechanism and tissue physiology

Nitric oxide (NO) is an intercellular signaling molecule; among its many and varied roles are the control of blood flow and blood pressure via activation of the heme enzyme, soluble guanylate cyclase. A growing body of evidence suggests that an additional target for NO is the mitochondrial oxygen-consuming heme/copper enzyme, cytochrome c oxidase. This review describes the molecular mechanism of this interaction and the consequences for its likely physiological role. The oxygen reactive site in cytochrome oxidase contains both heme iron (a₃) and copper (Cu_B) centers. NO inhibits cytochrome oxidase in both an oxygen-competitive (at heme a₃) and oxygen-independent (at Cu_B) manner. Before inhibition of oxygen consumption, changes can be observed in enzyme and substrate (cytochrome c) redox state. Physiological consequences can be mediated either by direct "metabolic" effects on oxygen consumption or via indirect "signaling" effects via mitochondrial redox state changes and free radical production. The detailed kinetics suggest, but do not prove, that cytochrome oxidase can be a target for NO even under circumstances when guanylate cyclase, its primary high affinity target, is not fully activated. In vivo organ and whole body measures of NO synthase inhibition suggest a possible role for NO inhibition of cytochrome oxidase. However, a detailed mapping of NO and oxygen levels, combined with direct measures of cytochrome oxidase/NO binding, in physiology is still awaited. mitochondria; cytochrome oxidase     

Mitochondrial Matrix Ca2+ Accumulation Regulates Cytosolic NAD+/NADH Metabolism,Protein Acetylation,and Sirtuin Expression

Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD⁺/NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades.     

Reactive oxygen species formation in the transition to hypoxia in skeletal muscle

Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 µM H₂O₂ applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O₂ to 0%, 5%, 21%, or 40% O₂, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O₂ exposure. This signal could be inhibited completely with 40 µM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H₂O₂, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular PO₂, a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O₂-sensing systems and responses to acute metabolic stress. dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat     

(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.     

The real‐time quantification of autofluorescence spectrum shape for the monitoring of mitochondrial metabolism

The cellular proportion of free and protein‐bound NADH complexes is increasingly recognized as a metabolic indicator and biomarker. Because free and bound forms exhibit different fluorescence spectra, we consider whether autofluorescence shape sufficiently correlates with mitochondrial metabolism to be useful for monitoring in cellular suspensions. Several computational approaches for rapidly quantifying spectrum shape are used to detect Saccharomyces cereviseae response to oxygenation, and to the addition of mitochondrial functional modifiers and metabolic substrates. Observed changes appear consistent with previous studies probing free/protein‐bound proportions, making this a potentially useful approach for the real‐time monitoring of metabolism. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

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.     

On-line monitoring of the denitrification process by measurement of NADH fluorescence

On-line monitoring method of the denitrification process was developed by NADH fluorescence measurements using the facultative microorganism, Ochrobactrum anthropi SY509. The NADH fluorescence signals showed a rapid drop and a rise at the initiation and termination points of the denitrification, respectively. This NADH fluorescence method could be applied successfully to the monitoring of the denitrification process in sequential batch and continuous operations while these rapid changes of fluorescence were not observed in a batch operation due to accumulation of some metabolites secreted from the microorganism.     

Temperature Dependent Alterations in the Pattern of Photochemical and Non-Photochemical Quenching and Associated Changes in the Photosystem II Conditions of the Leaves

The alterations in the PSII activity of leaves, subsequent toa mild or severe heat stress were characterized by monitoringthe Chl a fluorescence and thermoluminescence emission fromintact leaves. The Chl a fluorescence measurements were carriedout in leaves adapted to either ‘state I’ or ‘stateII’ since under these two conditions the photosyntheticapparatus is known to have distinctly different structure-functionrelationships. The pattern of Chl a fluorescence induction instate II-adapted leaves was different from that of state I-adaptedleaves due to the alterations in the extent of photochemical(q_Q) and non-photochemical (q_E) quenching during the time courseof induction. The pattern of changes in q_Q and q_E values wasalso altered by heat treatment depending on the severity ofheat stress; severe heat stress (47°C) suppressing theseparameters drastically. Mild heat treatment (42°C) did notaffect the ability of leaves to undergo state I to state IItransition whereas the severe heat stress totally abolishedsuch transition. The fluorescence and thermoluminescence characteristicsof the leaves that have been exposed to the severe heat stresssuggest that a large number of affected PSII units retain afunctional water-oxidizing complex at the donor side. (Received June 14, 1994; Accepted July 19, 1995)     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa     

ROS scavenging before 27 degrees C ischemia protects hearts and reduces mitochondrial ROS, Ca2+ overload, and changes in redox state

We have shown that cold perfusion of hearts generates reactive oxygen and nitrogen species (ROS/RNS). In this study, we determined 1) whether ROS scavenging only during cold perfusion before global ischemia improves mitochondrial and myocardial function, and 2) which ROS leads to compromised cardiac function during ischemia and reperfusion (I/R) injury. Using fluorescence spectrophotometry, we monitored redox balance (NADH and FAD), O₂^– levels and mitochondrial Ca²⁺ (m[Ca²⁺]) at the left ventricular wall in 120 guinea pig isolated hearts divided into control (Con), MnTBAP (a superoxide dismutase 2 mimetic), MnTBAP (M) + catalase (C) + glutathione (G) (MCG), C+G (CG), and N^G-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase inhibitor) groups. After an initial period of warm perfusion, hearts were treated with drugs before and after at 27°C. Drugs were washed out before 2 h at 27°C ischemia and 2 h at 37°C reperfusion. We found that on reperfusion the MnTBAP group had the worst functional recovery and largest infarction with the highest m[Ca²⁺], most oxidized redox state and increased ROS levels. The MCG group had the best recovery, the smallest infarction, the lowest ROS level, the lowest m[Ca²⁺], and the most reduced redox state. CG and L-NAME groups gave results intermediate to those of the MnTBAP and MCG groups. Our results indicate that the scavenging of cold-induced O₂^– species to less toxic downstream products additionally protects during and after cold I/R by preserving mitochondrial function. Because MnTBAP treatment showed the worst functional return along with poor preservation of mitochondrial bioenergetics, accumulation of H₂O₂ and/or hydroxyl radicals during cold perfusion may be involved in compromised function during subsequent cold I/R injury. hypothermic ischemia; mitochondrial Ca²⁺; reactive oxygen species     

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.     

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。