Quantitative analysis of brain NADH in the presence of hemoglobin using microfiber spectrofluorometry: a pre-calibration approach
Introduction
Mitochondria are the principal sites for adenosine triphosphate (ATP) production in aerobic cells. Dysfunction of mitochondria underlies a variety of central nervous system (CNS) disorders including stroke and brain trauma, as well as neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. The molecular mechanisms by which mitochondria contribute to these pathophysiological processes are only starting to be understood [1], [2], [3], [4]. One solution to this question is to measure the functional state of mitochondria directly in brain and then correlate the developing brain damage or functional changes with the progress of a particular disease.
Many studies have been carried out to monitor mitochondrial function both in vitro and in vivo [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. Among them are absorption spectrophotometric and spectrofluorometric techniques that measure changes of redox activity of mitochondrial cytochromes and the primary respiratory chain substrate, nicotinamide adenine dinucleotide (NADH) [15], [16], [17], [18]. Fluorometric techniques could also be a potential noninvasive diagnostic approach to monitor the cerebral intracellular functional state and oxygen supply. NADH is an important factor of oxidative phosphorylation, and some fluorometric methods were developed to measure changes of NADH levels more than 40 years ago [5]. Measuring NADH fluorescence as a practical method to monitor cell function is not only used in brain research [19] and tissue viability assessment [20], but also in clinical applications, such as kidney and liver transplantation [21], [22] and cancer detection [23].
The application of NADH fluorescence methods to in vitro and in vivo experiments is, however, limited to qualitative analysis because several problems are encountered in quantification of NADH concentration (or redox state). In blood-perfused tissues, the intensity of NADH fluorescence is not only determined by NADH concentration but also by changes in hemoglobin (Hb) concentration and its oxygenation/redox state. Thus, in blood-perfused tissues, mitochondrial NADH fluorescence will be determined both by the concentration of NADH and by the tissue content of Hb. Different methods have been used to compensate for the effect of Hb while estimating the redox state from measured NADH fluorescence intensity [7], [22]. However, previous studies could not quantitatively determine the NADH content directly. Efforts were usually based on indirect approaches. For instance, the enzymatic cycling method [24] applied to animal experiments measured NADH concentration by destroying living tissue into separated samples. Measuring other signal changes that were relevant to the change of NADH was another semi-quantification approach. Rampil et al. [7] used the ratio of NADH fluorescence intensity to ultraviolet (UV) diffuse reflectance intensity for the estimation of NADH in blood-perfused rat heart. Similar problems have also been encountered in the determination of different chemical components from the fluorescence spectra in general chemical analysis. Analytical solutions to these problems are mainly based on the principal component analysis (PCA) approach [25] or its modifications [26], [27], [28], [29]. In this type of approach, it is assumed that spectral data are obtained from samples of different components’ linear combinations, and the mathematical models are presented by linear algebraic operations of the eigenvectors of the fluorescence spectral matrices.
This report presents a direct method to quantify NADH content from measured NADH fluorescence emission spectra. The method is based on a pre-calibration process that creates a mathematical database model. Standard chemical solutions serve as pre-calibrated concentrations of NADH and Hb. Calibrated spectra data are then interpolated by a bi-cubic algorithm and are saved in a database. Quantitative analysis is performed by searching two-dimensionally for the nearest NADH/Hb index that has the minimum deviation from the experimental data. This method is a pattern-analysis-based approach. In following sections, we will describe the experimental setup, pre-calibration procedure, NADH fluorescence measurement, the mathematical model, and quantitative analysis of NADH, respectively. Repeatability and reliability tests and experimental results from metabolizing tissue will also be presented.
Section snippets
Solution preparation
Chemical solutions were employed for two purposes: (1) pre-calibration and (2) accuracy testing. A typical solution was composed of alcohol dehydrogenase (product no. A3263, Sigma, St. Louis, MO, USA), reduced-form β-NADH (product no. N8129, Sigma, St. Louis, MO, USA), and venous blood from the right atrium of the male Wistar rat, which is used as the resource of Hb. Before pre-calibration, alcohol dehydrogenase and blood were mixed with artificial cerebrospinal fluid (ACSF) [30] to mimic the
Mathematical model
From the one-dimensional function of NADH fluorescence (spectral amplitude vs. wavelength), it is very difficult to estimate quantitatively the concentration of NADH in the presence of Hb. However, at one particular wavelength, if we investigate the amplitudes of the spectra with different NADH and Hb concentrations from known chemical solutions (Table 1), the amplitude can be treated as a two-variable-indexed function. These two variables are the concentration of NADH and the concentration of
Data interpolation
In order to show clearly the spectra acquired from the 12 standard solutions described in Table 1, four groups of spectra are presented in Fig. 7. Each group (organized by column in Table 1) has a fixed NADH concentration (NADH concentration values are A=5, B=15, C=25, in four columns of Table 1, respectively) and different Hb values (Hb=0, 2, of NADH solution for each group).
After two-dimensionally interpolating amplitude values at one particular wavelength (such as the 12
Conclusion
It is well known that the initial step of the mitochondrial respiratory chain involves the oxidation of NADH to NAD+ by complex I [35]. Consequently, the redox state (NADH/NAD+) of mitochondrial NADH is closely associated with the respiratory state of mitochondria, providing a very sensitive measurement of oxygen availability or mitochondrial function. The different fluorescence properties of NADH and NAD+ molecules make it possible for the measurement of the NADH redox state since NADH emits
Summary
Mitochondria are the cells’ power source, and dysfunction of mitochondria has been linked to many neurological diseases. The primary respiratory chain substrate reduced-form nicotinamide adenine dinucleotide (NADH) is an important regulator of respiratory chain function in mitochondria. Because of its fluorescent properties, NADH has been used to qualitatively assess mitochondrial pathophysiology in cells and tissues. However, assessment of changes in tissue NADH has been limited to qualitative
Acknowledgements
This study was supported by the National Institutes of Health (NS38276 and NS05820) and the National Science Foundation (DUE-0127290). The authors are very grateful to Dr. Brant Watson for his valuable suggestions and to Chunyan Wu and Jun Yang for their programming assistance.
Liqun Qiu, M.S., is a Research Associate in the Department of Neurology, University of Miami School of Medicine. Liqun Qiu received her Master degree in Biomedical Engineering from the University of Miami in August 2003, and a Bachelor of Science degree, also in Biomedical Engineering, from the Zhe-Jiang University, P. R. China in 1997. Liqun Qiu's current research is focused on understanding mitochondrial dysfunction and electrophysiological change in brain under hypoxia and hypo-metabolic
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Liqun Qiu, M.S., is a Research Associate in the Department of Neurology, University of Miami School of Medicine. Liqun Qiu received her Master degree in Biomedical Engineering from the University of Miami in August 2003, and a Bachelor of Science degree, also in Biomedical Engineering, from the Zhe-Jiang University, P. R. China in 1997. Liqun Qiu's current research is focused on understanding mitochondrial dysfunction and electrophysiological change in brain under hypoxia and hypo-metabolic conditions.
Weizhao Zhao, Ph.D., is an Associate Professor of Biomedical Engineering and Neurology. He received his Ph.D. degree in Electrical and Computer Engineering in 1991, and Master degree in Electrical Engineering in 1987, both from the University of Miami, FL. Dr. Zhao teaches Biomedical Measurement, Medical Imaging related courses in the Department of Biomedical Engineering in the University of Miami College of Engineering. He is Director of the Image Analysis of Computing Laboratory of the Cerebral Vascular Disease Research Center in the University of Miami School of Medicine.
Thomas J. Sick, Ph.D., is currently a Professor of Neurology, Physiology, and Biophysics at the University of Miami School of Medicine. He received his Ph.D. degree in Physiology from Tulane University in 1979, and a Master of Science degree in Psychology from Tulane University in 1976. His undergraduate studies were conducted at Mount Saint Mary's College, MD, where he received a Bachelor of Arts degree in 1973. Dr. Sick's research revolves around studies of mitochondrial function in brain following neurological injury or disease.