Qiu Liqun, Zhao Weizhao, Sick Thomas
Department of Biomedical Engineering, Department of Neurology, University of Miami, PO Box 248294, Coral Gables, FL 33124-0621, USA.
Comput Biol Med. 2005 Oct;35(7):583-601. doi: 10.1016/j.compbiomed.2004.05.002.
Dysfunction of mitochondria links a variety of central nervous system disorders and other neurodegenerative diseases. The primary respiratory chain substrate reduced-form nicotinamide adenine dinucleotide (NADH) is an important regulator of respiratory chain function in mitochondria and, because of its fluorescent properties, has been used to assess mitochondrial pathophysiology in cells and tissues. However, assessment of changes in tissue NADH has been limited to qualitative analysis primarily because hemoglobin (Hb) interferes with NADH fluorescence measurements by absorbing both excitation and emission light. This report presents a computer-assisted approach to estimate tissue NADH and Hb concentrations quantitatively at the same time. The method is based on a two-dimensionally interpolated database model that is calibrated by fluorescence emission spectra with known-value standard chemical solutions. Quantitative concentrations for NADH and Hb can be determined by the corresponding known-value spectral data that have the minimum error to the sample spectrum obtained from an experiment. Repeatability and reliability tests are also presented in this report. Results demonstrate that this method can feasibly quantify the NADH content regardless of the Hb background in living hippocampal cells during hypoxia, suggesting that it has the potential to be applied to in vivo experiments in the future.
线粒体功能障碍与多种中枢神经系统疾病及其他神经退行性疾病相关。主要呼吸链底物还原型烟酰胺腺嘌呤二核苷酸（NADH）是线粒体呼吸链功能的重要调节因子，因其荧光特性，已被用于评估细胞和组织中的线粒体病理生理学。然而，组织NADH变化的评估主要限于定性分析，这主要是因为血红蛋白（Hb）通过吸收激发光和发射光干扰NADH荧光测量。本报告提出了一种计算机辅助方法，可同时定量估计组织NADH和Hb浓度。该方法基于二维插值数据库模型，该模型通过已知值标准化学溶液的荧光发射光谱进行校准。NADH和Hb的定量浓度可通过与从实验获得的样品光谱误差最小的相应已知值光谱数据来确定。本报告还进行了重复性和可靠性测试。结果表明，该方法能够在缺氧期间对活海马细胞中无论Hb背景如何的NADH含量进行可行的定量分析，这表明它未来有应用于体内实验的潜力。
