title: Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage.
abstract: The metabolic coenzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are the primary electron donor and acceptor respectively, participate in almost all biological metabolic pathways. This study develops a novel method for the quantitative kinetic analysis of the degradation reaction of NADH and the formation reaction of FAD in human plasma containing an uncalibrated interferent, by using three-way calibration based on multi-way fluorescence technique. In the three-way analysis, by using the calibration set in a static manner, we directly predicted the concentrations of both analytes in the mixture at any time after the start of their reactions, even in the presence of an uncalibrated spectral interferent and a varying background interferent. The satisfactory quantitative results indicate that the proposed method allows one to directly monitor the concentration of each analyte in the mixture as the function of time in real-time and nondestructively, instead of determining the concentration after the analytical separation. Thereafter, we fitted the first-order rate law to their concentration data throughout their reactions. Additionally, a four-way calibration procedure is developed as an alternative for highly collinear systems. The results of the four-way analysis confirmed the results of the three-way analysis and revealed that both the degradation reaction of NADH and the formation reaction of FAD in human plasma fit the first-order rate law. The proposed methods could be expected to provide promising tools for simultaneous kinetic analysis of multiple reactions in complex systems in real-time and nondestructively.
authors.affiliation: School of Chemistry and Chemical Engineering, Guizhou University, Guiyang 550025, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Kang
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. Electronic address: hlwu@hnu.edu.cn.
authors.forename: Hai-Long
authors.initials: HL
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Shou-Xia
authors.initials: SX
authors.lastname: Xiang
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Xiao-Hua
authors.initials: XH
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Yong-Jie
authors.initials: YJ
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Ru-Qin
authors.initials: RQ
authors.lastname: Yu
mesh_terms: D002138:Calibration; D005182:Flavin-Adenine Dinucleotide / Q000097:blood*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000097:blood*
chemical_list: D009243:NAD; D005182:Flavin-Adenine Dinucleotide
keywords: Chemical kinetics; Fluorescence; Second-order advantage; Three-way calibration