Attenuated Total Reflectance Fourier Transformation Infrared spectroscopy fingerprinted online monitoring of the kinetics of circulating Butyrylcholinesterase enzyme during metabolism of bambuterol

• Published in: Analytica chimica acta 2018-04, Vol.1005, p.70-80
• Main Authors: Eid, Sherif M., Abd El-Rahman, Mohamed K., Elghobashy, Mohamed R., Kelani, Khadiga M.
• Format: Article
• Language: English
• Subjects: Ability; Affinity; ATR FTIR; Bambuterol; Bronchodilator Agents - blood; Bronchodilator Agents - metabolism; Butyrylcholinesterase; Butyrylcholinesterase - blood; Butyrylcholinesterase - metabolism; Continuous flow; Enzyme Assays - instrumentation; Enzyme Assays - methods; Enzyme kinetics; Equipment Design; Fourier transforms; Humans; Infrared spectroscopy; Isotonicity; Kinetics; Metabolism; Online enzyme monitoring; Prodrugs - analysis; Prodrugs - metabolism; Reflectance; Spectroscopy, Fourier Transform Infrared - instrumentation; Spectroscopy, Fourier Transform Infrared - methods; Studies; Substrates; Temperature; Terbutaline; Terbutaline - analogs & derivatives; Terbutaline - blood; Terbutaline - metabolism
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2017.12.011

We have described a continuous flow ATR-FTIR method for measuring some of the Butyrylcholinesterase enzyme kinetics (Km and Vmax). This is done by developing a circulating system to be close as much as possible to the human circulation using human serum as a source of the enzyme with adjusted pH, isotonicity and temperature to give the maximum affinity of the enzyme towards its substrate (bambuterol). The experiment was running continuously for 90 min to monitor the production of terbutaline from the zero time of its appearance with a measured spectrum in each minute using ZnSe prism. The method was selective and successful for determination of Vmax to be 8.16 × 10−8 mol/min/ml and Km to be 2.28 × 10−5 mol, showing high affinity of the enzyme towards its prodrug substrate Bambuterol. This study critically probes the quantitative ability of the ATR-FTIR method for terbutaline, which was validated according to ICH guidelines showing high accuracy 100.39% and high selectivity towards the produced terbutaline, as the produced spectrums considered as fingerprint of each compound. [Display omitted] •A system for measuring Butyrylcholinesterase enzyme kinetics established using Human serum, HPLC pump, ATR-FTIR unit.•The system measures the product of bambuterol metabolism.•A Validated method for determination of terbutaline was established.•Characteristics and differences of the ATR-FTIR spectrums of BAM and TER were studied.•Finally the reaction Kinetics (Km and Vmax) of BAM metabolism were studied.