On-line Inhibition Study of Cytochrome P450 2C9 Isoform Reaction with Diclofenac
| Authors | |
|---|---|
| Year of publication | 2011 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts 8th International Interdisciplinary Meeting on Bioanalysis CECE 2011 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | in-capillary reaction, on-line capillary eletrophoresis, cytochrome P450, diclofenac, sulfaphenazole, drug metabolism |
| Description | During last two decades, capillary electrophoresis (CE) proved its position of a powerful and versatile analytical technique for enzyme assays exhibiting a number of advantages over conventional methods. These include fast, highly effective separations, low sample and other chemicals consumption, and high throughput by automation. In addition, the fuse-silica capillary can serve not only as a separation column but also as a nanoscale reaction chamber integrating production, separation and detection of analytes into single run. In-capillary enzymatic assays thus further intensify the testing economy and enable fully automated analyses performed by commercially available CE devices. Development of effective way for reactants mixing inside very limited space of a capillary constitutes a challenging task which prevents wider practical utilization of this attractive concept, nevertheless. Couple of approaches dealing with this limitation has been already presented; however, none of them is suitable for practical implementation. For this reason, the goal of this study was to introduce a generic methodology which enables interfusing of selectable reactants into homogenous reaction mixture inside a nanoscale capillary reactor. Principle based on combination of longitudinal and transverse diffusion was employed to fulfill these requirements. Conceptually, the solutions of reaction mixture components are injected by relatively low pressure as a series of consecutive narrow plugs having parabolic profiles due to laminar flow inside the capillary. Resulting character of plugs with rather longitudinal interfaces then enable creation of homogenous reaction mixture by both longitudinal and transverse diffusions within minute period. On-line inhibition study of cytochrome P450 2C9 reaction with diclofenac and sulfaphenazole was performed in order to prove the applicability of proposed system. Obtained values of Michaelis constant, 50 % inhibitory concentration and inhibition constant were in agreement with literature data determined by other techniques. Presented principle thus constitutes a promising tool for on-line inhibition studies of cytochrome P450 2C9 requiring frequent changes in reaction mixture composition. |
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