Structural and thermodynamic investigations of metabolites of the thromboxane synthase pathway

Abstract

Reversed-phase high-performance liquid chromatographic (RP-HPLC) separations represent methodological approaches for the investigation of reaction mixtures containing fatty acids. Therefore, these analytical methods are very useful tools for kinetic studies and quantifications. In this report, we summarize our earlier results about special determinations of equilibria of the thromboxane synthase products thromboxane B 2 (TXB 2) and 12- S-hydroxyheptadecatrienoic acid (HHT) performed by RP-HPLC. Intending to quantify these metabolites phenomena of substance instability, equilibrium reactions and surface adsorption striked out. Choosing optimized and modulated chromatographic parameters, e.g. composition of the mobile phase (pH, percentage of organic modifier) and column temperature combined with diode array detection allowed detailed, highly reproducible, precise and quantitative 3D analyses clarifying the emerged effects. Two hemiacetalic anomers of TXB 2, two cis– trans isomers of HHT and its partition equilibrium with contacted surfaces were quantified and characterized thermodynamically. Dissociation, equilibrium and rate constants, Gibbs free energy, standard enthalpy, and entropy of reaction were calculated based on experimental HPLC data.