05 Jun Fitting kinetic equations derived from metabolic pathways to LC/MS-MS integrated peak areas

Ismael Zamora(1), Laura Goracci(2), Guillem Plasencia(3)
1.Lead Molecular Design, S.L., Sant Cugat del Valles, Barcelona; 2.Molecular Discovery, London, UK; 3.Lead Molecular Design S.L., Sant Cugat de Valles, Spain

Introduction WebMetabase allows drawing metabolic pathways to describe how a parent compound is transformed into metabolites in in vitro or in vivo experiments. Assuming that reactions occur in a single compartment (like is the case for an in vitro incubation) and also that each arrow corresponds to a single-step irreversible chemical reaction (even though it’s not true, i.e. they are often catalyzed by enzymes) we can build a system of differential equations whose equations, when solved, can be fitted to the MS area integrated signals. This theoretical curve and the integrated signal MS area often fit well, and when they do not, information useful to improve the metabolic pathway can be extracted from it. Methods LC/MS of several compounds was processed by MassMetasite and loaded into WebMetabase. A metabolic pathway was built for each parent compound, and the system of differential equations, assuming single step irreversible chemical reactions, was built, solved, and fitted to the experimental integrated MS area using Maxima. Visual inspection of the plot of the theoretical MS area curve and experimental MS area points vs. time sometimes allows, when the fitting quality is bad, to derive information that can be used to change the position of the compound in the metabolic pathway and improve the fitting quality Preliminary Data Chemical kinetics theory is a good approximation of enzyme kinetics in in vitro assays because typically cytochromes do not have a high affinity for the compounds they metabolize, and also because the concentration of metabolites is much lower than the enzyme affinity, thus simplifying the Michaelis-Menten enzyme kinetics equation to a first order chemical kinetics equation. We used chemical kinetics analysis of metabolic pathway, as implemented in WebMetabase, as guidance to build metabolic pathways for several compounds. In several cases, the observed MS area vs. time of compounds included in the metabolic pathways fitted the theoretical equation that would correspond to them if the metabolic pathway scheme would consist of single-step irreversible chemical equations, thus confirming that the assumptions made were correct in these cases. There were some cases where experimental MS area vs. time points does not fit the theoretical chemical kinetics curve and could not be improved by rearranging them in the metabolic pathway. In these cases where fitting was bad or failed, probably one of the previous assumptions is false, for instance they are not formed or metabolized in single-step reactions, or chemical kinetics theory is not a good approximation of the real enzyme kinetics. Novel Aspect To the best of our knowledge, chemical kinetics theory has not been previously used to verify a metabolic pathway scheme.