Methods: Artemisinin sensitive and resistant P. falciparum parasites were tightly synchronized with D-sorbitol treatment. Fourteen flasks (with 3.5 x 106 average parasite equivalence) containing early ring-stage parasites were subsequently exposed to dihydroartemisinin (DHA) for six hours (controls were not drug exposed). The parasites were washed twice with drug free media and then lysed from the erythrocyte membrane using saponin. A series of wash steps were then performed to remove lysis products, which include contaminating erythrocyte metabolites, and frozen prior to analysis. This procedure was performed five times for each condition to provide replicates for metabolomic analysis. Parasite metabolites were then extracted from at least thirty microliters of pelletized parasite material using methanol and detected using liquid chromatography and mass spectrometry by a commercial metabolomics service (Metabolon, Inc.).
Results: Orotate, a metabolite product of pyrimidine biosynthesis, was reduced to undetectable levels in both sensitive and resistant parasites after DHA exposure. In addition, levels of both lipid and cholesterol-derived metabolites were significantly elevated in DHA-exposed sensitive parasites, compared to resistant parasites.
Conclusion: Untargeted metabolomic studies are achievable with low amounts of parasite material. Overall, the major metabolic effects of artemisinin treatment detected in our studies are consistent with previous targeted biochemical studies, lending credence to the appropriateness of our experimental design.
M. Carey, None
J. Guler, None