Alcohols induce beta-hematin formation via the dissociation of aggregated heme and reduction in interfacial tension of the solution.

The formation of the malarial pigment, a unique hemozoin crystal with unit cells comprised of heme dimers, has been proposed as an ideal target for antimalarial screening. The mechanism of beta-hematin formation (a synthetic crystal structurally identical to hemozoin) has been suggested that a hydrophobic interaction is needed to solubilize heme, but this hypothesis needs further evidence. Direct study of the process of hemozoin formation in the malarial food vacuole has not been performed, due to complicated groups of lipids and proteins. To overcome this difficulty and to explore the environmental conditions for beta-hematin formation, we systematically studied beta-hematin formation induced by a series of small normal alcohols (methanol, ethanol, n-propanol, and n-butanol), which are structurally similar. For the first time, the ability of beta-hematin inducer could be evaluated by its concentration that is required to enhance heme crystallization by 50% (EC(50) values). These values provide a rapid and convenient tool for comparing the ability of initiators in beta-hematin formation. Our results showed that the ability of alcohols to induce beta-hematin formation in the order: n-butanol>n-propanol>ethanol>methanol. The induction of beta-hematin formation by alcohols is related with their degree of hydrophobicity and ability to solubilize heme, suggesting that the dissociation of aggregated heme by alcohols is a major factor in beta-hematin formation. In addition, alcohols can reduce the surface tension of a solution, thus lowering the energy barrier for creating critical nuclei.

Leukocyte activation by malarial pigment.

Malarial pigment, a unique hemozoin crystal composed of unit cells of heme dimers, is present in large amounts in circulating monocytes and neutrophils and can persist unchanged in macrophages for several months. In the present study, we investigated the effect of hemozoin not only on macrophages, but also on neutrophils. We used beta-hematin (BH), a chemically synthetic crystal structurally identical to hemozoin, for these studies. In vitro, BH up-regulated the expression of tumor necrosis factor-alpha in whole blood and in isolated peritoneal macrophages, indicating that hemozoin is able to stimulate monocytes. BH stimulated murine peritoneal neutrophils to express macrophage inflammatory protein-2 (MIP-2), a homologue of human interleukin-8 that is used as a marker of neutrophil activation. Injecting BH into the peritoneal cavity resulted in a dose-dependent migration of neutrophils and a high level of myeloperoxidase activity of peritoneal cells. Finally, BH directly induced neutrophil chemotaxis in vitro. Taken together, these results suggest that the malarial pigment hemozoin can activate leukocytes and may participate in the pathology of severe malaria.

Inhibition assay of beta-hematin formation initiated by lecithin for screening new antimalarial drugs.

Measurement of heme crystallization provides a tool for screening new antimalarial drugs. Current assays for heme crystallization have employed initiators such as thermo, histidine-rich proteins, and lipids extracted from parasites and infected plasma. These initiators are unnatural or require laborious steps to prepare. In this study, we used a commercially available lipid, lecithin, a kind of phospholipid containing about 50% unsaturated fatty acids, as an initiator for heme crystal (beta-hematin) formation. We demonstrated that the inhibition of lecithin-based beta-hematin formation by antimalarial drugs is highly correlated with the preformed beta-hematin-based method. In addition, the lecithin-based assay is sensitive and convenient for large-scale screening of new novel antimalarials. We also indicated that dimethyl sulfoxide is an ideal solvent for preparation of heme stock solution, which is stable and can be used for 1 month.