Neutral lipids associated with haemozoin mediate efficient and rapid beta-haematin formation at physiological pH, temperature and ionic composition

Ambele, Melvin; Egan, Timothy

Neutral lipids associated with haemozoin mediate efficient and rapid beta-haematin formation at physiological pH, temperature and ionic composition

Journal Article

2012

Journal Title

Malaria Journal

Link to Journal

http://www.malariajournal.com/

Publisher

BioMed Central Ltd

Publisher

University of Cape Town

Department

Department of Chemistry

Faculty

Faculty of Science

License

http://creativecommons.org/licenses/by/2.0

Abstract

BACKGROUND: The malaria parasite disposes of host-derived ferrihaem (iron(III)protoporphyrin IX, Fe(III)PPIX) by conversion to crystalline haemozoin in close association with neutral lipids. Lipids mediate synthetic haemozoin (beta-haematin) formation very efficiently. However, the effect on reaction rates of concentrations of lipid, Fe(III)PPIX and physiologically relevant ions and biomolecules are unknown. METHODS: Lipid emulsions containing Fe(III)PPIX were prepared in aqueous medium (pH 4.8, 37degreesC) to mediate beta-haematin formation. The reaction was quenched at various times and free Fe(III)PPIX measured colorimetrically as a pyridine complex and the kinetics and yields analysed. Products were also characterized by FTIR, TEM and electron diffraction. Autofluorescence was also used to monitor beta-haematin formation by confocal microscopy. RESULTS: At fixed Fe(III)PPIX concentration, beta-haematin yields remained constant with decreasing lipid concentration until a cut-off ratio was reached whereupon efficiency decreased dramatically. For the haemozoin-associated neutral lipid blend (NLB) and monopalmitoylglycerol (MPG), this occurred below a lipid/Fe(III)PPIX (L/H) ratio of 0.54. Rate constants were found to increase with L/H ratio above the cut-off. At 16 muM MPG, Fe(III)PPIX concentration could be raised until the L/H ratio reached the same ratio before a sudden decline in yield was observed. MPG-mediated beta-haematin formation was relatively insensitive to biologically relevant cations (Na+, K+, Mg2+, Ca2+), or anions (H2PO4, HCO3, ATP, 2,3-diphosphoglycerate, glutathione). Confocal microscopy demonstrated beta-haematin formation occurs in association with the lipid particles. CONCLUSIONS: Kinetics of beta-haematin formation have shown that haemozoin-associated neutral lipids alone are capable of mediating beta-haematin formation at adequate rates under physiologically realistic conditions of ion concentrations to account for haemozoin formation.