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

• Main Authors: Ambele Melvin A, Egan Timothy J
• Format: Article
• Language: English
• Published: BMC 2012-10-01
• Series: Malaria Journal
• Subjects: Haemozoin; β-haematin; Neutral lipids; Kinetics; Rates; Autofluorescence
• Online Access: http://www.malariajournal.com/content/11/1/337

<p>Abstract</p> <p>Background</p> <p>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 (β-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.</p> <p>Methods</p> <p>Lipid emulsions containing Fe(III)PPIX were prepared in aqueous medium (pH 4.8, 37°C) to mediate β-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 β-haematin formation by confocal microscopy.</p> <p>Results</p> <p>At fixed Fe(III)PPIX concentration, β-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 μM 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 β-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 β-haematin formation occurs in association with the lipid particles.</p> <p>Conclusions</p> <p>Kinetics of β-haematin formation have shown that haemozoin-associated neutral lipids alone are capable of mediating β-haematin formation at adequate rates under physiologically realistic conditions of ion concentrations to account for haemozoin formation.</p>