title: Neutral lipids associated with haemozoin mediate efficient and rapid β-haematin formation at physiological pH, temperature and ionic composition.
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 (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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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(+), Mg(2+), Ca(2+)), or anions (H(2)PO(4)(-), HCO(3)(-), ATP, 2,3-diphosphoglycerate, glutathione). Confocal microscopy demonstrated β-haematin formation occurs in association with the lipid particles.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa.
authors.forename: Melvin A
authors.initials: MA
authors.lastname: Ambele
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Egan
mesh_terms: D055598:Chemical Phenomena; D003124:Colorimetry / Q000379:methods; D004583:Electrons; D005453:Fluorescence; D006420:Hemeproteins / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D013696:Temperature
chemical_list: D006420:Hemeproteins; D008055:Lipids; C020753:hemozoin; D006427:Hemin