Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials

 

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dc.contributor.author Combrinck, Jill en_ZA
dc.contributor.author Fong, Kim en_ZA
dc.contributor.author Gibhard, Liezl en_ZA
dc.contributor.author Smith, Peter en_ZA
dc.contributor.author Wright, David en_ZA
dc.contributor.author Egan, Timothy en_ZA
dc.date.accessioned 2015-12-07T08:51:42Z
dc.date.available 2015-12-07T08:51:42Z
dc.date.issued 2015 en_ZA
dc.identifier.citation Combrinck, J. M., Fong, K. Y., Gibhard, L., Smith, P. J., Wright, D. W., & Egan, T. J. (2015). Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials. Malaria journal, 14(1), 253. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/15644
dc.identifier.uri http://dx.doi.org/10.1186/s12936-015-0729-9
dc.description.abstract BACKGROUND: The activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes. METHODS: All major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5%v/v, pH7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method. RESULTS: A higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem. CONCLUSIONS: This higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials. en_ZA
dc.language.iso eng en_ZA
dc.publisher BioMed Central Ltd en_ZA
dc.rights This is an Open Access article distributed under the terms of the Creative Commons Attribution License en_ZA
dc.rights.uri http://creativecommons.org/licenses/by/4.0 en_ZA
dc.source Malaria Journal en_ZA
dc.source.uri http://www.malariajournal.com/ en_ZA
dc.subject.other Malaria en_ZA
dc.subject.other Plasmodium falciparum en_ZA
dc.subject.other Haem en_ZA
dc.subject.other Haemozoin en_ZA
dc.subject.other β-haematin en_ZA
dc.subject.other Colorimetry en_ZA
dc.subject.other 24-well plate assay en_ZA
dc.subject.other Flow cytometry en_ZA
dc.title Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials en_ZA
dc.type Journal Article en_ZA
dc.rights.holder 2015 Combrinck et al. en_ZA
uct.type.publication Research en_ZA
uct.type.resource Article en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Health Sciences en_ZA
dc.publisher.department Division of Clinical Pharmacology en_ZA
uct.type.filetype Text
uct.type.filetype Image


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This is an Open Access article distributed under the terms of the Creative Commons Attribution License Except where otherwise noted, this item's license is described as This is an Open Access article distributed under the terms of the Creative Commons Attribution License