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

Simple item page
dc.contributor.authorCombrinck, Jillen_ZA
dc.contributor.authorFong, Kimen_ZA
dc.contributor.authorGibhard, Liezlen_ZA
dc.contributor.authorSmith, Peteren_ZA
dc.contributor.authorWright, Daviden_ZA
dc.contributor.authorEgan, Timothyen_ZA
dc.date.accessioned2015-12-07T08:51:42Z
dc.date.available2015-12-07T08:51:42Z
dc.date.issued2015en_ZA
dc.description.abstractBACKGROUND: 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.identifier.apacitationCombrinck, J., Fong, K., Gibhard, L., Smith, P., Wright, D., & Egan, T. (2015). Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials. <i>Malaria Journal</i>, http://hdl.handle.net/11427/15644en_ZA
dc.identifier.chicagocitationCombrinck, Jill, Kim Fong, Liezl Gibhard, Peter Smith, David Wright, and Timothy Egan "Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials." <i>Malaria Journal</i> (2015) http://hdl.handle.net/11427/15644en_ZA
dc.identifier.citationCombrinck, 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.ris TY - Journal Article AU - Combrinck, Jill AU - Fong, Kim AU - Gibhard, Liezl AU - Smith, Peter AU - Wright, David AU - Egan, Timothy AB - 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. DA - 2015 DB - OpenUCT DO - 10.1186/s12936-015-0729-9 DP - University of Cape Town J1 - Malaria Journal LK - https://open.uct.ac.za PB - University of Cape Town PY - 2015 T1 - Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials TI - Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials UR - http://hdl.handle.net/11427/15644 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/15644
dc.identifier.urihttp://dx.doi.org/10.1186/s12936-015-0729-9
dc.identifier.vancouvercitationCombrinck J, Fong K, Gibhard L, Smith P, Wright D, Egan T. Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials. Malaria Journal. 2015; http://hdl.handle.net/11427/15644.en_ZA
dc.language.isoengen_ZA
dc.publisherBioMed Central Ltden_ZA
dc.publisher.departmentDivision of Clinical Pharmacologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsThis is an Open Access article distributed under the terms of the Creative Commons Attribution Licenseen_ZA
dc.rights.holder2015 Combrinck et al.en_ZA
dc.rights.urihttp://creativecommons.org/licenses/by/4.0en_ZA
dc.sourceMalaria Journalen_ZA
dc.source.urihttp://www.malariajournal.com/en_ZA
dc.subject.otherMalariaen_ZA
dc.subject.otherPlasmodium falciparumen_ZA
dc.subject.otherHaemen_ZA
dc.subject.otherHaemozoinen_ZA
dc.subject.otherβ-haematinen_ZA
dc.subject.otherColorimetryen_ZA
dc.subject.other24-well plate assayen_ZA
dc.subject.otherFlow cytometryen_ZA
dc.titleOptimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarialsen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Combrinck_Optimization_multi_well_colorimetric_assay_2015.pdf
Size:
2.11 MB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Journal Articles