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

Malaria Journal

Methodology

Timothy J. Egan¹  David W. Wright²  Kim Y. Fong²  Peter J. Smith³  Jill M. Combrinck³  Liezl Gibhard³ 

[1] Department of Chemistry, University of Cape Town, Private Bag, 7701, Rondebosch, South Africa;Department of Chemistry, Vanderbilt University, Station B 351822, 37235, Nashville, TN, USA;Division of Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, Cape Town, South Africa;

关键词: Malaria;    Plasmodium falciparum;    Haem;    Haemozoin;    β-haematin;    Colorimetry;    24-well plate assay;    Flow cytometry;   

DOI  :  10.1186/s12936-015-0729-9

 received in 2015-03-13, accepted in 2015-05-07,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundThe 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.MethodsAll major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5 % v/v, pH 7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method.ResultsA 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.ConclusionsThis 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.

【 授权许可】

Unknown   
© Combrinck et al. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.