Hemozoin Production Research Articles

In Vitro Investigation Unveiling New Insights into the Antimalarial Mechanism of Chloroquine: Role in Perturbing Nucleation Events during Heme to β-Hematin Transformation.

Malaria parasites generate toxic heme during hemoglobin digestion, which is neutralized by crystallizing into inert hemozoin (β-hematin). Chloroquine blocks this detoxification process, resulting in heme-mediated toxicity in malaria parasites. However, the exact mechanism of chloroquine's action remains unknown. This study investigates the impact of chloroquine on the transformation of heme into β-hematin. The results show that chloroquine does not completely halt the transformation process but rather slows it down. Additionally, chloroquine complexation with free heme does not affect substrate availability or inhibit β-hematin formation. Scanning electron microscopy (SEM) and X-ray powder diffraction (XRD) studies indicate that the size of β-hematin crystal particles and crystallites increases in the presence of chloroquine, suggesting that chloroquine does not impede crystal growth. These findings suggest that chloroquine delays hemozoin production by perturbing the nucleation events of crystals and/or the stability of crystal nuclei. Thus, contrary to prevailing beliefs, this study provides a new perspective on the working mechanism of chloroquine.

An assay procedure to investigate the transformation of toxic heme into inert hemozoin via plasmodial heme detoxification protein

Most antimalarial therapeutics, including chloroquine and artemisinin, induce free heme-mediated toxicity in Plasmodium. This cytotoxic heme is produced as a by-product during the large-scale digestion of host hemoglobin. Conversion of this host-derived heme into inert crystalline hemozoin is the only defense mechanism in Plasmodium against heme-induced cytotoxicity. Heme detoxification protein (HDP), a highly conserved plasmodial protein, is reported to be the most efficient biological mediator for heme to hemozoin transformation. Despite its significance, HDP has never been extensively studied for heme transformation into hemozoin. Therefore, we wish to develop a method to study the HDP-mediated transformation of heme into hemozoin. We have adopted, modified, and optimized the pyridine hemochrome assay to study HDP catalysis and use substrate and time kinetics to study the HDP-mediated transformation of heme into hemozoin. In contrast to the previously reported assay for HDP, we found that the new assay is more precise, accurate, and handy, making it more suitable for kinetic studies. HDP-mediated transformation of heme into hemozoin is not a single-step process, and involves a transient intermediate, most likely a cyclic heme dimer. The kinetics and the manner of HDP-mediated hemozoin production are dependent on the substrate concentration, and a small fraction of substrate remains untransformed to hemozoin irrespective of reaction time. Combining HDP as a catalyst and the pyridine hemochrome assay will facilitate the efficient screening of future antimalarials.

Sensitive detection of Plasmodium vivax malaria by the rotating-crystal magneto-optical method in Thailand

The rotating-crystal magneto-optical detection (RMOD) method has been developed for the rapid and quantitative diagnosis of malaria and tested systematically on various malaria infection models. Very recently, an extended field trial in a high-transmission region of Papua New Guinea demonstrated its great potential for detecting malaria infections, in particular Plasmodium vivax. In the present small-scale field test, carried out in a low-transmission area of Thailand, RMOD confirmed malaria in all samples found to be infected with Plasmodium vivax by microscopy, our reference method. Moreover, the magneto-optical signal for this sample set was typically 1–3 orders of magnitude higher than the cut-off value of RMOD determined on uninfected samples. Based on the serial dilution of the original patient samples, we expect that the method can detect Plasmodium vivax malaria in blood samples with parasite densities as low as sim5–10 parasites per microliter, a limit around the pyrogenic threshold of the infection. In addition, by investigating the correlation between the magnitude of the magneto-optical signal, the parasite density and the erythrocytic stage distribution, we estimate the relative hemozoin production rates of the ring and the trophozoite stages of in vivo Plasmodium vivax infections.

Enhanced Antimalarial Efficacy Obtained by Targeted Delivery of Artemisinin in Heparin-Coated Magnetic Hollow Mesoporous Nanoparticles.

Malaria is one of the deadliest infectious diseases threatening half of the world population. With the deterioration of the parasiticidal effect of the current antimalarials, novel approaches such as screening of more specific inhibitors and targeted delivery of drugs have been under intensive research. Herein, we prepare hollow mesoporous ferrite nanoparticles (HMFNs) of 200 nm with ferromagnetic properties using a one-pot hydrothermal reaction. A magnetically targeted drug-delivery system coloaded with artemisinin in the inner magnetite shell and heparin on the outer mesoporous shell (HMFN@ART@HEP) is developed. Specific targeting of the magnetic nanoparticles to the parasite-infected erythrocytes is achieved by the attraction between the HMFNs and hemozoin (paramagnetic), a vital metabolite of plasmodium in the erythrocytic stage. With the hemozoin production reaching the maximum during the schizont period of the parasite, HMFN@ART@HEPs are adsorbed to the infected red blood cells (iRBCs), which not only interferes with the release of merozoites but also significantly enhances the inhibitory efficacy due to the increased local concentration of artemisinin. Subsequently, the heparin coated on the surface of the nanoparticles can efficiently interfere with the invasion of freshly released merozoites to new RBCs through the specific interaction between the parasite-derived ligands and heparin, which further increases the inhibitory effect on malaria. As a cluster of heparin, heparin-coated nanoparticles provide stronger blocking capability than free heparin, resulting from multivalent interactions with surface receptors on merozoite. Thus, we have developed a HMFN-based delivery system with considerable antimalarial efficacy, which is a promising platform for treatment against malaria.

Rapid and quantitative antimalarial drug efficacy testing via the magneto-optical detection of hemozoin

Emergence of resistant Plasmodium species makes drug efficacy testing a crucial part of malaria control. Here we describe a novel assay for sensitive, fast and simple drug screening via the magneto-optical detection of hemozoin, a natural biomarker formed during the hemoglobin metabolism of Plasmodium species. By quantifying hemozoin production over the intraerythrocytic cycle, we reveal that hemozoin formation is already initiated by ~ 6–12 h old ring-stage parasites. We demonstrate that the new assay is capable of drug efficacy testing with incubation times as short as 6–10 h, using synchronized P. falciparum 3D7 cultures incubated with chloroquine, piperaquine and dihydroartemisinin. The determined 50% inhibitory concentrations agree well with values established by standard assays requiring significantly longer testing time. Accordingly, we conclude that magneto-optical hemozoin detection provides a practical approach for the quick assessment of drug effect with short incubation times, which may also facilitate stage-specific assessment of drug inhibitory effects.

Enhanced In-Vitro Hemozoin Polymerization by Optimized Process using Histidine-Rich Protein II (HRPII).

Conductive biopolymers, an important class of functional materials, have received attention in various fields because of their unique electrical, optical, and physical properties. In this study, the polymerization of heme into hemozoin was carried out in an in vitro system by the newly developed heme polymerase (histidine-rich protein 2 (HRP-II)). The HRP-II was produced by recombinant E. coli BL21 from the Plasmodium falciparum gene. To improve the hemozoin production, the reaction conditions on the polymerization were investigated and the maximum production was achieved after about 790 μM at 34 °C with 200 rpm for 24 h. As a result, the production was improved about two-fold according to the stepwise optimization in an in vitro system. The produced hemozoin was qualitatively analyzed using the Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM). Finally, it was confirmed that the enzymatically polymerized hemozoin had similar physical properties to chemically synthesized hemozoin. These results could represent a significant potential for nano-biotechnology applications, and also provide guidance in research related to hemozoin utilization.

Identification of proteins for controlled nucleation of metal-organic crystals for nanoenergetics

Abstract

Quantitative characterization of hemozoin in Plasmodium berghei and vivax

The incidence and global distribution of chloroquine resistant (CR) Plasmodium vivax infection has increased since emerging in 1989. The mechanism of resistance in CR P. vivax has not been defined. The resistance likely relates to the formation and disposition of hemozoin as chloroquine's primary mechanism of action involves disruption of hemozoin formation. CR P. berghei strains, like CR P. vivax strains, are confined to reticulocyte host cells and reportedly they do not accumulate appreciable intraerythrocytic hemozoin. Reports comparing hemozoin production between P. vivax strains and CR to chloroquine sensitive (CS) P. berghei are absent. Here we compare in vivo patterns of hemozoin formation and distribution in blood, spleen and liver tissue of male Swiss mice infected with CS or CR P. berghei not treated with chloroquine and CR P. berghei also treated with chloroquine. Light microscopy, laser desorption mass spectrometry and a colorimetric hemozoin assay detect trace hemozoin in the blood of CR P. berghei infected mice but significant hemozoin accumulation in liver and spleen tissue. Field emission in lens scanning electron microscopy reveals CR P. berghei hemozoin crystals are morphologically smaller but similar to those formed by CS parasites. CR P. berghei produces approximately five-fold less total hemozoin than CS strain. Lipid analysis of CS and CR P. berghei sucrose gradient purified bloodstage hemozoin indicates a similar lipid environment around the isolated hemozoin, predominately monopalmitic glycerol and monostearic glycerol. In contrast to CR and CS P. berghei, colorimetric hemozoin analysis of P. vivax strains indicates similar amounts of hemozoin are produced despite differing chloroquine sensitivities. These results suggest CR P. berghei forms significant hemozoin which accumulates in liver and spleen tissues and that the P. vivax chloroquine resistance mechanism differs from P. berghei.

Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei

Malaria parasite resides within the host red blood cells, where it degrades vast amount of haemoglobin. During haemoglobin degradation, toxic free heme is liberated which subsequently gets converted into hemozoin. This process is facilitated by action of various proteins viz. heme detoxification protein (HDP), and histidine rich proteins II and III (HRP II & III). Out of these, HDP is the most potent in hemozoin formation and plays indispensible role for parasite survival. Despite this, the detailed study of HDP from rodent and simian parasite has not been performed till date. Here, we have cloned and sequenced hdp gene from different malaria parasites Plasmodium vinckei, Plasmodium yoelii, Plasmodium knowlesi, and Plasmodium cynomolgi. Furthermore, HDP from P. vinckei (PvHDP) was over-expressed and purified for detailed characterization. The PvHDP is cytosolic, expressed throughout the intra erythrocytic stages and its expression is higher in late trophozoite and schizont stages of parasite. The PvHDP interacts with free heme (KD=89 nM) and efficiently converts heme into hemozoin in a time and concentration dependent manner. Moreover, PvHDP showed activity in acidic pH and over a broad range of temperature. Histidine modification of PvHDP using DEPC showed reduction in heme binding and hemozoin formation, thus emphasizing the importance of histidine residues in heme binding and subsequent hemozoin production. Furthermore, applicability of PvHDP to screen anti-plasmodial agents (targeting heme to hemozoin conversion) was also determined using chloroquine, and mefloquine as reference antimalarials. Results showed that these drugs inhibit heme polymerization effectively in a concentration dependent manner. In conclusion, our study identified and biochemically characterized HDP from rodent malaria parasite P. vinckei and this will help to develop a high throughput assay to evaluate new antimalarials targeting hemozoin pathway.

Hemozoin Induces Lung Inflammation and Correlates with Malaria-Associated Acute Respiratory Distress Syndrome

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Erythrocyte membranes convert monomeric ferriprotoporphyrin IX to β-hematin in acidic environment at malarial fever temperature

Hemozoin production makes it possible for intraerythrocytic malaria parasites to digest massive quantities of hemoglobin but still avoid potential ferriprotoporphyrin IX (FP) toxicity, which they cannot decompose further. Some antimalarial drugs, such as chloroquine, work by inhibiting this production, forcing the parasite to starve to death. As part of the efforts to identify possible biological mechanisms of FP polymerization, we have used normal human erythrocyte membranes as a model, to promote β-hematin (β-h) synthesis. Hemin in 35% aqueous dimethyl sulfoxide (DMSO) was reacted with isolated erythrocyte membranes and incubated overnight in sodium acetate buffer, pH 4.8, at 41°C. Infrared spectroscopy and electron microscopy showed that β-h was produced. Hemin in 10% was less effective as the substrate than when it was in 35% DMSO. A high malarial temperature seemed to be necessary, because FP polymerization was less at 37°C than at 41°C. Production was partially inhibited by chloroquine. These observations are of interest because other investigators have reported that membrane lipids mediated FP polymerization, but whole membranes were ineffective. On the other hand, our hypothesis is that the transport vesicles (TV) in malaria parasites could provide the receptor for FP and the lipids that promote hemozoin formation. Erythrocyte membranes may not be directly involved, but Plasmodium species transport hemoglobin in membrane-bound TV into food vacuoles, where hemoglobin catabolism is completed and hemozoin crystals are stored.

Evidence for the Contribution of the Hemozoin Synthesis Pathway of the Murine Plasmodium yoelii to the Resistance to Artemisinin-Related Drugs

Plasmodium falciparum malaria is a major global health problem, causing approximately 780,000 deaths each year. In response to the spreading of P. falciparum drug resistance, WHO recommended in 2001 to use artemisinin derivatives in combination with a partner drug (called ACT) as first-line treatment for uncomplicated falciparum malaria, and most malaria-endemic countries have since changed their treatment policies accordingly. Currently, ACT are often the last treatments that can effectively and rapidly cure P. falciparum infections permitting to significantly decrease the mortality and the morbidity due to malaria. However, alarming signs of emerging resistance to artemisinin derivatives along the Thai-Cambodian border are of major concern. Through long-term in vivo pressures, we have been able to select a murine malaria model resistant to artemisinins. We demonstrated that the resistance of Plasmodium to artemisinin-based compounds depends on alterations of heme metabolism and on a loss of hemozoin formation linked to the down-expression of the recently identified Heme Detoxification Protein (HDP). These artemisinins resistant strains could be able to detoxify the free heme by an alternative catabolism pathway involving glutathione (GSH)-mediation. Finally, we confirmed that artemisinins act also like quinolines against Plasmodium via hemozoin production inhibition. The work proposed here described the mechanism of action of this class of molecules and the resistance to artemisinins of this model. These results should help both to reinforce the artemisinins activity and avoid emergence and spread of endoperoxides resistance by focusing in adequate drug partners design. Such considerations appear crucial in the current context of early artemisinin resistance in Asia.

TheIn VivoDiagnosis of Malaria: Feasibility Study Into a Magneto-Optic Fingertip Probe

An in-depth analysis is presented into the feasibility of implementing a new magneto-optic test diagnostic for malaria in the form of a noninvasive finger probe. The diagnostic principle quantifies the malaria pigment hemozoin concentration in the blood stream by measuring the fractional change in transmitted optical intensity arising from the Cotton-Mouton effect on application of a magnetic field. Hemozoin is, to varying degrees, responsive to probing in this manner whether free in the blood plasma following lysis or, more commonly, contained in intracellular vacuoles of erythrocytes or leukocytes. Measurements made on living tissue and sophisticated phantoms, which mix solid and liquid phase media to mimic fingertip physiology, demonstrate that hemozoin concentrations less than 0.02 μg/ml may be readily detectable when blood, caused to pool in the capillary loop arterioles of the extensively vascularized nail bed, is interrogated through the fingernail. On conservative assumptions regarding the relationship between hemozoin production and parasitemia and taking account of the effect of locale on hemozoin responsivity the study indicates that an instrument built on this premise may in principle offer rapid non-invasive detection of malaria at parasitemia levels below 100 parasitized red blood cells per ml.

In Vitro Activity of Artemisinin in Combination with Clotrimazole or Heat-treated Amphotericin B against Plasmodium falciparum

Currently available artemisinin-based combination therapies (ACTs) for malaria are inadequate. There remains an enormous unmet need for alternate artemisinin-based combination therapies. One of the fastest methods to identify promising artemisinin-based combination therapies is to look for synergistic or additive antimalarial interaction between artemisinin and an alternate drug against P. falciparum in vitro. Amphotericin B and clotrimazole are known drugs for treatment of human fungal infections. We repurposed clotrimazole or heat-treated amphotericin B in fixed ratio combination with artemisinin for antimalarial properties. Isobologram results show synergistic/additive interaction in both of the cases at therapeutically safe concentrations. Artemisinin, clotrimazole, and their synergistic combinations also decrease hemozoin production in parasitized erythrocytes. New permeation pathways induced in infected cells remain unaffected by drug combinations as indicated by sorbitol lysis. It would be interesting to extend the studies' in vivo system.

Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy

We previously customized a Nipkow spinning disk confocal microscope (SDCM) to acquire 4D data for live, intraerythrocytic malarial parasites [Gligorijevic B, McAllister R, Urbach JS, Roepe, PD. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria. Biochemistry 2006;45:12400–10]. We reported that chloroquine (CQ) treatment did not appear to affect progress through the cell cycle, and suggested that toxicity may be manifested post-schizogony. We now use SDCM, synchronized cell culture and continuous vs. bolus drug dosing to investigate stage specific CQ effects in detail. We develop a novel, extremely rapid method for counting schizont nuclei in 3D. We then quantify schizont nuclei and hemozoin (Hz) production for live parasite cultures pulsed with CQ at different stages in the cell cycle and find that bolus treatment of rings affects the multiplicity of nuclear division. We quantify parasitemia and merozoite development in subsequent cycles following bolus CQ exposure and find that a portion of CQ toxicity is manifested post-schizogony as “delayed death”. Using these methods and others we compare CQ sensitive (CQS) vs. resistant (CQR) strains as well as transfectants that are CQR via introduction of mutant PfCRT. Surprisingly, we find that PfCRT confers resistance to CQ administered at the very early ring stage of development, wherein a digestive vacuole is not yet formed, as well as at the schizont stage, wherein Hz production is thought to plateau. Taken together, these data force a rethinking of CQ pharmacology and the mechanism of CQR.

Comparison of Plasmodium falciparum growth in sickle cells in low oxygen environment and candle-jar

The first successful in vitro cultivation of Plasmodium falciparum in sickle cells in a gas mixture containing 3% oxygen, 4% carbon dioxide and 93% nitrogen has been reported recently, contradicting earlier claims that the parasite does not multiply continuously in sickle cell trait (HbAS) and sickle cell anemia (HbSS) erythrocytes at low oxygen tension. The present study extends that report by growing three P. falciparum strains in erythrocytes from four different sickle cell trait and four sickle cell anemia donors. Because P. falciparum is known to grow normally in sickle cells when incubated in a candle-jar estimated to contain 15–18% oxygen, we have also compared the growth at 3% oxygen with that in a candle-jar. For convenience, we also refer to the 3% oxygen and the candle-jar as low and high oxygen environment, respectively. The three P. falciparum strains were first grown continuously in low oxygen environment for at least 1 month in erythrocytes from one HbAS carrier. These stock cultures were then used to infect erythrocytes from additional three HbAS carriers and four HbSS patients. Results of the experiments showed that parasite growth and hemozoin production in HbAS erythrocytes in low oxygen environment were comparable to those obtained in the candle-jar. There was growth retardation in HbSS erythrocytes in low oxygen environment, but some of the parasites survived and eventually produced high parasitemia levels. Continuous cultivation of different P. falciparum strains in HbAS erythrocytes is necessary for investigation of possible molecular differences between malaria parasites in sickle cells and those in HbAA erythrocytes.

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1–2% with Pfhrp-2 and 0.25–0.5% with lipids. We added lipids after 9 h in a 12 h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9 h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron–carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.

Trans Expression of a Plasmodium falciparum Histidine-rich Protein II (HRPII) Reveals Sorting of Soluble Proteins in the Periphery of the Host Erythrocyte and Disrupts Transport to the Malarial Food Vacuole

The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. A candidate heme polymerase, the histidine-rich protein II (HRPII), is proposed to be delivered to the fv by ingestion of the infected-red cell cytoplasm. Here we show that 97% of endogenous Plasmodium falciparum (Pf) HRPII (PfHRPII) is secreted as soluble protein in the periphery of the red cell and avoids endocytosis by the parasite, and 3% remains membrane-bound within the parasite. Transfected cells release 90% of a soluble transgene PfHRPIImyc into the red cell periphery and contain 10% membrane bound within the parasite. Yet these cells show a minor reduction in hemozoin production and IC(50) for chloroquine. They also show decreased transport of resident fv enzyme PfPlasmepsin I, the endoplasmic reticulum (ER) marker PfBiP, and parasite-associated HRPII to fvs. Instead, all three proteins accumulate in the ER, although there is no defect in protein export from the parasite. The data suggest that novel mechanisms of sorting (i) soluble antigens like HRPII in the red cell cytoplasm and (ii) fv-bound membrane complexes in the ER regulate parasite digestive processes.

On the mechanism of hemozoin production in malaria parasites: activated erythrocyte membranes promote beta-hematin synthesis.

The ferriprotoporphyrin IX (FP) molecules released by intraerythrocytic malaria parasites during hemoglobin digestion are converted to beta-hematin and are stored in the parasites' food vacuoles. It has been demonstrated in cell-free medium that the incorporation of FP into beta-hematin under physiological conditions requires a catalyst from parasite lysates or pre-formed beta-hematin. In the present studies, lysates of Plasmodium falciparum-infected erythrocytes were suspended in 1 M NaOH and were washed with phosphate buffer, pH 7.6. When the cell extracts were incubated with hematin in 0.5 M sodium acetate buffer, pH 5, for 20 hr at 37 degrees C, a large quantity of beta-hematin was formed. To determine whether parasite components were necessary for the beta-hematin formation, normal erythrocyte ghosts were similarly treated with 1 M NaOH and then incubated with hematin. In repeated experiments it was found that, on the average, 70% of the hematin was converted to beta-hematin. Membranes treated with HCl or CH(3)COOH also promoted the formation of beta-hematin, while untreated membranes were ineffective. The possibility that metabolic activities in the food vacuoles of malaria parasites may activate membrane fragments, from hemoglobin vesicles, to promote beta-hematin formation is discussed in this paper.

In vitro activity of riboflavin against the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum digests hemoglobin and polymerizes the released free heme into hemozoin. This activity occurs in an acidic organelle called the food vacuole and is essential for survival of the parasite in erythrocytes. Since acidic conditions are known to enhance the auto-oxidation of hemoglobin, we investigated whether hemoglobin ingested by the parasite was oxidized and whether the oxidation process could be a target for chemotherapy against malaria. We released parasites from their host cells and separately analyzed hemoglobin ingested by the parasites from that remaining in the erythrocytes. Isolated parasites contained elevated amounts (38.5% +/- 3.5%) of oxidized hemoglobin (methemoglobin) compared to levels (0.8% +/- 0.2%) found in normal, uninfected erythrocytes. Further, treatment of infected cells with the reducing agent riboflavin for 24 h decreased the parasite methemoglobin level by 55%. It also inhibited hemozoin production by 50% and decreased the average size of the food vacuole by 47%. Administration of riboflavin for 48 h resulted in a 65% decrease in food vacuole size and inhibited asexual parasite growth in cultures. High doses of riboflavin are used clinically to treat congenital methemoglobinemia without any adverse side effects. This activity, in conjunction with its impressive antimalarial activity, makes riboflavin attractive as a safe and inexpensive drug for treating malaria caused by P. falciparum.