Data for this Review were identified by searches of Medline, Current Contents, and references from relevant articles and other publications. All articles retrieved with the search terms “haemozoin” or “malaria pigment” were reviewed for their relevance. The search terms “malaria or Plasmodium” were combined with “diagnosis”, “prognosis”, “immunology”, and “drug or therapy”, and the results were screened for relevant articles relating to haemozoin. Mainly English language papers were
ReviewHaemozoin: from melatonin pigment to drug target, diagnostic tool, and immune modulator
Introduction
The presence of a black pigment in the brains and spleens of patients with malaria on necropsy was first reported by Giovanni Maria Lancisi in 1717.1 In 1847, Meckel reported a necropsy he had done on a woman who had died in a hospital for the insane. He noted large amounts of a black-brown pigment in the blood and internal organs, particularly in the liver and spleen,2 which he believed to be melanin (panel). However, it was not until 2 years later that Rudolf Virchow conclusively linked this pigment with malaria.3 Later, Laveran examined the fresh unstained blood of patients with malaria and recognised pigmented bodies in erythrocytes that established the relation between the parasite and the pigment.4 Only later was it found that haemozoin was not a melanin pigment, but that its colour arises from haem.5, 6 Almost a century passed before the chemical composition and molecular structure of this material were finally determined.7, 8, 9
During the intraerythrocytic phase, malaria parasites feed on host-cell haemoglobin,10 liberating free haematin (aquaferriprotoporphyrin IX), a known toxin in higher organisms, which is also likely to be highly toxic to the parasite. The parasite solves this problem by detoxifying the haem, turning it into the brownish-yellow malaria pigment, haemozoin.11, 12, 13
Despite the detailed description of malaria pigment found at necropsy almost 200 years ago,2 haemozoin has not been in the limelight for long. A PubMed search in May, 2006, with the terms “haemozoin or malaria pigment” retrieved only 300 publications among 48 500 identified when using the terms “malaria” or “plasmodium”. However, interest seems to be increasing with the recognition that haemozoin may be of great importance in diverse aspects of malaria (figure 1).
Section snippets
Haemozoin structure and biogenesis
Synthetic β-haematin formation resembles a mineralisation process, and haemozoin formation has been suggested to be a type of biomineralisation or biocrystallisation.24, 25, 26 The mechanism of haemozoin formation in vivo has been the subject of much debate. Early suggestion of a haem polymerisation enzyme is no longer feasible,14 because the product is not a polymer. Some have suggested that histidine-rich protein (HRP) either nucleates or catalyses haemozoin formation.27 Recently, however,
Haemozoin in the human body
At the end of the intraerythrocytic cycle, up to 80–90% of all haem iron is localised in the digestive food vacuole of the parasite. At least 95% of this haem is converted to haemozoin.36 The approximate size of the haemozoin crystals is thought to be 1 μm×0·4 μm×0·2 μm,37 and approximately three to five crystals are produced per parasite.38 However, little certainty exists about the number of digestive food vacuoles per parasite, or the number, size, shape, and surface structure of haemozoin.
Biocrystallisation as an antimalarial drug target
The first synthetic drug with antimalarial activity was methylene blue,48, 49 and modifications of the molecule eventually gave birth to one of the most widely used antimalarial drugs of the last century, chloroquine. Chloroquine is cheap and safe, was a reliable first-line therapeutic and prophylactic drug, and was used extensively for over 40 years. The first cases of chloroquine resistance were reported from South America50 and southeast Asia.51 Chloroquine resistance is not caused by
Optical pigment detection to diagnose malaria
Haemozoin is birefringent (figure 3) and paramagnetic.78 This birefringence allows haemozoin detection by darkfield or polarisation microscopy (figure 2 and figure 3).79 Darkfield microscopic methods were the focus of further developments in the 1980s,80, 81, 82 and we are occasionally reminded of the usefulness of polarising microscopy.83 However, neither method was ever widely used for diagnosis. Possibly, use of such equipment in endemic areas was not financially feasible, whereas the lack
Diagnosis of malaria during pregnancy
Placental malaria is notorious for being difficult to diagnose microscopically.96 This is thought to be because of the sequestration of parasites in the placenta. The amount of placental haemozoin has been reported as a marker of parasitaemia.97, 98, 99 Obviously, the determination was done after birth by use of part of the maternal placenta. However, in placental malaria, haemozoin is also found in the peripheral blood, especially in phagocytic blood leucocytes. On the basis of this concept,
Application of magnetism to malaria
Haemozoin has well-known paramagnetic properties, and application of a magnetic field to a blood sample has been shown to accumulate intraerythrocytic malaria parasites.78, 100 The method was further refined by use of a steel-wool-packed plastic tube inserted into a permanent magnet to retain the paramagnetic forms of the parasites.101 A recently reported fluidic method applied this principle to a microscopic slide, which leads to the deposition of the parasitised erythrocytes on the slide.39
Haemozoin-containing leucocytes as markers of disease severity and prognosis
The peripheral parasitaemia does not always show the severity of the disease. Many clinicians have seen patients with similar parasitaemias, whereby one patient had only mild symptoms and the other patient developed severe, even fatal, malaria (figure 4). One explanation of this could be a different total parasite burden, whereby the patient with severe malaria has many more sequestered parasites, but both patients have the same peripheral parasitaemia.103 Several studies have shown that the
Haemozoin as a key factor of immune modulation
Malaria infections are characterised by an altered immune status, and the severe forms seem to be caused by activation or an overactivation of the immune system.21, 110 The search for parasite products that trigger this response (ie, “malaria toxins”) has led to the identification of some candidate substances.21, 110 However, despite a rather small number of studies (approximately 40), recent reviews have pointed out that haemozoin seems to be another factor in immune modulation.21, 22, 23
Interference of haemozoin with host metabolism
In malaria patients, plasma lipid profiles seem to be altered in a characteristic way.112, 113, 114, 115, 116, 117 Fasting plasma lipids are characterised by low total cholesterol, very low concentrations of high-density lipoprotein cholesterol, apolipoprotein A1 below detection concentrations, and raised triglycerides. These alterations are transient and seem to be largely limited to the parasitaemic phase, and at least the temporary depletion of high-density lipoprotein can be explained as
Immune suppression or immune activation
In consideration of the evolution of the complex and intricate life cycle of Plasmodium spp, it seems noteworthy that the parasite has developed the well-designed haemozoin formation route to detoxify haem. But does haemozoin really (over)activate the immune system? An (over)activation could have potentially detrimental consequences for the parasite. First, the activated immune system would be expected to result in a more rapid clearance of the parasite from the host. Second, an overactivated
Fate of haemozoin in the human body
Very little is known about the kinetics of haemozoin elimination. On necropsy, the livers and spleens of patients with malaria have a dark, black appearance, even if their death was not malaria related.1 This raises the intriguing question of where, how, and for how long haemozoin is deposited in the human body? If haemozoin is stored for long periods, does it lose its immunomodulatory properties? Could it be possible that recurrent or chronic malaria infections cause a chronic “haemozoin
Conclusions
Although the finding of haemozoin has helped to unravel the cause of malaria, for almost a century the only practical application was the use of its birefringent properties for darkfield microscopy. However, recent developments and research have led to several novel methods for the diagnosis of malaria, or to determine disease severity, based on the detection of malaria pigment. Furthermore, it seems that haemozoin may be one of the key factors involved in malaria-associated immunopathology,
Search strategy and selection criteria
References (145)
- et al.
The state of ferriprotoporphyrin IX in malaria pigment
J Biol Chem
(1987) - et al.
Excess haemoglobin digestion and the osmotic stability of Plasmodium falciparum infected red blood cells
Blood
(2003) - et al.
Quinoline anti-malarial drugs inhibit spontaneous formation of beta-haematin (malaria pigment)
FEBS Lett
(1994) - et al.
Interactions between malaria parasites and the host immune system
Curr Opin Immunol
(2005) - et al.
Pigment biocrystallization in Plasmodium falciparum
Trends Parasitol
(2002) - et al.
Histidine-rich protein 2 of the malaria parasite, Plasmodium falciparum, is involved in detoxification of the by-products of haemoglobin degradation
Mol Biochem Parasitol
(2001) Theories on malarial pigment formation and quinoline action
Int J Parasitol
(2002)- et al.
Involvement of lipids in ferriprotoporphyrin IX polymerization in malaria
Biochem Biophys Acta
(1999) - et al.
Structural and morphological characterization of haemozoin produced by Schistosoma mansoni and Rhodnius prolixus
FEBS Lett
(2005) - et al.
Haemozoin (β-haematin) biomineralization occurs by self-assembly near the lipid/water interface
FEBS Lett
(2006)
Characterisation of synthetic beta-haematin and effects of the antimalarial drugs quinidine, halofantrine, desbutylhalofantrine and mefloquine on its formation
J Inorg Biochem
The shape and size of haemozoin crystals distinguishes diverse Plasmodium species
Mol Biochem Parasitol
Differences in automated depolarization patterns of Plasmodium falciparum and P vivax malaria infections defined by the Cell-Dyn CD4000 haematology analyser
Trans R Soc Trop Med Hyg
Clearance kinetics of parasites and pigment-containing leukocytes in severe malaria
Blood
Inhibition of heme crystal growth by antimalarials and other compounds: implication for drug discovery
Biochem Pharmacol
The role of drug accumulation in 4-aminoquinoline antimalarial potency. The influence of structural substitution and physicochemical properties
Biochem Pharmacol
An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials
Biochem Pharmacol
Standardization of the physicochemical parameters to assess in vitro the β-hematin inhibitory activity of antimalarial drugs
Exp Parasitol
A colorimetric high-throughput β-hematin inhibition screening assay for use in the search for antimalarial compounds
Anal Biochem
Activity of piperaquine and other 4-aminoquinoline antiplasmodial drugs against chloroquine-sensitive and resistant blood-stages of Plasmodium falciparum. Role of β-haematin inhibition and drug concentration in vacuolar water and lipid-phases
Biochem Pharmacol
Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P vinckei petteri and P yoelii nigeriensis in vivo
Biochem Pharmacol
Current drug development portfolio for antimalarial therapies
Curr Opin Pharmacol
Inhibition of heme crystal growth by antimalarials and other compounds: implications for drug discovery
Biochem Pharmacol
Separation of malaria-infected erythrocytes from whole blood: use of a selective high-gradient magnetic separation technique
Lancet
Improvement in dark-field microscopy for the rapid detection of malaria parasites and its adaptation to field conditions
Trans R Soc Trop Med Hyg
Automated malaria diagnosis using pigment detection
Parasitol Today
Performance evaluation of automated depolarization analysis for detecting clinically unsuspected malaria in endemic countries
Trans R Soc Trop Med Hyg
Placental haemozoin and malaria in pregnancy
Placenta
Haemozoin as a marker of placental parasitization
Trans R Soc Trop Med Hyg
Polarisation microscopy increases the sensitivity of haemozoin and Plasmodium detection in the histological assessment of placental malaria
Acta Trop
A simple method for the purification of malaria pigment
Mol Biochem Parasitol
About the noxious emanations of swamps and their remedies
About black pigment in spleen and blood of an insane woman
Zeitschr für Psychiatrie
About the pathophysiology of blood
Arch Pathol Anatomie
A newly discovered parasite in the blood of patients suffering from malaria. Parasitic etiology of attacks of malaria (1880)
About the chemical nature of malaria pigment
Giornale della R Accademia di Medicina di Torino
Malarial pigment (so-called melanin): its nature and mode of production
J Exp Med
An iron-carboxylate bond links the heme units of malaria pigment
Proc Natl Acad Sci USA
The structure of malaria pigment beta-haematin
Nature
Mode of action of chloroquine on Plasmodium berghei and P cynomolgi
Nature
Morphological effects of chloroquine on Plasmodium berghei in mice
Nature
Purification and some properties of malarial pigment
Ann Trop Med Parasitol
Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites
Nature
Plasmodium haemozoin formation mediated by histidine-rich proteins
Science
Quinoline antimalarials
Exp Opin Ther Patents
Avoiding misdiagnosis of malaria: a novel automated method allows specific diagnosis, even in the absence of clinical suspicion
Emerg Infect Dis
Intraleucocytic malaria pigment and prognosis in severe malaria
Trans R Soc Trop Med Hyg
Association of intraleukocytic Plasmodium falciparum malaria pigment with disease severity, clinical manifestations, and prognosis in severe malaria
Am J Trop Med Hyg
Regulating immunity to malaria
Parasite Immunol
Malaria pigment paralyzes dendritic cells
J Biol
Cited by (105)
Developing an archaeology of malaria. A critical review of current approaches and a discussion on ways forward
2023, International Journal of PaleopathologySynthesis and antimalarial activity of novel 3-substituted chloroquine derivatives
2024, Canadian Journal of Chemistry