Major artifacts encountered in studying biological samples containing ferric protoporphyrin IX

Heme (ferric protoporphyrin IX, FP) dissolves very rapidly into the lipid phase of membranes, and a large number of studies have focused attention on its possible toxic effect in whole cells or isolated membranes. However, because of its molecular structure and reactivity, different problems can be encountered during the course of studying biological samples containing FP. In this article, we discuss important interferences by FP and artifacts that can affect the experimental values. First, FP interferes with the Lowry's protein determination; therefore, membranes containing FP are overestimated in their protein content determined by this procedure. Second, freezing membranes at -20°C artifactually increases the local concentration of FP, thereby enhancing FP-induced lipid peroxidation. Third, in the presence of thiol compounds such as N-acetyl cysteine, FP is degraded to products that interfere with the thiobarbituric acid assay, one of the most widely used methods to measure the extent of lipoperoxidation

Novel Antimalarial Aminoquinolines: Heme Binding and Effects on Normal or Plasmodium falciparum-Parasitized Human Erythrocytes ▿

Two new quinolizidinyl-alkyl derivatives of 7-chloro-4-aminoquinoline, named AM-1 and AP4b, which are highly effective in vitro against both the D10 (chloroquine [CQ] susceptible) and W2 (CQ resistant) strains of Plasmodium falciparum and in vivo in the rodent malaria model, have been studied for their ability to bind to and be internalized by normal or parasitized human red blood cells (RBC) and for their effects on RBC membrane stability. In addition, an analysis of the heme binding properties of these compounds and of their ability to inhibit beta-hematin formation in vitro has been performed. Binding of AM1 or AP4b to RBC is rapid, dose dependent, and linearly related to RBC density. Their accumulation in parasitized RBC (pRBC) is increased twofold compared to levels in normal RBC. Binding of AM1 or AP4b to both normal and pRBC is higher than that of CQ, in agreement with the lower pKa and higher lipophilicity of the compounds. AM1 or AP4b is not hemolytic per se and is less hemolytic than CQ when hemolysis is accelerated (induced) by hematin. Moreover, AM-1 and AP4b bind heme with a stoichiometry of interaction similar to that of CQ (about 1:1.7) but with a lower affinity. They both inhibit dose dependently the formation of beta-hematin in vitro with a 50% inhibitory concentration comparable to that of CQ. Taken together, these results suggest that the antimalarial activity of AM1 or AP4b is likely due to inhibition of hemozoin formation and that the efficacy of these compounds against the CQ-resistant strains can be ascribed to their hydrophobicity and capacity to accumulate in the vacuolar lipid (elevated lipid accumulation ratios)

Altered Lipid Composition of Surfactant and Lung Tissue in Murine Experimental Malaria-Associated Acute Respiratory Distress Syndrome.

Malaria-associated acute lung injury (MA-ALI) and its more severe form malaria-associated acute respiratory distress syndrome (MA-ARDS) are common, often fatal complications of severe malaria infections. However, little is known about their pathogenesis. In this study, biochemical alterations of the lipid composition of the lungs were investigated as possible contributing factors to the severity of murine MA-ALI/ARDS. C57BL/6J mice were infected with Plasmodium berghei NK65 to induce lethal MA-ARDS, or with Plasmodium chabaudi AS, a parasite strain that does not induce lung pathology. The lipid profile of the lung tissue from mice infected with Plasmodium berghei NK65 developing MA-ALI/ARDS, but not that from mice without lung pathology or controls, was characterized by high levels of phospholipids -mainly phosphatidylcholine- and esterified cholesterol. The high levels of polyunsaturated fatty acids and the linoleic/oleic fatty acid ratio of the latter reflect the fatty acid composition of plasma cholesterol esters. In spite of the increased total polyunsaturated fatty acid pool, which augments the relative oxidability of the lung membranes, and the presence of hemozoin, a known pro-oxidant, no excess oxidative stress was detected in the lungs of Plasmodium berghei NK65 infected mice. The bronchoalveolar lavage (BAL) fluid of Plasmodium berghei NK65 infected mice was characterized by high levels of plasma proteins. The phospholipid profile of BAL large and small aggregate fractions was also different from uninfected controls, with a significant increase in the amounts of sphingomyelin and lysophosphatidylcholine and the decrease in phosphatidylglycerol. Both the increase of proteins and lysophosphatidylcholine are known to decrease the intrinsic surface activity of surfactant. Together, these data indicate that an altered lipid composition of lung tissue and BAL fluid, partially ascribed to oedema and lipoprotein infiltration, is a characteristic feature of murine MA-ALI/ARDS and possibly contribute to lung dysfunction

The Lipid Moiety of Haemozoin (Malaria Pigment) and P. falciparum Parasitised Red Blood Cells Bind Synthetic and Native Endothelin-1

Endothelin1 (ET-1) is a 21-amino acid peptide produced by the vascular endothelium under hypoxia, that acts locally as regulator of vascular tone and inflammation. The role of ET-1 in Plasmodium falciparum malaria is unknown, although tissue hypoxia is frequent as a result of the cytoadherence of parasitized red blood cell (pRBC) to the microvasculature. Here, we show that both synthetic and endothelial-derived ET-1 are removed by parasitized RBC (D10 and W2 strains, chloroquine sensitive, and resistant, resp.) and native haemozoin (HZ, malaria pigment), but not by normal RBC, delipidized HZ, or synthetic beta-haematin (BH). The effect is dose dependent, selective for ET-1, but not for its precursor, big ET-1, and not due to the proteolysis of ET-1. The results indicate that ET-1 binds to the lipids moiety of HZ and membranes of infected RBCs. These findings may help understanding the consequences of parasite sequestration in severe malaria

Differential induction of malaria liver pathology in mice infected with Plasmodium chabaudi AS or Plasmodium berghei NK65

Cerebral malaria and severe anaemia are the most common deadly complications of malaria, and are often associated, both in paediatric and adult patients, with hepatopathy, whose pathogenesis is not well characterized, and sometimes also with acute respiratory distress syndrome (ARDS). Here, two species of murine malaria, the lethal Plasmodium berghei strain NK65 and self-healing Plasmodium chabaudi strain AS which differ in their ability to cause hepatopathy and/or ARDS were used to investigate the lipid alterations, oxidative damage and host immune response during the infection in relation to parasite load and accumulation of parasite products, such as haemozoin.status: publishe

Effects of malaria heme products on red blood cell deformability.

In falciparum malaria, the deformability of the entire erythrocyte population is reduced in proportion to disease severity, and this compromises microcirculatory blood flow through vessels partially obstructed by cytoadherent parasitized erythrocytes. The cause of rigidity of uninfected erythrocytes in not known but could be mediated by malaria heme products. In this study, we show that red blood cell deformability (RBC-D), measured by laser-assisted optical rotational cell analyzer, decreased in a dose-dependent manner after incubation with hemin and hydrogen peroxide but not with hemoglobin or beta-hematin. Hemin also reduced mean red cell volume. Albumin decreased and N-acetylcysteine (NAC) both prevented and reversed rigidity induced by hemin. Hemin-induced oxidative damage of the membrane seems to be a more important contributor to pathology than cell shrinkage because the antioxidant NAC restored RBC-D but not red blood cell volume. The findings suggest novel approaches to the treatment of potentially lethal malaria