Inhibition by anti-malarial drugs of haemoglobin denaturation and iron release in acidified red blood cell lysates--a possible mechanism of their anti-malarial effect?

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4-5.5, 37 degrees C) human red blood cell lysates were used to simulate this process, measuring the denaturation of haemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, their rates decreasing with increasing pH, and increasing in the presence of protease. Both processes were inhibited by the quinoline-containing anti-malarial drugs (QCDs) chloroquine, quinine, mefloquine and amodiaquine at concentrations well below those expected in the acidic food vacuole of the parasite. Spectrophotometric analysis indicated that chloroquine complexes with heme in acid-denatured haemoglobin. Other weak bases as well as verapamil and diltiazem, known to reverse the resistance of malarial parasites to chloroquine, were without effect indicating that the action of QCDs is specific. Based on our previous results and the present report, we suggest that iron release in acidified lysates is mediated through the formation of ferryl (Fe(IV)) radicals. QCDs possibly complex with this radical, as they do with heme, and prevent its contact with an adjacent heme molecule which is required for ring opening and iron release. These results may suggest that one of the anti-malarial effects of QCDs is to deprive the parasite of an adequate iron supply. Addition of iron to cultures of Plasmodium falciparum was expected to circumvent the deprivation of iron and reduce the anti-malarial effect of QCDs. However, adding iron as penetrating fructose or nitrilotriacetate complexes did not alter the parasite's susceptibility to chloroquine. Ascorbate markedly increased the release of iron in acidified lysates, and this effect was not reduced by chloroquine. Ascorbate was found to decrease parasite susceptibility to chloroquine, suggesting that iron deprivation may be an important factor in the anti-malarial action of QCDs.