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

Abstract

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.

FIG. 1

Peroxidase activity associated with methemoglobin formed in vitro and in vivo. (A) Uninfected erythrocytes (RBC) were lysed hypotonically, the cytosolic fraction was incubated in the absence or presence of potassium ferricyanide (to form methemoglobin), and the proteins were separated by SDS-PAGE, transferred to nitrocellulose, and assayed for peroxidase activity or immunoblotted for hemoglobin content. Molecular masses are indicated in kilodaltons. (B) Trophozoite-infected erythrocytes were lysed with sorbitol, and the supernatant fraction (sup.) was separated from isolated parasites in the pellet fraction. Both fractions were assayed for peroxidase activity and hemoglobin content as described for panel A. (C) Uninfected erythrocytes (U) and parasites at the indicated times of growth were isolated by lysing infected erythrocytes with saponin and analyzed for peroxidase activity and hemoglobin content as described for panel A. Hemozoin was also determined, as indicated at the bottom of panel II.

FIG. 2

Effects of riboflavin on the peroxidase activity of methemoglobin, hemozoin formation, and hemoglobin uptake. Cultures of ring-stage parasite-infected erythrocytes were incubated in the absence or presence of 100 μM riboflavin for 24 h, and parasites were isolated by saponin lysis. (A) Peroxidase activity and hemoglobin content were determined as described in the legend to Fig. 1A. (B) Levels of hemozoin produced (open bars) were determined by conversion to hematin. The amount of hemoglobin ingested (by the parasite) (solid bars) was derived by subtracting the amount of hemoglobin released (by sorbitol lysis) from the cytosol of infected erythrocytes from the total hemoglobin content found in uninfected erythrocytes. The bars represent the mean of three independent determinations ± the standard error of the mean.

FIG. 3

Effects of riboflavin on the morphology of the fv. Infected erythrocytes were treated with or without riboflavin (100 μM) for the indicated times and prepared for electron microscopy by embedding in Spurr's resin, and thin sections were obtained. (A) Mock-treated infected erythrocyte. (B and C) Infected erythrocytes treated with riboflavin for 24 and 48 h, respectively. hz, hemozoin; p, parasite; e, erythrocyte. Scale bars, 5 μm. (D) fv diameters measured in ultrathin sections from cells that were mock treated or treated with riboflavin for 24 or 48 h. The bars represent the mean ± standard error of the mean. A total of 48 sections were measured.

FIG. 3

Effects of riboflavin on the morphology of the fv. Infected erythrocytes were treated with or without riboflavin (100 μM) for the indicated times and prepared for electron microscopy by embedding in Spurr's resin, and thin sections were obtained. (A) Mock-treated infected erythrocyte. (B and C) Infected erythrocytes treated with riboflavin for 24 and 48 h, respectively. hz, hemozoin; p, parasite; e, erythrocyte. Scale bars, 5 μm. (D) fv diameters measured in ultrathin sections from cells that were mock treated or treated with riboflavin for 24 or 48 h. The bars represent the mean ± standard error of the mean. A total of 48 sections were measured.

FIG. 3

Effects of riboflavin on the morphology of the fv. Infected erythrocytes were treated with or without riboflavin (100 μM) for the indicated times and prepared for electron microscopy by embedding in Spurr's resin, and thin sections were obtained. (A) Mock-treated infected erythrocyte. (B and C) Infected erythrocytes treated with riboflavin for 24 and 48 h, respectively. hz, hemozoin; p, parasite; e, erythrocyte. Scale bars, 5 μm. (D) fv diameters measured in ultrathin sections from cells that were mock treated or treated with riboflavin for 24 or 48 h. The bars represent the mean ± standard error of the mean. A total of 48 sections were measured.

FIG. 3

Effects of riboflavin on the morphology of the fv. Infected erythrocytes were treated with or without riboflavin (100 μM) for the indicated times and prepared for electron microscopy by embedding in Spurr's resin, and thin sections were obtained. (A) Mock-treated infected erythrocyte. (B and C) Infected erythrocytes treated with riboflavin for 24 and 48 h, respectively. hz, hemozoin; p, parasite; e, erythrocyte. Scale bars, 5 μm. (D) fv diameters measured in ultrathin sections from cells that were mock treated or treated with riboflavin for 24 or 48 h. The bars represent the mean ± standard error of the mean. A total of 48 sections were measured.

FIG. 4

Proposed model for hemoglobin oxidation and riboflavin action in P. falciparum-infected erythrocytes.