Cytometric quantification of singlet oxygen in the human malaria parasite Plasmodium falciparum.
The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen ((1)O(2)). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of (1)O(2) by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While (1) O(2) is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of (1)O(2) we have established a new cytometric method that allows the stage specific quantification of (1)O(2). Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200-600 pixels for rings, 700-1,200 pixels for trophozoites and 1,400-2,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous (1)O(2) of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that (1)O(2) derives predominantly from the digestion of hemoglobin.
Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht Straβe 74, D-20359 Hamburg, Germany.
Sabine
S
Butzloff
Matthew R
MR
Groves
Carsten
C
Wrenger
Ingrid B
IB
Müller
D000818:Animals; D002138:Calibration; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008018:Life Cycle Stages / Q000187:drug effects; D008288:Malaria / Q000469:parasitology*; D010271:Parasites / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D010569:Perylene / Q000031:analogs & derivatives / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D026082:Singlet Oxygen / Q000378:metabolism*
D026082:Singlet Oxygen; D010569:Perylene; C026363:cercosporin