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Intracellular ferriprotoporphyrin IX is a lytic agent
Abstract:
Human erythrocytes were treated with menadione to oxidatively denature hemoglobin and release ferriprotoporphyrin IX (ferriheme, FP) intracellularly. The high affinity of FP for chloroquine was used to detect its release. After incubation for 1 hr at 37 degrees C and pH 7.4 with 0.5 mM menadione, erythrocytes bound 14C-chloroquine with an apparent dissociation constant of 10(-6)M. Untreated erythrocytes did not bind chloroquine with high affinity. At a chloroquine concentration in the medium of 2 microM, for example, menadione-treated erythrocytes bound 70 mumole chloroquine/kg and untreated erythrocytes bound 13.4 mumole/kg. The intracellular location of FP released by menadione was verified by finding that Tween 80 did not prevent chloroquine binding. By contrast, Tween 80 inhibited the binding of chloroquine to erythrocytes treated with extracellular FP. The hemolytic response to menadione was characteristic of the hemolytic response to FP. Thus, 5 microM chloroquine caused hemolysis to increase to 60% from baseline values of 5% in experiments using erythrocytes treated either with 0.5 mM menadione or with 5 microM FP; and, in both cases, the potentiating effect of chloroquine was inhibited by 1 microM mefloquine or 10 microM quinine. Higher concentrations of menadione caused hemolysis in the absence of chloroquine. We conclude that FP released by menadione exists intracellularly in a form that is accessible to bind chloroquine and to express its lytic activity.
Insights
Menadione treatment releases ferriheme (FP) inside red blood cells, which binds to chloroquine. This intracellular ferriheme also causes red blood cell damage, similar to extracellular ferriheme.
Area of Science:
- Hematology
- Biochemistry
- Toxicology
Background:
- Hemoglobin denaturation can lead to the release of ferriprotoporphyrin IX (ferriheme, FP).
- The interaction between ferriheme and drugs like chloroquine is of toxicological interest.
Purpose of the Study:
- To investigate the intracellular release of ferriheme (FP) from human erythrocytes induced by menadione.
- To determine the binding characteristics of chloroquine to menadione-induced intracellular FP.
- To assess the hemolytic activity associated with intracellular FP and its modulation by antimalarial drugs.
Main Methods:
- Human erythrocytes were treated with menadione to induce oxidative denaturation of hemoglobin.
- 14C-chloroquine binding assays were performed to quantify chloroquine affinity for treated erythrocytes.
- Hemolysis assays were conducted to evaluate the lytic effects of menadione and FP, with and without chloroquine, mefloquine, and quinine.
Main Results:
- Menadione-treated erythrocytes exhibited high-affinity binding of 14C-chloroquine (Kd = 10(-6)M), indicating intracellular FP release.
- Tween 80 confirmed the intracellular localization of menadione-induced FP, as it did not inhibit chloroquine binding.
- Menadione and extracellular FP induced similar hemolytic responses, potentiated by chloroquine and inhibited by mefloquine and quinine.
Conclusions:
- Menadione treatment effectively releases ferriheme (FP) intracellularly within erythrocytes.
- The released intracellular FP binds chloroquine with high affinity and contributes to red blood cell lysis.
- The findings suggest that intracellular FP shares similar toxicological properties with extracellular FP.