In vitro effects of co-incubation of blood with artemether/lumefantrine & vitamin C on the viscosity & elasticity of blood

The combination of artemether and lumefantrine is used as an oral antimalarial agent. Artemether interacts with components of blood to generate free radicals which may damage the malaria parasite and alleviate symptoms of malaria. Lumefantrine eliminates residual parasites, decreases parasite burden, and resolves clinical symptoms of the disease1. The antimalarial agents are sometimes co-administered with vitamin C (ascorbic acid) which is predominantly associated with antioxidant properties234, possibly to oppose oxidative stress and consequent haemolysis and anaemia associated with malaria infection5. Vitamin C has been shown to potentiate the effects of the antimalarial agent exifone in vitro6. The practice of vitamin C supplementation in patients receiving antimalarial therapy may also be supported by the low serum concentration of vitamin C in Plasmodium infection7. There are however, recommendations against administration of vitamin C to patients with malaria, to avoid possible impairment of therapeutic action of antimalarial drugs8. This is supported by reports of the interference of antiplasmodial action of antimalarial agents in the presence of antioxidants910.

The mechanism of action of artemether/lumefantrine involves its haemolytic effects on erythrocytes11. This combination has also been shown to reduce blood viscosity, elasticity and erythrocyte aggregation in vitro12. We undertook this in vitro study to investigate the effects of vitamin C on artemether/lumefantrine induced changes in whole blood viscosity and elasticity in healthy human volunteers.

Material & Methods

Participants were 12 healthy female volunteers (students with mean age 20.8±1.5 yr) with normal haemoglobin genotype (HbAA) attending the University of the West Indies, Mona, Jamaica. The study was restricted to female participants as glucose-6-phosphate dehydrogenase (G6PD) deficiency affects haemolysis. The G6PD enzyme deficiency is mainly expressed in males as females are carriers of the gene13. Individuals were excluded if they had sickle cell disease (HbSS) or trait (HbAS), or if they had been taking medication for any illness within the last three months. All participants signed a consent form confirming voluntary participation in the study. Ethical approval was obtained from the University of the West Indies / Faculty of Medical Sciences Ethics Committee. The study was conducted in the Department of Basic Medical Sciences at the University of the West Indies, Mona, in April 2013.

Sample preparation: The blood (3 ml) was collected into vacutainer tubes containing K+ EDTA anticoagulant and kept at 25°C until used for analysis. A Coartem® (artemether 80 mg/lumefantrine 480 mg) tablet (Novartis Pharmaceutical Company, Beijing, China) was dissolved in 80 ml physiological saline (0.9%) solution to prepare stock solution. Soluble vitamin C (Redoxon®, Bayer Inc., UK) tablets were dissolved in 0.9 per cent saline solution to prepare vitamin C solution (1 mg/ml). Blood (0.5 ml) samples from all participants were placed in each of four Eppendorf tubes. Aliquots (30 µl) of Coartem® solution were pipetted into three of those tubes to constitute 0.06/0.36 mg/ml Coartem® in each blood sample, based on therapeutic adult dose of 320/1920 mg artemether/lumefantrine. The fourth tube had no drug added and served as the control sample. Vitamin C solution (10 or 50 ml) was added to each of two tubes containing blood and Coartem®, such that the concentrations of vitamin C were equivalent to adult doses of 100 or 500 mg, respectively. The samples were gently mixed and allowed to equilibrate for five minutes before tests were done.

Viscometry: Blood samples were added to the sample cup of a BioProfiler (Vilastic Scientific Inc., Texas, USA). The haematocrit reading obtained using a Coulter Counter (Coulter Corporation, Florida, USA) was entered into the BioProfiler which measured the viscosity and elasticity of whole blood at a frequency of 2 Hz, shear rate of 62.8 per second. The haemorheologic measurements at those settings approximate microcirculatory flow patterns. Readings were taken at a projected haematocrit of 45 per cent and temperature of 37°C14. Tests were performed within one hour of blood collection.

Statistical analysis: Statistical analysis of differences in group means was performed by analysis of variance (ANOVA) and Duncan's multiple range test using SigmaPlot 11.0 software (Systat Software Inc., San Jose, CA, USA).

Results & Discussion

The viscosity of control blood sample was 4.72 ± 0.38 mPa.s. In the presence of antimalarial drug alone, blood viscosity significantly (P<0.05) decreased to 3.78 ± 0.17 mPa.s. Further significant (P<0.05) reduction to 3.20 ± 0.10 mPa.s was observed with 100 mg vitamin C and to 2.67 ± 0.05 mPa.s (P<0.01) with 500 mg vitamin C, respectively. The elasticity of blood in the control sample was 0.33 ± 0.04 mPa.s; whereas in samples exposed to antimalarial drug alone the elasticity was significantly (P<0.05) lower at 0.24 ± 0.03 mPa.s. There were further significant (P<0.05) reduction to 0.21 ± 0.02 mPa.s in samples exposed to antimalarial drug with 100 mg vitamin C and to 0.13 ± 0.02 mPa.s (P<0.01) in samples with 500 mg vitamin C.

The results of the present study in relation to the effects of artemether/lumefantrine on blood viscosity and elasticity were consistent with an earlier study12. Free radicals generated due to artemether increase haemolysis, resulting in a decrease of the haematocrit11. The haematocrit is the major determinant of whole blood viscosity15. It is, therefore, likely that in the present study, artemether/lumefantrine generated the free radicals that caused increased haemolysis resulting in reduced hematocrit and ultimately reduced whole blood viscosity and elasticity.

The effect of vitamin C on viscosity in this in vitro study was consistent with the findings in human studies where blood viscosity was significantly inversely associated with acute ingestion and blood concentration of vitamin C161718. Vitamin C was also shown to potentiate the effects of a therapeutic agent on rheological indicators including decreasing blood viscosity and erythrocyte aggregation, and increasing deformability of erythrocytes19. Our in vitro results represent the situation in vivo, and suggest that co-administration of vitamin C with artemether/lumefantrine may increase the risk of haemolysis and possible anaemia in patients with malaria.

In erythrocytes, vitamin C exhibits pro-oxidant or antioxidant activity depending on the presence of glutathione or the source of vitamin C, whether intracellular or extracellular2021. Vitamin C is also reported to induce oxidative stress in Plasmodium-infected erythrocytes22. The effects observed in the present study may also be related to the doses (100 and 500 mg) of the vitamin used.

In conclusion, the results of the present study showed that the co-incubation of blood with vitamin C and artemether/lumefantrine in vitro significantly increased the haemolytic effects of the latter. This observation warrants further scientific validation, especially in relation to the practice of vitamin C supplementation with antimalarial therapy.