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ORIGINAL ARTICLE
IJBC 2009; 4: 151-157 Studies of Methemoglobin Concentrations of
Three Human Erythrocyte Genotypes (Hb AA, Hb
AS, and Hb SS) in the Presence of Five Anti-
malarial Drugs
Paul C. Chikezie1, Comfort C. Monago2, Augustine Uwakwe2
1. Department of Biochemistry, Imo State University, Owerri, Imo State, Nigeria.
2. Department of Biochemistry, University of Port Harcourt, Port Harcourt, Rivers State, Nigeria.
Corresponding author: Augustine Uwakwe, Department of Biochemistry, University of Port Harcourt, Port Harcourt, Rivers State, Nigeria. (Phone: +23408055404143, E-mail: uwadik@gmail.com) Abstract
Background: Malaria remains the world's most devastating human parasitic infection. Our goal was to assess the
capacity of increasing concentrations of five antimalarial drugs (FansidarTM, HalfanTM, Quinine, CoartemTM and
Chloroquine phosphate) to elicit the generation of methemoglobin in three human erythrocyte genotypes (Hb AA,
Hb AS and Hb SS).
Materials and Methods: Spectrophotometric method was used for determination of plasma methaemoglobin
concentration in the presence of 0.2 g%, 0.4 g%, 0.6 g% and 0.8 g% (w/v) of the five antimalarial drugs.
Results: The five antimalarial drugs showed a concentration dependent variability to cause the elevation of plasma
methemoglobin concentration in the three genotypes. Specifically, CoartemTM, exhibited the highest propensity to
elevate plasma methemoglobin concentration. However, the other four antimalarial drugs showed a statistically
significant (P<0.05) but minimal effect to cause elevation of plasma methemoglobin concentration. For instance,
with Hb AS blood sample and at drug concentration of 0.8g%, methaemoglobin concentrations (percentage) 0f
3.03±1.82, 2.65±0.45, 6.41±1.21, and 3.02±0.98 were obtained for halfan, quinine, coartem and chloroquine
phosphate, respectively. The control value was 2.17±1.82% of methemoglobin.
Conclusion: The oxidative potentials of these four antimalarial drugs and their metabolites in the red cells did not
overwhelm the erythrocyte methemoglobin reducing capacity that could elicit the presentation in vitro toxic
methemoglobinemia.
Keywords: Chloroquine, Coartem, Erythrocyte, Fansidar, Genotype, Halfan, Quinine, Methemoglobin.
chloroquine.3 This drug combination effectively Malaria remains the world's most devastating blocks two enzymes involved in the biosynthesis of human parasitic infection, affecting more than 500 folinic acid within the parasite.4 Artemether million people and causing 1.7 to 2.5 million deaths represents a major advance for the treatment of each year.1 Nearly all human malaria is caused by severe, multi-drug resistant falciparum malaria.2 four species of obligate intracellular protozoa of the The drug is a more potent derivative of artemisinin, genus Plasmodium.2 Antimalarial drugs are administered in the form of artemisinin categorized by the stage of parasite they affect and combination therapy (ACT), artemether– the clinical indications for their use. lumefantrine combination drug therapy FansidarTM is a combination of pyrimethamine (CoartemTM). Other regimens for malarial (250mg) and sulphadoxine (50mg) commonly used chemoprophylaxis are the aminoquinolines such as for prophylaxis and treatment of certain strains of chloroquine phosphate and its analogs. Quinoline Plasmodium falciparum that are resistant to blood schizontocides behave as weak bases IRANIAN JOURNAL OF BLOOD AND CANCER Volume 1 Number 4 Summer 2009 Chikezie et al. concentrated in food vacuoles of susceptible genotype were collected from patients attending Plasmodia where they increase pH, inhibit the clinic at the Federal Medical Centre (FMC), Owerri peroxidase activity of haem and disrupt its and Imo State University Teaching Hospital nonenzymatic polymerization to hemozoin. The (IMSUTH), Orlu, Imo State, Nigeria. All Blood failure to inactivate haem then kills the parasite via samples were obtained by venipunture and stored oxidative damage to membranes, digestive in EDTA anti-coagulant test tube. proteases, and possibly other critical biomolecules Anti-malarial Drugs
of the parasite.5,6 Five antimalarial drugs were used in this study: Concisely, methemoglobin is formed when FansidarTM (Swiss (Swipha) Pharmaceuticals Nigeria ferrous iron (Fe2+) of deoxyhemoglobin is converted Ltd), CoartemTM, (Beijing Norvatis Pharmaceutical to the ferric iron (Fe3+) state on exposure of Company, Beijing, China) Chloroquine phosphate erythrocytes to oxidizing agents and oxygen free (May and Baker, Pharmaceutical Company, Nigeria radicals.7,8 Ferric iron (Fe3+) state hemoglobin does Plc), HalfanTM (Smithkline Beecham Laboratories not bind reversibly with oxygen. Studies have Pharmaceutical Company, France) and Quinine shown that methemoglobin is formed continuously in plasma but rarely exceeds 1.5% of total plasma Five percent (5.0 g%, w/v) stock solution of the hemoglobin.8,9 Basically two enzymes, Diaphorase I five antimalarial drugs were prepared by dissolving and diaphorase II, in synergy with red blood cell 2.5 grams of each drug in 50 ml of distilled water. non–enzymatiic antioxidants, ascorbic acid, Serial dilutions were made to obtain corresponding glutathione, and other sulfydryl derivatives serve to concentrations in the order; 0.8 g%, 0.6 g%, 0.4 g% minimize erythrocyte methemoglobin level.10-14 and 0.2 g% (w/v). Cyanotic presentation is typically observed at methemoglobin concentration greater than 15% Determination of Plasma Methaemoglobin
and is often one of the earliest clinical evident features of methemoglobinemia.7 The analysis of plasma methemoglobin Ali and Kadaru15 (2005) described in vitro concentration was carried out using the processing of donor blood with spectrophotometric method described by Tietz sulphadoxine/pyrimethamine drugs combination (1976).9 The analysis was done within 60 minutes of for the eradication and prevention of transfusion- collecting the blood samples. The principle of this induced malaria. In furtherance of their reports, our determination is based on the fact that hemoglobin present study seeks to ascertain methemoglobin and methemoglobin absorb light at different concentration of three human erythrocyte wavelengths, at 540 nm and 630 nm as their genotypes, Hb AA, Hb AS, and Hb SS in the presence respective peak absorbance.9 The approach of five commonly prescribed antimalarial drugs. The employed the establishment procedure of lysing present research findings will provide an insight whole blood in distilled water. into the capacity of these five antimalarial drugs to interfere and alter the redox status of hemoglobin In a test tube containing 5.0 ml of distilled water, molecule. Therefore, our results may provide a 0.02 ml of whole blood was added. The mixture was subset of preliminary data for effective, successful, allowed to stand for 60 mintues at room and safe utilization of these antimalarial drugs for in temperature, and the absorbance was read at two vitro blood processing exercise. different wavelength maximums (λmax), 540 nm Materials and Methods
and 630 nm, using a spectrophotometer (Model 6400, Jenway). Collection of Blood Samples
Test
Five milliliters (ml) of confirmed human red The effect of each of the five antimalarial drugs blood cell genotypes, Hb AA and Hb AS were on plasma methemoglobin concentration was obtained from subjects/volunteers within the age carried out by introducing 0.02 ml of the specified bracket of 18-35 years old. Blood samples of Hb SS concentrations (0.2-0.8 g% w/v) of each drug IRANIAN JOURNAL OF BLOOD AND CANCER
Table 1.
The percentage of plasma methemoglobin of total hemoglobin concentration in the presence of increasing experimental concentrations of
the five antimalarial drugs in three human erythrocyte genotypes.

Drugs (g %)
FansidarTM
Genotype AA* AS SS AA
3.48±0.75b* 3.31±2.45b 4.98±3.72d 2.18±0.88c 2.20±2.10C 3.72±2.18d 2.17±0.21d 2.25±0.97c 3.83±2.11d 3.81±0.74d 3.32±2.41d 4.01±2.11d 2.63±0.69d 2.61±1.84d 4.81±2.61d 5.63±2.51c 2.35±1.10b 2.30±1.31c 3.81±3.22b 2.48±0.58c 2.50±1.61b 4.78±3.21a 4.98±0.89c 4.62±2.33c 5.63±1.88c 2.65±0.35c 2.89±1.44b 4.82±1.66c 2.55±0.61d 3.76±2.01a,b 5.66±3.12b 2.30±0.55b 2.41±2.01b 3.75±1.52c 2.62±0.29b 2.71±1.54a 4.13±1.08C 5.31±0.42b *Means in the column with the same letter are not significantly different at P< 0.05 according to LSD. AA, AS, and SS are the different blood hemoglobin genotypes Chikezie et al. solution into separate test tubes. This was followed sample. Although this value represented an by the addition of 5 ml of distilled water and 0.02 increase in methemoglobin concentration in the ml of whole blood sample. The mixture was allowed genotype (Hb AS), it was not significantly different to stand for 60 mintues at room temperature, after (P<0.05) from the control samples. which, the absorbance was read at 540 nm and 630 An overview of the results presented in table 1 nm using a spectrophotometer (Model 6400, shows a general tendency of the five antimalarial Jenway). The percentage plasma methemoglobin drugs to elevate plasma methemoglobin was obtained with the formula: concentration in all three human erythrocyte genotypes in a concentration dependant manner. It Percentage Methaemoglobin (Fe3+) = is worthwhile to note that CoartemTM amongst the 540 2 + (A five antimalarial drugs showed the highest propensity to elicit increased plasma where A540 and A630 are absorbance at λmax of methemoglobin concentration in the three 540 nm and 630 nm, respectively (Tietz, 1976). genotypes. Specifically, at 0.8% concentration of Statistical Analysis
CoartemTM, plasma methemoglobin concentrations The experiment was designed in a completely in the three human erythrocyte genotypes were randomized method and data collected were significantly different (P< 0.05) when statistically analyzed by the analysis of variance procedure compared with the control sample of the three while treatment means were separated by the Least Significance Difference (LSD) incorporated in the Statistical Analysis System (SAS) package of 9.1 Discussion
The pattern of variability of basal plasma methemoglobin concentrations of the control samples amongst the three human genotypes, The percentage of plasma methemoglobin of which was in the order: HbSS> HbAS > HbAA total hemoglobin concentration in the presence of (table1), was in concordance with earlier reports.16 increasing experimental concentrations of the five They noted that the primary reason for the antimalarial drugs is presented in table 1. In the relatively raised concentration of oxidized absence of the five antimalarial drugs (control hemoglobin (methemoglobin) in Hb SS erythrocytes sample) the mean ± S.D of plasma methemoglobin was the higher production of superoxide ion by concentration (%) showed a genotype dependent these erythrocytes compared to those of Hb AA and variability. However, there was no statistically Hb AS erythrocytes. Furthermore, Orjih et al17 significant difference (P<0.05) in plasma (1985) and Uwakwe18 (1991) reported higher than methemoglobin concentration between Hb AA and normal level of erythrocyte endogenous oxidant Hb AS genotypes. (haemin) in Hb SS genotype. Haemin has a profound Quinine at 0.2% concentration exhibited the capacity to activate certain erythrocyte redox lowest capacity to generate methemoglobin in enzymes e.g NADH methemoglobin reductase,18 human Hb AA enthrocyte genotype with value at and its presence at high concentration is 2.17 ± 0.21 percent, which however was not attributable to the high level of haemolytic significantly different (P<0.05) from the control phenomenon peculiar to this hemoglobin variant sample of the same genotype. Observation showed cells.17 There is also the case of certain that 0.2% of another quinoline derivative, HalfanTM methemoglobinopathies found in association with generated 3.72 ± 2.48 percent of methemoglobin in Hb SS erythrocytes. Hb SS erythrocyte, being the lowest methemoglobin These are Hb MBoston, Hb MIwate, HbMHydepark, and concentration in this class of human erythrocyte HbMHammersmith which are noted to have tendency genotype in the presence of the antimalarial drug. towards spontaneous oxidation in vivo. Basically, In a similar way, the heterozygous genotype Hb AS these structurally and functionally defective presented 2.20 ± 2.10 percent methemoglobin hemoglobins are resistant to enzymatic reduction when 0.2% of HalfanTM was added to the blood and exhibit high molecular stability.19 Therefore, IRANIAN JOURNAL OF BLOOD AND CANCER Studies of Methemoglobin Concentrations of Three Human Erythrocyte Genotypes … their presence could as well result to the generation Although the introduction of quinine in the three of significantly higher concentration of human red blood cell genotypes caused moderate methemoglobin than what is observed in normal but significant increase in plasma methemoglobin hemoglobin erythrocyte.20 concentration, the data presented in table 1 did not Early studies have noted that certain xenobiotics indicate quinine to be capable of inducing toxic or are capable to elicit the formation and elevation of drug–induced methemoglobinemia in vitro. This erythrocyte methemoglobin concentration, thereby observation, by implication, was in concordance distorting the normal plasma hemoglobin (Fe2+)/ with the reports of Laurence et al (1997).22 They methemoglobin (Fe3+) ratio. Callister (2003) noted that quinine antimalarial action was more reported the nitrates and anilines as the most specific upon plasmodia parasites than on the common causes of methemoglobin toxicity in elements of blood. Furthermore, amongst all the 4- humans.8 This physiologic dysfunctional state aminoquinoline derivatives, Ursula (1998) through (methemoglobinemia) is presented as clinical in vivo investigations, established primaquinine and cyanosis when plasma methemoglobin chloroquinine but not quinine as two antimalarial concentration exceeds 15%.7 agents that can induce toxic methemoglobinemia.7 Our present findings showed that at the four The clinical presentation is exacerbated in increasing experimental concentrations of individuals with impaired activity or deficiency of FansidarTM, plasma methemoglobin concentration glucose-6-phosphate dehydrogenase.24 However, was significantly elevated in a concentration reports from in vivo studies showed that the dependent manner in the three human erythrocyte administration of quinine above a critical dose genotypes (table 1). However, the oxidative resulted in the diffusion of substantial quantity of potential of FansidarTM at the four increasing the drug into the interior of the erythrocyte and experimental concentrations (0.2% - 0.8%) were not predisposed the red cells to hemolysis elicited by high enough to engender the oxidation of a tissue lytic factor.23 significant quantity of ferrous state hemoglobin The antimalarial activity of HalfanTM (Fe2+) to ferric state hemoglobin (Fe3+) that is (halofantrine) is similar to that of the quinolines, diagnostic of toxic methemoglobinemia. These since it forms a complex with ferritoporphyrin IX results agreed with the reports of Ursula (1998) and which is toxic to the malarial parasites.24 In a similar Callister (2003).7,8 They noted that sulfonamide, a way, the results of our present study (table 1) component of the drug FansidarTM, was capable of showed that HalfanTM like the quinolines does not elevating plasma methemoglobin concentration. possess the capacity to profoundly elevate plasma This property is probably related to the chemical methemoglobin in the three human erythrocyte characteristics of sulfadoxine. Sulfadoxine is acidic genotypes that is diagnostic of methemoglobinemia in nature and present in plasma in anionic form and presented as clinical cyanosis. which gives it high oxidative potentials.4 It is The relative high concentration of plasma worthwhile to note that for the test experiment, methemoglobin in the three human erythrocyte values of plasma methemoglobin concentrations of genotypes in the presence of increasing the three human erythrocyte genotypes when experimental concentrations of CoartemTM related to the blood volume and the experimental (artemether) when compared to the other four concentration of FansidarTM administered, seem to antimalarial drugs may not be unconnected with suggest a safe drug combination to minimize in the generation of free radicals which is associated drug-induced methemoglobinemia. with the metabolism of this drug in the red blood Furthermore, the fact that pyrimethamine is cells. The interaction of artemether with haem iron present at a relatively higher concentration in the caused the cleavage of the drug endoperoxide drug combination may also suggest pyrimethamine bridge which engendered the formation of was not profoundly associated with methemoglobin metabolites (free radicals) of high oxidative generation as earlier described. However, animal potentials.2 The red blood cells methemoglobin experiment studies have shown pyrimethamine can reduction systems may have been overwhelmed by interfere with hematopoiesis.21 these metabolically generated oxidizing species. In Volume 1 Number 4 Summer 2009 Chikezie et al. contrast, toxicity of the artemether endoperoxide is these drugs may reveal paradoxical results and well tolerated and safe in human subjects when observation with respect to the capacity of these administered up to seven days at therapeutic antimalarials to distort and elevate plasma methemoglobin concentrations. This paradoxical presentation obviously implied that the red blood cells antioxidant/reduction References
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Limbird LE, Gilman AG, editors. Goodman and Gilman's Pharmacological basis of therapeutics. 10th Ed. Newyork: The five antimalarial drugs caused a McGraw– Hill; 2001. concentration dependent increase in plasma 3. Bray PG, Mungthin M, Ridley RG, Ward SA. Access to methemoglobin concentration in the three human hematin; the basis of chloroqunine resistance. erythrocyte genotypes. CoartemTM (artemether), Mol. Pharmacol. 1998; 54:170–9. when compared to the other four antimalarial drugs 4. Milhous WK, Norman FW, Jean HB, Robert ED. In (FansidarTM, HalfanTM, Quinine and Chloroquine Vitro activities and mechanism of resistance of PhosphateTM) showed a profound capacity to distort antimalarial drugs. Antimicrob Agents Chemother. 1985; and elevate plasma methemoglobin concentration. 5. Bates MD, Meshnick SR, Sigler CI, Leland P, The other four antimalarial drugs showed a Hollingdale MR. 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Doi:10.1016/j.bbrc.2004.09.124

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