INTERACTION OF ANTIMALARIAL DRUGS (PYRIMETHAMINE AND SULPHADOXINE) WITH NORMAL AND SICKLE HAEMOGLOBINS: A UV-VISIBLE STUDY

ABSTRACT

Crude haemoglobins were extracted from blood samples of identified individuals of normal (AA), sickle trait carrier (AS), and sickle (SS) by employing centrifugation techniques. The crude haemoglobins were dialysed at 4oC for 12hr against 50mM Tris-HCl buffer of pH 7.2. The effects of pyrimethamine and sulphadoxine on the haemoglobins in the presence and absence of sodium dodecyl sulphate (SDS) were studied at pH 5.0 and 7.2 with uv-visible titration spectrophotometry. The study showed that sodium dodecyl sulphate at pH 5.0 unfolded the studied proteins. These can be related to destabilization of haemoglobin structure by proteases such as plasmepsins and falcipains in the acidic environment of malaria parasite food vacuole due to malaria parasite infection. Pyrimethamine and sulphadoxine at pH 5.0 and 7.2 decreased the concentration of oxyhaemoglobin and increased the concentrations of methaemoglobin and deoxyhaemoglobin of the studied proteins. The results also show how haemoglobins are deoxygenated due to interaction with sodium dodecyl sulphate. Deoxygenation of haemoglobin as a result of their interaction with SDS can be likened to pathological condition whereby malaria parasites infection reduced the oxygen tension of erythrocytes of their host. HbS had the highest interaction with sulphadoxine and pyrimethamine followed by HbAS while HbA had the least interaction. Formation of methaemoglobin is associated with lipid oxidation. Increase in absorbance at 275 nm observed in this study refers to dynamic motion of the studied proteins and their deviation from normal structure and function. The interaction of haemoglobins with sulphadoxine-pyrimethamine combination at pH 5.0 caused a large perturbation of the protein conformation that was reflected in modest spectral shift of the soret band.

CHAPTER ONE

INTRODUCTION

One of the main causes of death today is malaria, especially in numerous parts of Asia, Sub-Saharan Africa and the America (Esparza, 2005). Of the four Plasmodia that cause malaria, Plasmodium falciparum is responsible for the majority of illness and death in mankind (Duraisingh and Refour, 2005; Idro et al., 2005; Okie, 2005; Worrall et al., 2005). In Sub- Saharan Africa, this disease has a profound impact on children and infants, whilst millions have already died from AIDS (Acquired Immunodeficiency Syndrome) (Esparza, 2005; Harms and Feldmeier, 2005). In addition to this, malaria adds in mortality while the spread of chloroquine resistant strains of the plasmodium parasites across Africa increases (Farooq and Mahajan, 2004; Mahajan et  al.,  2005).  Approximately,  three  million  people,  of whom more  than  half  are children,  die  of malaria  caused  by P.  falciparum annually (Duraisingh and  Refour, 2005). Mortality and morbidity increase every year with over 500 million people infected with P. falciparum, presenting clinical symptoms of mild to severe malaria.

There exist several reasons for the increase in the occurrence of malaria including:

i An increase of the protozoan parasite’s resistance to anti-malarial drugs,

ii The development of the anopheles mosquito vectors’ resistance to numerous insecticides

iii The growth and the widespread migration of vulnerable populations to vastly endemic areas

(Abdel-Hameed, 2003; Gregson and Plowe, 2005).

Malaria can be treated with various anti-malarial drugs. Sulphadoxine and pyrimethamine combination therapy was introduced into clinical practice for the prevention and treatment of malaria in the late 1960s as a follow-up on the drug chloroquine (Schultz et al., 1994). The first product by the name of Fansidar® containing sulphadoxine and pyrimethamine in combination was produced by Roche in 1971. Since the expiration of the Fansidar® patent, numerous generic products have been produced worldwide, contributing towards cheaper anti-malaria therapy.

1.1 Malaria

1.1.1 A Health Problem

Of  all  the  communicable  illnesses,  Malaria,  known  as  the  globe’s  greatest  tropical parasitic infection, claims the most lives aside from tuberculosis (TB). Being a health problem in over 90 countries and inhabited by approximately 2400 million people, an expected 300 – 500 million clinical cases occur per annum and more than one million lives are claimed yearly (Greenwood et al., 2005). In the year 2001, the three illnesses TB, HIV and malaria contributed to approximately 5.7 million deaths, of which the majority was young children together with men and women of their productive years, and in developing countries, deemed liable for 23% of fatalities (Theobald et al., 2006). The  great  impact  of  these  diseases  and  the   globally  inadequate  responses  has counteracted health gains produced over the last ten years and contributes towards poverty considerably in numerous low- and middle-income countries (Theobald et al., 2006). The World Health  Organization (WHO)  states that  90% of  fatalities  worldwide are  in  Africa,  mainly children under the age of 5 years.

1.1.2 History of Malaria

Scientific studies on this parasitic infection made their first  major advance in 1880. Charles Louis Alphonse Laveran, a doctor in the French armed forces working in Algeria, observed the parasites in red  blood cells obtained from malaria patients, claiming that this protozoan causes malaria, making it the first time for protozoa to be identified as the cause of disease. The two Italian scientists Angelo Celli and Ettore Marchiafava named the protozoan Plasmodium. A year later, the Cuban doctor Carlos Finlay who was treating patients in Havana for  yellow  fever,  was  the  first  to  suggest that  the  disease  was  transmitted  to  humans  by mosquitoes. But it was Sir Ronald Ross from Britain, working in India during that time, who in 1898, showed that mosquitoes transmitted malaria. He showed that malaria was transmitted to birds via certain species of mosquitoes and the malaria parasites were isolated from mosquitoes’ salivary glands that fed on birds infected with the parasites (Wikipedia, 2013).

1.1.3The Life Cycle of the Malaria Parasite

Protozoa of the  genus Plasmodium cause  malaria.  Four  species  of Plasmodium are responsible for the disease in humans: P. falciparum, P. malariae, P. ovale and P. vivax. Of these, P. falciparum may cause the condition known as cerebral malaria, which is responsible for the majority of fatal outcomes. The pathogenesis and life cycle of the malaria parasite are complex (Miller et al., 2002), consisting of two stages: a sexual stage (sporogony), which occurs within the mosquito, and an asexual stage (schizogony), which occurs in the host (Frederich et al., 2002). The illness results when an infected female anopheles mosquito feeds on the blood of an uninfected vertebrate host. Mosquitoes inject parasites (sporozoites) into the subcutaneous tissue, less frequently directly into the bloodstream. In less than one hour, the sporozoites travel to the liver, invade the hepatocytes and undergo exoerythrocytic schizogony. After some time, depending on the plasmodium species, the schizonts are transformed into merozoites, which after release into the blood stream invade erythrocytes. After significant reorganization of the membrane proteins of an occupied erythrocyte (Parker et al., 2004), the merozoites undergo erythrocytic schizogony, which comprises young rings (12 hr after erythrocyte infection), mature rings (18 hr), early trophozoites (24 hr), mature trophozoites (30 hr), early schizonts (36 hr) and mature schizonts (42 hr). At the end of erythrocytic schizogony, the parasites return into the merozoite form but enormously multiplied causing splattering of the erythrocyte. The released merozoites  invade  new  red  blood  cells  and  start  a  new  erythrocytic  schizogony  cycle. Erythrocytic schizogony occurs every 2–3 days, depending on the plasmodium species. Each cycle is accompanied by typical malaria symptoms, such as fever, chills, headache and exhaustation. After several cycles, some of the merozoites undergo sexual development and are transformed into gametocytes. The gametocytes remain in the erythrocytes and are consumed by an anopheles mosquito. In the mosquito, female and male gametocytes join and form zygote. Within 18 to 24 hr, the zygote transforms into a slowly motile ookinete. Between 7 and 15 days, depending on the plasmodium species and the ambient temperature, a single oocyst forms more than 10,000 sporozoites. The motile sporozoites migrate into the salivary glands and accumulate in the acinar cells.  When infected, the mosquito bites a susceptible vertebrate host; a new parasite cycle commences (Opsenica and Solaja, 2009).

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