INTRODUCTION

Malaria is a parasitic infectious disease, caused by Plasmodium species that infect the red blood cell (RBC) when an infected female anopheles mosquito inoculate them into the human host during a blood meal [1]. Transfusion of infected blood or its products can also transmit the parasite to the human host [2]. Five (5) Plasmodium species can infect and cause malaria in humans. They can be transmitted from one human host to another. These include Plasmodium falciparum, P. ovale, P. knowlesi, P. vivax, and P. malariae [3].

If the female anopheles mosquito ingests the sexual stages of plasmodium while feeding on infected blood, she becomes infected. The plasmodium develops in the mosquito and is passed into another human host during the next blood meal. In humans, the parasite first stage develops in the liver, from where more developed plasmodia are released into the bloodstream to infect RBCs, reproduce and cause RBCs to lyse and release another set of parasites [4]. If many cells lyse, it could lead to jaundice from the build-up of bilirubin from the hemoglobin (Hb) in the dead red cells [5].

16. falciparum is more virulent among the plasmodium species that cause malaria in man and it causes more severe disease. It accounts for the majority of malarial deaths [6].

Malaria is among the commonest and clinically important parasitic diseases in the world. The parasites cause illness in about 200 to 500 x 10⁶ persons yearly [7] and account for about 500,000 deaths worldwide annually, mostly in children [8]. Most infections and deaths caused by malaria are in Asia and Africa [9]. Patients who have been properly diagnosed of being infected with malaria are administered antimalarial drugs for treatment [10].

The therapeutic aims are to completely cure uncomplicated malaria as soon as possible and to limit it from worsening to severe disease. Uncomplicated malaria is defined as when a patient shows symptoms of malaria and positive parasitological test (microscopy or Rapid Diagnostic Test) but shows no signs of severe malaria [3].

Antimalarial drugs, for example, Artesunate, are also prescribed for prophylaxis to people traveling to areas where malaria is prevalent. The type of drugs prescribed is dependent on the drug resistance in the areas [11].

Among the most common antimalarial drugs in use for malaria treatment are artemisinin derivatives, amodiaquine, chloroquine, atovaquone/proguanil, quinine, mefloquine, mepacrine or quinacrine, sulfadoxine/pyrimethamine, and primaquine [8]. Among them, Artemisinin is the most rapid-acting class, acting against the asexual stage gametocytes and blocking the sexual stage for both uncomplicated and severe malaria.

Artemisinin is a natural antimalarial drug derived from the plant Artemisia annua. Artemether and artesunate are members of the artemisinin antimalarials (sesquiterpene lactone compounds), developed to improve the bioavailability of artemisinin [3]. They are used to treat severe malaria for speedy clearance of all asexual stages of P. falciparum, with faster fever resolution than with other antimalarial drugs [12, 13].

Artesunate is a water-soluble, hemisuccinate derivative of dihydroartemisinin. It can be administered through parenteral (IM, IV) and enteral (oral) routes. For severe malaria, parenteral administration is recommended for initial treatment [3, 14].

The World Health Organization (WHO) recently approved Artemisinin-based Combination Therapy (ACT) as a first-line drug to treat uncomplicated falciparum malaria [6, 15] due to the increasing rate of malaria parasites resistance to quinine and other antimalarial drugs.

The ACT combines artemisinin derivative with another antimalarial drug with a different mode of action and longer-acting [3]. It is the recommended standard for the treatment of malaria in Nigeria [16]. It involves the simultaneous use of two or more drugs with separate modes of action and different targets in the parasite to kill the blood schizonts.

ACT regimens are strongly recommended, based on high-quality evidence, to provide treatment for 3 days with artemisinin derivative [3]. If properly followed, this gives efficient clearance of asexual and early transmission stages of parasites, while the infectious mature sexual stages are unaffected. To halt transmission, the ACT is often combined with Primaquine [15, 16]. The following artemisinin derivatives have been developed and distributed worldwide to combine with other drugs for treating malaria. Viz artemether, artesunate, dihydroartemisinin, artemisinin, and arteether. Many of them are associated with cases of idiosyncratic liver injury [8].

Upon administration, artesunate is hydrolyzed to Dihydroartemisinin (DHA), which accumulates in the P. falciparum-infected cells. DHA is the active metabolite and lasts longer than the artesunate in the infected RBCs. The plasma half-life of artesunate is between 3-29 minutes while that of DHA is between 40-95 minutes [15]. DHA binds to the plasma membrane of the infected RBC and suppresses the sarcoplasmic/endoplasmic reticulum calcium pump of P. falciparum [16].

Among the artemisinin-based drugs, artesunate is the most commonly used today. It mediates its effect by reducing the transmission of sexual stages (gametocyte) of the parasite. WHO recommends a complete course of 5 or 7 days, starting with 4.0mg per kg (body weight) for the first three days (combined with mefloquine), followed by 2.0mg per kg for the remaining days [15].

During infection of the RBCs, the plasmodium degrades Hb to get essential amino acids (aas) needed by the parasite for energy and to synthesize its protein. Haem is released from the iron-porphyrin complex of the degraded Hb. The two major pathways utilized in the mechanism of action of artemisinin involve inhibition of calcium transporters (PfATP618) of the parasite [16] and the breakdown of iron (or haem) [15].

Reactive oxygen species formed from the free ferrous ion (Fe²⁺) generated from haem breakdown inhibit parasite’s cell division [14] by cleaving the endoperoxide bridge.

The antimalarial drug inhibits replication of the parasite and formation of food vacuole by diffusion in the infected RBCs and the drug becomes protonated. Since the food vacuole has a PH of about 4.7 (acidic), the drug cannot diffuse out. It caps the hemozoin molecule and prevents more haem biocrystallization and consequently leads to accumulation of haem. The drug then binds to haem to form haem-drug complex, which is toxic to the cell and interrupts the function of the cell membrane. The action of the toxic complex ultimately leads to RBC lysis and autodigestion of parasite cells [4]. Haem build-up leads to hyperbilirubinaemia due to the release of excess bilirubin from RBC lysis [5].

The liver plays vital biochemical roles in several synthetic, digestive, detoxifying, metabolic, and excretory functions in the body. It has some reserve capability, being a large organ while performing its biochemical functions. The liver can prevent the decrease in protein concentrations, except there is pronounced liver damage [17]. In many individuals that have liver diseases, there can be normal liver functions, though there is extensive damage. Liver disease in cases like that may be detected only by tests that detect liver injury and this is commonly accomplished by estimating the plasma concentrations of liver enzymes, that are released to the blood in a somewhat pulsatile fashion from hepatocytes, depending on the degree of liver injury. Liver functions can also be assessed by the measurement of serum bilirubin and albumin concentrations in blood. The biosynthetic function involves the regulation of protein, carbohydrate, and lipid metabolism. Albumin is synthesized only by the liver and it’s commonly estimated in serum to assess the function of the liver [18].

The excretory function can be assessed by measuring the serum levels of bilirubin. Bilirubin is a byproduct of haem turnover from RBC lysis. It is excreted in bile and urine. If the rate of RBCs lysis is faster than normal, there is a tendency for bilirubin to accumulate in the blood and may cause jaundice [5]. Metabolism of bilirubin starts with the lysis of RBCs in the body. Hb is degraded to haem and globin. Bilirubin is formed from the haem fraction and is bound to albumin in the blood, transported to the liver to be conjugated with glucuronic acid, forming the conjugated (direct) bilirubin, and is released into bile that is stored in the gall bladder or taken directly to the small intestines where it is acted upon by intestinal bacteria. A small fraction of the end products undergo hepatic-duodenal re-uptake and later reappears in urine [5].

Under normal circumstances, the process of conjugation is very efficient and ensures a low amount of unconjugated bilirubin in plasma. In certain diseases with either an increased rate of bilirubin turnover or reduced conjugation of bilirubin, unconjugated hyperbilirubinaemia occurs. Conjugated hyperbilirubinaemia may result from reduced secretion of direct bilirubin into bile (as seen in hepatitis), or from diminished bile movement into the intestine (as seen in obstruction of bile ducts) [15]. Haemolysis can be seen in several medical procedures and diseases, such as malaria and the administration of certain drugs. Among the drugs are antimalarial medications [14].

Accumulation of bilirubin may also result from inadequate or impaired removal by the liver or leaking into the bloodstream after removal [18]. When the level rises to 3mg/dl or higher, it discolors other tissues, white parts of the eye, and skin yellow, a condition known as jaundice. Serum bilirubin estimation involves measurement of both total and direct bilirubin in the screening or monitoring of diseases of the liver or gall bladder [16]. In chronic liver diseases, the patients may exhibit high levels of bilirubin after blood transfusion, due to the high turnover of the transfused RBCs [14]. Unconjugated hyperbilirubinaemia may be due to unrestrained formation of bilirubin or reduced metabolic process by the liver [18]. 

Albumin is a small spherical protein (mw 66.3KDa). It accounts for about 50% of total plasma protein by mass. It has relatively high concentrations in plasma and due to its small size, it is the main protein constituent in extravascular fluids of the body [19]. Albumin is produced only in the liver. Measurement of albumin in the plasma is used as a good marker to evaluate its synthesis by the liver [20]. In apparently healthy individuals, about 0.2g to 0.3g of serum albumin is formed daily per kg body weight. Its serum concentration is preserved by the rate of synthetic and catabolic processes and its distribution between extravascular and vascular compartments. In inflammatory conditions or insufficient protein consumption, albumin synthesis may be suppressed. A decrease in albumin synthesis may lead to a serum albumin decrease of greater than 3.0g/L [20, 21].

Any chemical substance (drugs or toxins) that has either slight or pronounced hepatotoxic effects on the liver may affect its synthetic ability. The majority of the drugs bind to albumin and are taken to the liver for detoxification. Some drugs also bind to globulin [21]. For the fact that albumin is synthesized by the liver, a decrease of albumin concentration in serum may be indicative of liver injury or disease. Decreased albumin concentration may also come from comprehensive protein wasting, malnutrition, and extensive leakage in urine resulting from renal impairment [22].

Many substances like drugs, toxins, lipids, and hormones are transported in the blood, bound to albumin, and taken to the liver where they are detoxified by being converted to water-soluble forms for excretion [23]. Decreased albumin concentrations lead to lowered plasma oncotic pressure, which lets fluid out from interstitial spaces to the peritoneum, and produces ascites [21]. Acute liver damage does not usually cause low albumin concentrations in serum. It usually takes many weeks of consistent decrease of albumin synthesis to observe a drop in the amount of albumin in serum. Cirrhosis causes chronic liver damage and is the commonest reason for low albumin concentration. In chronic liver disease, before remarkable liver disease and cirrhosis set in, the albumin concentration in serum can remain normal. In late-stage liver injury, the serum albumin concentration may be <35.0g/L [21, 22]. Measurement of the amount of albumin in serum can give a clue of a patient’s status of a diseased liver or the body’s inability to absorb adequate protein in diets [22].

This research was designed and performed to evaluate the effects of artesunate (antimalarial drug) on some liver function parameters, namely bilirubin and albumin, in rats treated with the drugs, and to determine if high bilirubin levels would affect albumin estimation and to assess if serum albumin estimation is a reliable marker for liver function.

MATERIALS AND METHODS

Test animals

The study was conducted on thirty (30) Swiss Wistar rats of 3 to 5 months old, of both genders, and having body weights ranging from 100-360g. The rats were commercially procured and housed/caged in wire gauzed cages and they were fed on finisher mash and tap water for 7 days under atmospheric conditions.

Drug preparation and dosing

On the 8^th day, the rats were separated into 5 groups of 6 rats each. Rats in the first group [group one (1)] were used as control. Rats in the other groups (2-5) were placed on oral administration (by compulsion) of artesunate. 100mg of artesunate tablets were dissolved in 100ml of distilled water and administered to the rats based on their body weights as as shown in Table 1 for 7 days and they were also fed on the same finisher mash throughout the period. On the 15^th day, no drug was administered and the blood samples were collected.

Table 1. Drug dosage administered to the rats

Collection of blood

After 7 days of artesunate administration, each rat was euthanized with chloroform, pinned to the dissecting board, and dissected open along the ventral sagittal axis and the blood was quickly collected from the heart by aortic puncture (using a syringe with a sharp needle) while the animal was still breathing. 3ml - 5ml of blood was collected from each rat into a plain glass sample bottle and allowed to clot before separation, avoiding haemolysis that could interfere with the tests. The cells (residues) were discarded while the sera (supernatant) were used for the test - to estimate the biochemical parameters, namely albumin, and bilirubin.

Estimation of bilirubin

Randox reagents from Randox laboratories Ltd, Antrim, United Kingdom, were purchased & used for the biochemical assays in line with Cheesbrough [23]. The serum bilirubin was estimated using the Jendrassik and Grof method, based on the principle that bilirubin reacts with diazotized sulphanilic acid in the presence of hydrochloric acid and sodium nitrite to form a pink-colored azobilirubin complex. The conjugated (direct) bilirubin reacts directly with the diazotized sulphanilic acid, while the unconjugated (indirect) bilirubin requires an accelerator (caffeine) to form the azobilirubin. The pink acid azobilirubin is converted to blue azobilirubin by an alkaline (sodium) tartrate reagent. The optical density of the colored complex was read spectrophotometrically at 580nm (for total bilirubin) and 540nm (for conjugated bilirubin) [23, 24].

Estimation of albumin

Serum albumin was estimated using the bromocresol green (BCG) binding method. BCG is an indicator that is yellow under acidic conditions (pH 3.5 - 4.2), at which albumin specifically binds with BCG to form a blue-green colored complex, which absorbance is read in the spectrophotometer at 580nm [24].

RESULTS AND DISCUSSION

The results are presented in the table below.

Table 2. The Mean ± SD of Serum Bilirubin and Albumin Concentrations in the Different Groups of Rats Administered with Varying Doses of Artesunate