CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
ARTESUNATE
Artesunate is part of the Artemisinin group of drugs that treat malaria. It is a semisynthetic derivative of Artemisinin that is water-soluble and may therefore be
given by injection. It is sometimes abbreviated as AS. ARTESUNATE is used
primarily as treatment for Malaria (Boulangier et al 2007). Artemisinin and its
derivatives are the most important class of antimalarial drug effective for both
uncomplicated and severe malaria. Besides, Artemisinin and its derivatives have
been shown to possess anticancer, antiviral and anti-inflammatory activities
(Looareesuwan et al, 1992). Artesunate is prepared from Dihydroartemisinin
(DHA) by reacting it with Succinic acid anhydride in basic medium. Pyridine as
base/solvent, sodium bicarbonate in chloroform and catalyst DMAP (N,N –
dimethylaminopyridine) and triethylamine in 1,2 –dichloromethane have been
used, with yields of up to 100%. A large scale process involves treatment of DHA
in dichloromethane with a mixture of pyridine, a catalytic amount of DMAP and
succinic anhydride. The dichloromethane mixture is stirred for 6-9 hour to get
Artesunate in quantitative yield. The product is further re-crystalized from
dichloromethane, alpha – Artesunate is exclusively formed (M.P 135c) (White,
2008).
Artemisinin are derived from a biological active chemical called Quighaosu
or Artemisinin. These compounds have impressive parasiticidal properties In vivo
and Invitro. They rapidly arrest parasite more quickly than other antimalarial
drugs. Artemisinin, which is the parent compound, is the antimalarial principle of
1
these compounds and is derived from the leaves of a plant called Sweet Worm
Wood (Artemisia annua). Artemisinin was isolated by the Chinese Scientist from
Artemisia annua leaves. This antimalarial principle is highly crystalline and does
not dissolve in polar or non-polar solvents; hence, it is modified chemically to
yield these derivatives: Artesunate, Artemether, Arteether, Artelinic acid and
dihydroartemisinin. Artesunate is effective against Plasmodium Falciparum
resistant to other operationally used antimalarial drugs. Serious concern has been
raised about uncontrolled used of these drugs, because, at the moment they are the
last resort in the combat against multi-drug resistant P. falciparum malaria. The use
of these drugs should be controlled and restricted to multi-drug resistance on
severe malaria in order to preserve their efficacy and avoid emergency of resistant
strains. In malaria endemic areas such as Nigeria, self-medication is quiet common
and the purchase of antimalarial in the open market is rampant. The possibility of
administering overdose and misappropriation in the usage of antimalarial are very
common. Drugs, though useful in the treatment of disease conditions, could be
produce untoward effects in the individual. The untoward or toxic effect may be
harmful to the patient. Studies on brain stem showed that, preclinical evidence of
the brain toxicity in animals. Ferotoxicity studies based on animals are going on.
However, not much investigations or information have been documented in the
adverse effect of Artesunate on the liver since it is the organ of metabolism of
drugs and other substances. Therefore, the present study is aimed at investigating
the possible toxic potentials of the drug on the liver, using the hepatospecific
enzyme markers.
2
TOXICITY OF ARTESUNATE
Artemisinin derived drugs are available for the treatment of malaria, except for two
case reports; no major side effects have been reported in humans at doses used for
the treatment of malaria but it is still unknown whether the higher dose required for
the treatment of cancer patients could cause major side effects. Invivio studies
showed that dose of Artemisinin – related endoperoxides of at least 5 times higher
than those used for malaria therapy are required in order to induce an effect. The
safety of such doses has not yet been evaluated in phase clinical trials.
A first case report, describes a boy who received Artesunate Suppositories
and died 13 days. He had received a dose 7-fold higher than the maximum
recommended dose which reportedly led to toxicity of the brainstem.
In a second report a woman with recently resected early breast carcinoma
described symptom of toxic brainstem encephalopathy: since the neuro toxicity has
also been seen in animals, the authors of the case report ascribe the toxicity to
Artemisinin consumption, although she received also chemotherapy and a mixture
of other herbs on top. On the other hand, a review of the toxicity of Artemisinin
derivatives suggested that the toxicity seen in laboratory animals does not
necessarily occur in humans due to the difference in pharmacokinetic profile after
different routes of administration. The oral administration used I human is unlikely
to cause the neurotoxicity seen after intra oral muscular administration in rats.
3
METABOLOISM OF ARTESUNATE IN THE LIVER
The metabolism of Artemisinin in human liver microsome is primarily mediated
by cytochrome P-450 mono oxygenase enzyme (CYP) 2B6, with a secondary
contribution of CYP2A6 to Artemisinin metabolism is likely of minor importance.
There is a large body of evidence suggesting that Artemisinin influences the CYP
activity which could result in drug – drug interactions. An induction of activity by
Artemsinin was reported for CYP2A5, CYP2A6, CYP2B1, CYP2B6, CYP2B10,
CYP2C19, and CYP3A4. In addition, Artemisinin activates the constitutive
androstane receptor and pregnane X receptor, which explains the unregulation of
CYP2B6 and CYP3A4. The data regarding CYP1A2 are contradictory, whereas
Artemisinin inhibits CYP2D6. Artemisinin leads to an auto induction of drug
metabolism, which reduces its own bioavailability.
In various clinical studies, Artemisinin has been administered alone or in
combination with other antimalarial drugs.
EFFECTS OF ARTESUNATE IN THE LIVER
Artemisinin derivatives are tolerated well by patients. Mild and hematological and
electrocardiographic abnormalities, such as neutropenia and first degree heart
block, have been observed infrequently. Neurotoxic effects have been repeatedly
reported in experimental rats etc. Affected areas in the brainstem are recticular
system with regard to autonomic control, the vestibular system, the auditory
system (trapezoid nucleus), and the red nucleus, which is important for coordination. A longer exposure time to a lower peak blood concentration of an
4
artemisinin derivative is more neurotoxic than a shorter duration of exposure and a
higher peak blood concentration.
ETHANOL AND THE BODY
Ethanol also called Ethyl Alcohol, Pure Alcohol, Grain Alcohol or Drinking
Alcohol, is a volatile flammable colorless liquid. It is a powerful psychoactive drug
and one of the oldest recreational drugs. In common usage, it is commonly referred
to as Alcohol or Spirits (ABITTAN, 1999). Ethanol is the principal psychoactive
constituent in alcoholic beverages, with depressant effects on the central nervous
system (CNS). It has a complex mode of action and affects multiple systems in the
brain, most notably ethanol acts as an agonist to the GABA receptors. Similar
psychoactive includes those that also interact with GABA receptors, such as
gamma-hydroxybutyric acid (GHB). Ethanol is metabolized by the body as an
energy providing nutrients, as it metabolizes into acetyl-coA, an intermediate
common with glucose and fatty acid metabolism that can be used for energy in the
citric acid cycle or for biosynthesis (Macdonald, 1999).
Ethanol is metabolized extremely quickly by the body. Unlike foods, which
require time for digestion, Ethanol needs no digestion and is quickly absorbed
(Nutt et al, 2007). About 20% of ethanol is absorbed directly across the walls of an
empty stomach and can reach the brain. Once ethanol reaches the stomach, it
begins to break down with the alcohol dehydrogenase enzyme. This process
reduces the amount of ethanol entering the body by approximately 20% (Nutt et
al., 2007). Ethanol is rapidly absorbed in the upper portion of the small intestine.
The ethanol laden blood then travels to the liver via the veins and capillaries of the
digestive tract, which affects nearly every liver cell. The liver cells are the only cell
5
in the body that produces enough alcohol dehydrogenase to oxidize ethanol at an
appreciable amount (Lieber, 1999).
METABOLISM OF ETHANOL IN THE BODY
Ethanol affects every parts of the body; its most impact is upon the liver. More
than 90% of ethanol that enters the body is completely oxidized to acetic acid. This
process primarily occur in the liver, the remainder of the ethanol is not metabolized
and it is excreted either in the sweat, urine or given off in ones breathe. The liver
cell normally prefer fatty acids as fuel, and package excess fatty acids as
triglycerides (Sands et al., 1999), which then routes to other tissues of the body.
However, when ethanol is present, the liver cells are forced to first metabolize the
ethanol letting the fatty acids accumulate sometimes in huge amount (Sands et al.,
1999). This explains the fact while heavy drinkers tend to develop fatty livers (fat
deposition in the liver). The liver is able to metabolize about half ounce of ethanol
per hour (approximately one drink, depending on a person body size, food intake,
etc.) (Arnoid, 2005). If more ethanol arrives in the liver than the enzymes can
handle, the excess ethanol travels to all parts of the body, circulating until the liver
enzymes are finally able to process it. The major pathway involved in the liver and
in particularly at higher alcohol concentrations is the oxidation of alcohol by the
microsomal (small spherical vesicles) – cytochrome P-450 system (MESOS)
system. In addition to these routes, there is catalase-dependent oxidation of ethyl
alcohol and oxidation of it by the stomach when it is first ingested (Abittan and
Lieber, 1999). These latter two routes of metabolism are minor in comparison to
the ADH and MEOS system. As mentioned above perhaps the major route of
metabolism of the ethyl alcohol is its oxidation in the liver catalyzed by the
6
cytosolic enzymes alcohol dehydrogenase (ADH). It catalyzes the following
reaction.
CH3CH2OH +NAD+ → CH3CHO + NADH + H+
This reaction produces acetaldehydes, a highly toxic substance.
The second step of ethanol metabolism is catalyzed by acetaldehyde
dehydrogenase. This enzyme converts acetaldehyde to acetic acid, which is a
normal metabolic in humans and hence is non-toxic. ADH has broad specificity,
catalyzing various alcohols and steroids and catalyzing the oxidation of fatty acids.
It is also not a solitary enzyme, in that there are five different ADH genes, two of
which are ADH2 and ADH3 shown polymorphism (variations) of importance is
that fact that the ability of people to oxidize ethyl alcohol is dependent upon the
genetic make-up of the individual. People with alleles (types) of (ADH2) and
ADH3 may protect those having those having these genes from developing
alcoholism. These genes are common in the Asian population and convert alcohol
to acetaldehyde; this toxic compound builds up and makes people who drink too
much uncomfortable and ill. Therefore, these carriers are discouraged from
consuming large amount of alcohol. A similar situation is found in the second step
of ethanol metabolism which is catalyzed by acetaldehyde dehydrogenase (this
enzyme converts acetaldehyde to acetic acid, which is a normal metabolite in
humans and hence is non-toxic). A person who drinks too much builds up
acetaldehyde in their system and feels bad or is sick. This response discourage
drinking, thus preventing the development of alcohol abuse, dependence, and
alcoholism. Another system in the liver which oxidizes ethanol vice the enzyme
cytochrome P45011E1 (CYP2E1) is called the MEOS system. The reaction
catalyzed by MEOS is:
7
CH3CH2OH + NAPH + O2 → CH3CHO + NADP + H2O.
Though the minor significance in comparison to ADH metabolism of ethanol; the
MEOS system seems to play an increasingly important role at higher
concentrations of ethanol. It is not surprising that there are variations in the
P450E1 enzyme which lead to differences in the rate of ethanol metabolism. This
may have implications for tissue damage from ethanol, particular in the liver
(Abittan and Lieber, 1999). During ethanol metabolism, NAD becomes
unavailable for many other vital body processes for which it is needed, including
glycolsis, the TCA circle and the electron transport chain (Sands et al, 2005).
Without NAD, the energy pathway is blocked and alternative routes are taken, with
serious physical consequences. The accumulation of hydrogen atoms shifts the
body’s balance toward acid (Sands et al, 2005). The accumulation of NADH shifts
the TCA circle, resulting in a buildup of pyruvate and acetyl-coA. Excess acetylcoA results in fatty acid synthesis and fat begins to clog the liver (Lieber,
1999).Accumulation of fat in the liver can be observed after a single night of heavy
drinking.
EFFECT OF ETHANOL ON THE LIVER
Ethanol induced oxidative stress in the liver cells plays a major role in the
development of alcoholic liver disease. This condition results from several process
related to ethanol metabolism (Shaw et al., 1983). Changes in the NAD/NADH
ratio resulting from alcohol breakdown by the MEOS, this is particularly important
after chronic alcohol consumption, which stimulates the activity of the MEOS
(Shaw et al., 1983). Reduced level of the antioxidant GSH in the liver. GSH is a
8
small molecule consisting of three amino acids, including cysteine, Acetaldehyde
the first product of alcohol breakdown, and bind to GSH and specifically to
cysteine, thereby removing active GSH from the liver cells (Shaw et al., 1983). In
addition, alcohol itself inhabits the production of new GSH.
Both increased ROS production and GSH depletion lead among other
harmful effects, to the abnormal breakdown of fat molecules (i.e. lipid per
oxidation). This process results in the formation of toxic compounds that can
stimulates scaring and damage liver cells. Thereby contributing to alcoholic liver
disease. Accordingly, it is associated with alcohol metabolism one approach to
achieve this is to ensure that the cells have adequate levels of antioxidants.
Particularly GSH that can capture RDS and break them down or convert them to
less harmful molecules because GSH depletion plays a key role in alcoholic liver
injury, it is therapeutically important to increase GSH levels in the liver (Shaw et
al., 1983). GSH cannot be administered discreetly; however, because the molecules
cannot penetrate directly into liver cells similarly, the amino acid cysteine, which
is most important for ensuring adequate GSH levels, cannot be used as a
supplement because it cannot enter the liver cells. Therefore, clinicians have tried
to administer precursors of cysteine such as the compound acetyl cysteine or the
molecule S – adenosylmethionine (SAME) which can reach the cells and be
converted to cysteine (Shaw et al., 1983). Another important antioxidant is vitamin
E. Alcoholics with cirrhosis often have low vitamin E levels, within the normal
range therefore administration of vitamin E moreover, studies, in baboons have
found that animals with normal vitamin E levels in the liver still develop fibrosis or
even cirrhosis (Lieber et al., 1999). Vitamin E also showed no possible effect in a
trial of patients with alcoholic cirrhosis who received supplements of the
compound (de la maza et al., 1995). These observations suggests that although
9
vitamin E deficiency increases the liver vulnerability to ethanol, normal vitamin E
levels may not be able to prevent the development of alcoholic liver disease,
particularly fibrosis.
METABOLIC FATES OF NADH
The metabolic pathways for the disposal of excess NADH and the consequent
blocking of other normal metabolic pathways are as follows:
Pyruvic acid to lactic acid
The conversion of pyruvic acid to lactic acid requires NADH.
Pyruvic acid + NADH + H → Lactic acid + NADH
Pyruvic acid normally made by transamination of amino acids, is intended for
conversion into glucose by gluconeogenesis. The pathway is inhibited by low
concentration of pyruvic acid, since it has been converted to lactic acid. The final
result may be acidosis from lactic acid build up and hypoglycemia from lack of
glucose synthesis (McMurry, 2004).
SYNTHESIS OF LIPIDS
Excess NADH may be used as a reducing agent in two pathways. One to synthesis
glycerol and the other to synthesis fatty acids. As a result, heavy drinkers may
initially be over weight (McMurry, 2004).
10
ELECTRON TRANSPORT CHAIN
The NADH may be used directly in the electron transport chain to synthesize ATP
in fatty acid spiral and citric cycle. Fats may accumulate or acetyl-coA may
accumulate with the resulting production of ketone bodies. Accumulation of fat in
the liver can be alleviated by secreting lipids into the blood stream. The higher
lipid levels in the blood may be responsible for heart attacks (McMurry, 2004).
Toxicity of alcohol may be by acetaldehyde although the liver converts
acetaldehyde into acetic acid, it reaches a saturation point where some of it escapes
into the blood stream. The accumulation acetaldehyde exerts its toxic effects by
inhibiting the mitochondria reactions and functions. The alcoholic is a victim of a
vicious circle; a highacetaldehydelevel impairs mitochondria function and ceases
further liver damage hepatitis and cirrhosis (Lierber et al, 1994). Acetaldehyde in
the brain may inhibit enzymes designed to convert certain nerve transmitters from
aldehydes to acids. The nerve transmitters that accumulate may react with
acetaldehyde to from compounds which are similar to certain morphine type
compounds (Lieberet al., 1994).
NORMAL LIVER
The liver is the largest in the body, weighing 1.2 to 1.8kg in the adult. It occupies
the entire right upper quadrant of the abdomen, extending into the left upper
quadrant, in line with the nipple (Fawcett, 1994). In an anterior view, it is a
roughly triangular shaped organ and is covered by smooth glistening capsule. The
color is uniformly dark red to Maroon, with no little to no variation in color. The
gallbladder is attached to the inferiorsurface and be visible view along the lower
11
border. The liver is divided into the right robe and left lobe (Fawcett, 1994). The
liver is composed of lobules attached to each other by a scanty amount of
connective tissue; the connective tissue septa between the lobules holds branches
of the hepatics artery and the portal vein, as well as bile ducts. Each lobule of the
liver is a spherical structure, a few millimeters in diameter. The outline of these
lobules is extremely irregular in man, but is clearly demarcated and polygonal in
pigs (Gray, 1995).
CATEGORIES OF LIVER DAMAGE
Cholestasis is the result of damage to the bile ducts caused by diseases such as
primary biliary cirrhosis. (PBC) obstetric cholestasis (OC) and primary sclerosing
cholangitis (PSC). Obstruction of the common bile duct (Huff, 1984) the main
bileduct from the liver may result in jaundice, caused conditions such as gallstones
or a tumor infective damage such as hepatitis A, B, C, D, and E (Huff,1984).
Chemical damage such as poisoning and substance abuse – paracetamol
overdose, recreational drugs and alcohol. (Levin, 1995). Genetic or hereditary
damage such as Crigler – Najar Syndrome, Dubin-Johnson Syndrome,
haemochromatosis and Wilson’s disease. Vascular damage such as Budd-Chiari
syndrome (Levin, 1995).Autoimmune hepatitis such as PBC, PSC and
(AIH).Congental damage such as choledochal cyst, Caroli’s syndrome and
Gilbert’s syndrome.Metabolic damage ssuch as galactosaemia, fatty liver disease,
nonn alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis
(NASH) (Levin, 1995).
12
LIVER AND ITS MARKERS
Due to the widespread of hepatitis B virus (HBV) infections, hepatocellular
carcinoma (HCC), a liver cancer with low survival rates if not detected and treated
early (Jacobs et al, 2002). Liver cancer is usually a secondary cancer, caused by the
spread of tumor cells from elsewhere in the body. In liver cancer HCC manifests as
a primary cancer, which has been linked to hepatitis B and C infection and
cirrhosis. Noticeable symptoms do not usually appear until the cancer has
progressed, so it is rarely caught early, when intervention would be most effective
and survival rates are typically low (Jacobs et al. 2002). RASSFIA is a tumor
suppressing gene, in the blood of HCC patients. Healthy subjects show no signs of
the altered gene researchers have known that the DNA of HCC tumor cells lack a
functioning copy of RASSFIA (Jacobs et al, 2002). RASSFIA is hypermethylated
‘meaning the RASSFIA gene has been physically altered by cancer-related
processes that added clusters of carbon and hydrogen atoms, called methyl groups,
to portion of the DNA within the gene. Hypermethylation is epigenetic, the gene is
altered by environmental circumstances and is not inherent since cells’ protein
making system can’t access the hypermethylation effectively knocks out the tumor
– suppressing RASSFIA gene, which is then unable to stop cells from becoming
cancerous.
\
13
ALANINE TRANSAMINASE OR SERUM GLUTAMIC
PYRUVATE TRANSMINASE (SGPT)
It is an enzyme present in hepatocytes (liver cells). When a cell is damaged, it
leaks this enzyme into the blood, where it is measured. ALT rises dramatically in
acute liver damage, such as viral hepatitis or paracetamol (acetaminophen)
overdose. Elevations are often measured in multiples of the upper limit of normal
(ULN) (Banerjee et al, 1986).
ALKALINE PHOSPHATASE (ALP)
Alkaline phosphatase (ALP) is an enzyme in the cells living ducys of the liver.
ALP levels in plasma will rise with rise with large bile duct obstruction, intra
hepatic cholestasis or infiltrative disease of the liver. ALP is also present in bone
and placental tissues, so it is higher in growing children (as their bones are being
remodeled) and elderly patients with Paget’s disease (Banerjee et al., 1986).
14
AIMS AND OBJECTIVES
Aims:
To study the extent of damage of the liver through the assessment of liver enzyme
activity and other induces in the serum of experimental animals in other to monitor
the effects of ethanolalone and that of ethanol and artesunate solution on the liver.
Objectives:
- To monitor the effect of artesunate and ethanol on the liver.
- Compare parameters between cases and control serum; that is to know
whether there are any significant differences between the cases and the
control.
15
CHAPTER TWO
2.0 MATERIAL AND METHOD
2.1 MATERIALS
2.1.1
CHEMICALS AND REAGENT
Absolute Ethanol, Artesunate, and Distilled Water.
2.1.2
EXPERIMENTAL ANIMALS
The Rats were all purchased from the animal house, department of
pharmacology, University of Jos, Plateau State, Nigeria. They were kept in the
quarantine room and were fed with standard feed from Grand Cereals and Oil Mills
(GCOML), Bukuru Jos, Plateau State, Nigeria. They were also given water in a
plastic bottle for two weeks to attain a very good body weight once in every 24
hours.
2.1.3
APPARATUS
A digital Adventurer Ohaus weighing balance with sensitivity of 0.000
and model number: ARC 120, centrifuge tubes, syringes and needles, Pasteur
pipettes, test tube racks, specimen bottles (plane, free of anti-coagulant),
refrigerator, cotton wool, cannula, conical flask (250 ml), measuring cylinder,
beaker (250 ml), glass rod, mortar and pestle, spatula, tray, razor blade and pins.
16
2.1.4 METHODS
ANIMAL TREATMENT
The rats were induced for 7 and 21 days with solution of artesunate,
distilled water and ethanol and ethanol. At the end of the seven (7) days and
fourteen (14) days of the treatment, anesthesia was induced using chloroform to
anaesthetize them, prior to sacrifice; they were then sacrificed. Blood samples were
collected by direct cardiac puncture andthen transferred into sample bottles free of
anti-coagulant.
RAT GROUPING
The rats were grouped into four (4), from group I – IV of four (4) rats per group,
carrying different color code marks on the head, tail, back, hind and limbs. The
color code markings were made on the whole of the rats grouped both with the
color.
PREPARATION OF SOLUTION
0.57g of Artesunate was weighed and dissolved into a conical flask containing
3.04% v/v of ethanol and was made up to 100ml with distill water and was poured
into a clean grease free container well labeled
2.5% v/v (control 1) of ethanol
METHOD OF PREPARATION
0.57g of artesunate was weighed using an Adventurer Ohaus digital weighing
balance and was dissolved into 3.04% v/v of ethanol measured using a calibrated
1ml micropipette and a distilled water (96.6ml) was also measured using
17
measuring cylinder in order to make it up to 100ml, mix and allowed to stand for
72 hours in order to turn into solutions.
3.04% v/v ethanol was measured using 1ml micropipette and 96.6ml of distilled
water measured using measuring cylinder to make it up to 100ml and was poured
into a conical flask, shake and then transferred into a labeled container as control 1.
2.5%v/v of ethanol was measured using 1ml micropipette and 97.5ml of distilled
water was measured using measuring cylinder to make up to 100ml and was
poured into a conical flask, mix and then transferred into a well labeled container
as control 2.
Administration (4 tablets dosage 20mg/120mg, body weight – 35kg & above. The
weights of the experimental rats were used to calculate the amount of dosage
administered, to the experimental animals. The drug solution was administered to
the animals by oral compulsion for a period of 7 and 14 days.
DRUGS
The artesunate tablets were bought from La Med Pharmacy Jos, Plateau State and
is manufactured by CIPLA Limited, MIDC patalganga M.S. 410 220 INDIA. The
drug solution was made with distilled water and ethanol and was administered to
the animals by oral compulsion for period of 7 and 14 days.
18
EXPERIMENTAL DESIGN
The experimental animals were divided into 4 group of 4 albino wistar rat each.
Group 1-2 were the treatment group, while the group 3 & 4 was the control group.
The drug was administered to the group as follows; The group 1: 2.0mg/kg
Artesunate in solution per body weight for seven (7) days. Group 2: 2.2mg/kg
Artesunate in solution per body weight for 14 days. Control 1: 3.04% v/v
suspension of the drug solution for 7 and 14 days. Control 2: 2.5% v/v of
suspension of the drug for 7 and 24 days sacrificed 24 hours after the last dose on
the 7th and 14th day. Milliters of the blood sample was collected by direct cardiac
puncture into a plane sample bottle free of anti-coagulant and centrifuged at
12.000rpm for five (5) minutes using centrifuge machine and the serum was
separated for analysis.
ANALYTICAL METHODS
Estimation of Alanine aminotransferase (ALT) activities were done using,
Estimation of Alkaline phosphatase (ALP) using and total protein activities using
method.
2.1.5 PRACTICALS/TESTS
LIVER FUNCTION TEST (LFT)
This is the test which is used to assay and tell the condition or state of the liver. As
earlier discussed, the liver is seen as the main site of metabolism of xenobiotic
19
which may include drugs and toxicants. The test comprises of the following;
Alanine amino transferase (ALT), Total protein, and Alkaline phosphatase (ALP).
ALKALINE PHOSPHATASE (ALP)
This was analyzed using
. This assay operates via or with respect to Beer
lamber law. The alkaline phosphatase acts upon the AMP – buffed sodium
thymolphthalein monophosphate. The addition of an alkaline reagent stops enzyme
activity and simultaneously develops a blue chromogen, which is measure
photometrically. The results are automatically printed out from the machine.
TOTAL PROTEIN
Protein forms a purple colored complex with cupric ions in alkaline solution. The
reaction takes its name from the simple compound biuret which reacts in the same
way. The intensity of the purple color is measured at 540nm/yellow green filter
and compared with a standard serum of known protein concentration and can be
determined photometrically.
ALANINE AMINO TRANSFERASE (ALT)
The enzymatic reaction sequence employed in the assay of ALT is as follows:
L – Alanine + 2 – oxoghutarate → pyruvate + L – Glutamate.
Pyruvate + NADH + H+ → Lactate + NAD+ +H2O.
20
The pyruvate formed in the first reaction is reduced to lactate in the presence of
lactate dehydrogenase and NADH. The activity of ALT determined by measuring
the rate of oxidation of NADH at 340nm. Endogenous sample pyruvate is
converted to lactate by LDH during the lag phase prior to measurement.
21
CHAPTER THREE
ALT
Paired Samples Statistics
Std.
Std.
N
Deviation
Mean
Pair 1 control 81.3333 3
14.57166
8.41295
sample 120.666 3
35.90729
20.73108
Pair 2 control 81.3333 3
14.57166
8.41295
sample2 115.000 3
25.51470
14.73092
7.78888
3.89444
Mean
Error
7
0
Pair 3 controlo 90.0000 4
2
sample 232.000 4
224.58851 112.29426
0
Pair 4 controlo 90.0000 4
7.78888
3.89444
21.31314
10.65657
2
sample2 112.750 4
0
22
Paired Samples Correlations
Correla
Pair 1 Control
(3.04%v/v)
N
tion
Sig.
13
.550
.629
.951
.201
&
sample (7 days)
Pair 2 control
(3.04%v/v)
13
&
sample 2 (14 days)
Pair 3 Control 2 (2.5%v/v) 4
-.209 .791
& sample (7 days)
Pair 4 Control 2 (2.5%v/v) 4
-.888 .112
& sample 2 (14
days)
ALP
23
Paired Samples Statistics
Std.
Pair 1
Pair 2
Pair 3
Pair 4
Mean
N
Std. Deviation Mean
Control
157.6667
3
80.64945
46.56298
Sample
95.6667
3
17.78576
10.26861
Control
157.6667
3
80.64945
46.56298
sample2
164.0000
3
22.60531
13.05118
control2
117.5000
4
59.23119
29.61559
Sample
91.0000
4
17.26268
8.63134
control2
117.5000
4
59.23119
29.61559
sample2
180.2500
4
37.37535
18.68767
Error
Paired Samples Correlations
N
Correlation
Sig.
Pair 1
control & sample
3
-.999
.027
Pair 2
control & sample2
3
-.132
.916
Pair 3
control2 & sample
4
-.331
.669
Pair 4
control2 & sample2
4
.214
.786
N
Std. Deviation Std. Error Mean
TOTAL PROTEIN
Paired Samples Statistics
Mean
24
Pair 1
Pair 2
Pair 3
Pair 4
Control
63.6667
3
9.29157
5.36449
Sample
70.6667
3
5.77350
3.33333
Control
63.6667
3
9.29157
5.36449
sample2
68.6667
3
4.61880
2.66667
control2
62.7500
4
2.21736
1.10868
Sample
69.0000
4
5.77350
2.88675
control2
62.7500
4
2.21736
1.10868
sample2
67.5000
4
4.43471
2.21736
Paired Samples Correlations
N
Correlation
Sig.
Pair 1
control & sample
3
.994
.069
Pair 2
control & sample2
3
.590
.598
Pair 3
control2 & sample
4
.651
.349
Pair 4
control2 & sample2
4
-.153
.847
25
CHAPTER FOUR
DISCUSSION CONCLUSION RECOMMENDATION
4.1 Discussion:
artesunate comes in 50mg tablets. The commonly used effective adult dose in Nigeria is
6.8mg/kg in three divided daily doses. This informed the use of grade daily doses of 2.02.2mg/kg body weight in the experimental rats exposed to ethanol. This dose range gave the
opportunity of studying the effect of the drug solution. The administration of 2.0-2.2 mg/kg of
artesunate in alcoholic solution caused increase in the activities of alanine aminotransferase
(ALT) and alkaline phosphatase (ALP) (P<0.05) when compared with control. The significant
increase in the mean value of the serum hepato specific markers are the other dose level
(p>0.05) when compared withy the control. The current investigation or study suggests toxicity
of the liver cells of the experimental animal upon artesunate in alcoholic solution administration.
The findings in this study agree with the work of Ngokere et al (2004), in which artesunate
administration caused significant increase in the liver marker enzymes in rat. They also agree
with woodrow et al, (2005) a transient rise in the liver transaminases. The results are also in
agreement withy other result. The liver cell damage may have been caused by free radicals
generate by artesunate in alcoholic solution, which are also responsible for their anti malaria
actions. The deleterious effects were considered to be caused by free radicals produced during
peroxide formation. The level of hydroxyl and peroxide radicals induced by artesunate
treatments may be responsible for the hepatotoxicity to the experimental animal.
4.2 CONCLUSION:
The results of the experiment indicated that artesunate solution in ethanol administered to the
experimental animals caused a significant increase in hepatic biochemical parameters analyzed
for these work. This shows that the treatment with artesunate in ethanol solution. Some level
of damage to the organs and consequently their normal functions which also affect other
organs of the body leading to opportunity infections and death. Therefore artesunate in alcoholic
solution indicate more damage to hepatic biochemical parameters.
4.3 RECOMMENDATION
26
Based on the experiment conducted and the findings, artesunate oral intake or injection in
the presence of ethanol consumption poses damage to health. One should avoid the consumption
of ethanol and artesunate at the same time to prevents liver damage .further investigations
should be employed to known other specific organs which also can be harmed by artesunate in
alcoholic solution.
27
REFERENCE
Abittan, C.S
and Lieber , C.S. (1999). Pharmacology and Metabolism of alcohol, Including
its metabolic effects and interactions with other drugs. Clin Dermatol. 17.365-379
Adjuik. M., Agnamey, P. Rabiker, A. et al. (2002). “Amodiaquine –artesumate versus
amodiacuire for uncomplicated plasmodium falciparum malaria in African children: a
randomized, multi centre trial”. Lancet 359(9315): 1365-72.
Adjuk. M., Agnamey, P., Babikar, A. et al. (2004). “Artesumate combination for treatment of
malaria: Meta-analysis, 363:9-17.
Barradell. L.B., Ditton, A. Artesumate (1995). A review of its pharmacology and therapeutic
efficacy in treatment of malaria drugs. 50: 714-41.
Boulangier. D., Dieng. Y., Cisse. B., et al. (2007). Anti-schistosomal efficacy of artesunate
combination therapies administered as curative treatments for malaria attacks. Trans R
SOC Trop. Med. Hyg. 101 (2):113-16.
Boggan, B. (2003). Metabolism of Ethylalcohol
in the body. Lecture notes and
seminar 1-2pp.
De La maza, M.P, petermann ,M .M, Bunoutt D, and Hirsch , S. (1995). Effectsof long term
Vitamin E supplementation in alcoholic cirrhotics. Journal of the American college of
nutrition 14, 192.126.
Demetrious. J.A.., et al. (1995). Enzymes in clinical chemistry principles and technics, 2nd ed.
Hagerstown (MD), Harper and Row . 927.
Davies T.M.E. , Karunajeewa H.A, Ilett K.F (2005) Artemisinin based combination therapies
for uncomplicated malaria .Med J Aust , 182:181-5
Henry. J.B.(1984). Clinical diagnosis and management by laboratory methods, 17th ed. WB
Saunders and Co,P. 1437.
28
International Federation of Clinical chemistry, (1980). J. clin. Chem. clin. Bio 18: 5231.
Kochmar, J.F., Moss, D.W. and Tietz, N.W. (1976). Fundamentals of clinical chemistry, (ed), P.
604, W.B. Saunders and company, Philadelphia, PA.
Kochmar, J.F., Moss, D.W. and Tietz, N.W. (1976). Fundamentals of clinical chemistry, (ed), P.
604, W.B. Saunders and company, Philadelphia, PA.
South East Asian Quinine Artesunate Malaria Trial (SEAQUAMAT) (2005). “Artesmate versus
Quinine for treatment of severe falciparum malaria: a randomized trials” the Lancet 366
(9487): 7171-725.
Svensson, U.S, Ashton. M. (1999). Identification of the human cytochrome p450 enzymes
involved in the in vitro metabolism of artemisinin. Br. J. Clin. Pharmacol. 48: 528-35.
Lieber , C. S letevre, A. Spritz,
N, et al (1999). Difference in
metabolism of long and medium chain
acids
hepatic
fatty acids. The role of fatty
chain length in the production of the alcoholic fatty liver. Journal Clinical
Investigation 46:1451-460.
Li XQ,
Bjor kmar A, Andersson TB , Gustafsson LL, Masimirembwa C.M., (2003)
Identification of human cytochrome p450s that metabolise anti parasitic and drugs and
predictions of in vivo drug hepatic clearance from in vitro data. Eur J Clin pharmacol
59:429-42.
Malloy , E. Evelyn, K. (1932). Colorimetric method for the determination of serum Oxaloacetic
and glutamic pyruvate transaminase. Am Clin pathol . 28 :56-63.
Meshaick, S.R. (2002). Artemisinin mechanisms of action, resistance and toxicity Int. J.
Parasitol, 32: 1655-60.
MC Murry .J. (2004).Organic chemistry 6th edition (United states : Thompsm), 587-854.
Meremikwu. M., Alaribe. A., Ejemot, R., et al. (2006). Artemether –lumefantrine
versus artesunate plus amodiaquine for treating uncomplicated childhood malaria in Nigeria
randomized controlled trial” Malary J. 5:43.
29
Ngokere , A .A, Ngokere T.C Ikwudinma, A.P. (2004). Acute study of histomorphological and
Biochemical changes caused by Artesunate in visceral organs of the Rat, Journal of
Experimental and Clinical Anatomy, 32 (2):11-16
Price , R.N (1999). Adverse effects in patients with acute falciparum malaria treated with
artemisinin derivatives. American Journal ,of tropical medicine and hygiene. 60 (4): 547555.
Petras J.M, Young G.D, Bauman R.A et al. (2000). Arteether- induced brain injury in Macaca
mulatta. I . The precerebellar
nuclei, paramedian reticular nuclei, and Perihypoglossal
nuclei . Anat Embryol , 201: 383-97
Quighaosu Anti Malaria
Coordinating
Research Group (1979). Anti Malaria Studies in
Quighaosu. chinese medical journal (England) , 92.811-816
Toovey. S. (2006) are currently deployed artemisinin neurotoxcity? Toxicol lett. 166: 95-104.
Teja-Isava harm. P, Watt. G., Eamsila. C. et al. (2001). Comparative pharmakinetics and effects
kinetics of orally administered arteunate of healthy volunteers and patients with
uncomplicated falciparum malaria. Am. J. Trop. Med. Hyg. 65:717-21.
Tietz. N. (1976). Fundamentals of clinical chemistry 602-609.
Tu, Y. (1999). The development of new anti-material drugs. ginighaosu and dihydroginghaosu.
Clin. Med. J. (Engl) 112:976-9.
Utzinger. J., Xiao, S.H., Yenner, M, Keiser, J. (2001) Artemisinins for schistosomiasis and
beyond. Curr opin investig drugs; 18:105-16.
WHO (2000) Management of Severe Malaria. A. Practical hand book.
White , N .j (1999). Assessment of the pharmacodynamic properties of Anti- malaria drugs in
vivo . Anti- microbial Agents for chemothereapy 41:143-1422.
30
White ,N. (1994). Clinical Pharmacokinetice and pharamacodynamic of artesunate derivatives.
Transaction of the Royal society of tropical Medicine and hygiene, (suppl1) S41-S43
Woodrow , C.J., Haynes,
R.K ., Krishna, S. (2005). Artemisinin Mechanism of action. Post
Graduate Medical Journal, 81 ( 952): 71-78.
WHO (1994-1995). The role of Artemisinin and its derivatives in the current
treatment of
malaria (WHO/MAL/ 94.1076) WHO, geneva.
Widmann, F.K. (1980). Clinical interpretation of laboratory test
(9th edition). American
Association of publishers, Washington Dc.pp: 293-295
Young, D.S. (1990). Effects of drugs on clinical
Washington D.C.
Young, D.S., (1975). Clin. Chem 21:5
31
laboratory tests. AACC press,
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