ABSTRACT
Malaria
is a significant public health problem in the world especially in in
sub-Saharan Africa. One of the key contributory factors to the development and
progression of malaria and its complications is oxidative stress, a condition
characterized by increased production of free radicals or impaired antioxidant
defence system. Glucose-6-phosphate dehydrogenase (G-6-PD) produces NADPH which
intends regenerate reduced glutathione (GSH), an antioxidant which helps in the
removal of free radicals thereby preventing oxidative stress, Hence, this study was aimed to investigate
the relationship between G-6-PD, oxidative stress and malaria infection
inpatients visiting ESUT Teaching Hospital Parklane, Enugu. The recruited patientsfollowing
their informed consent were screened for malaria by RDT and Microscopy methods
and their baseline parameters including age, gender, etc. were obtained using a
questionnaire. Whole blood was collected and used for the determination of
malaria infection, oxidative stress, lipid peroxidation and protein oxidation,
anaemia and as well as the G-6-PD status in patients. A total of 101 patients were recruited
including 30 male and 71 female among which 86 had malaria positive while 15
tested malaria negative. Comparing RDT and Microscopy techniques in diagnosing
Malaria, showed RDT to have a low performance in in diagnosing malaria using microscopy as
standardwith a sensitivity of 10.47% and
of accuracy 23.76%. All the baseline characteristics of study
participants was not significantly different (p = 0.946) among the malaria and
non- malaria patients. Among the G6PD deficient patients,17.9%were found to be anaemicwhile 13.1% were non-anaemic whereas among the
non-deficient patients, 39.3% were anaemic while 29.8% were non-anaemic. As such,
there was no significant relationship (p
= 0.946) betweenG6PD
deficiency and among the malaria
patients.Comparison of anaemia and oxidative stress
indices among malaria patients showed significantly (p<0.05) low level of haemoglobin and haematocrit concentrations,
but there no significant difference (p>0.05) of MDA and protein oxidation
level between anemic and non-anaemic patients with malaria. Interaction between anaemia and G6PD deficiency on
study parameters, showed no significant (p<0.05) relationship on haemoglobin,
haematocrit, MDA and protein oxidation level in malaria patients. In
conclusion, this study showed the no association between anaemia, oxidative
stress and G-6-PD deficiencyamong malaria patients. Further studies are needed
to ascertain these findings as oxidative stress is implicated in the
pathogenesis of malaria.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of study
Malaria
is a mosquito-borne infectious disease affecting humans and other animals
caused by parasitic protozoans (a group of single-celled microorganisms)
belonging to the Plasmodium type
(WHO, 2014). According to the World Health Organization (WHO), malaria is a
significant public health problem in more than 100 countries and causes an
estimated 200 million infections each year, with more than 500 thousand deaths
annually. Over 90% of these deaths occur in sub-Saharan Africa, where the
disease is estimated to kill one child every 30 seconds (WHO, 2011). In other
areas of the world, malaria causes substantial morbidity, especially in the
rural areas of some countries in Asia and South America. Malaria causes symptoms that typically
include fever, tiredness, vomiting, and headaches. In severe cases it can cause
yellow skin, seizures, coma, or death (Caraballo, 2014). Symptoms usually begin
ten to fifteen days after being bitten, If not properly treated, people may
have recurrences of the disease months later. The disease is most commonly
transmitted by an infected female Anopheles mosquito. The mosquito bite
introduces the parasites from the mosquito's saliva into a person's blood (WHO,
2014). The parasites travel to the liver where they mature and reproduce. Five
species of Plasmodium can infect and
be spread by humans. (Caraballo, 2014). Most deaths are caused by Plasmodium falciparum.
The role of oxidative stress during malaria
infection is still unclear. Some authors suggest a protective role, whereas
others claim a relation to the physiopathology of the disease (Sohail et al., 2007).
However, recent studies suggest that the generation of reactive oxygen and
nitrogen species (ROS and RNS) associated with oxidative stress, plays a
crucial role in the development of systemic complications caused by malaria.
Malaria infection induces the generation of hydroxyl radicals (OH•)
in the liver, which most probably is the main reason for the induction of
oxidative stress and apoptosis (Guha et
al., 2006). Additionally, Atamna et
al. (1993) observed that erythrocytes infected with P. falciparum
produced OH• radicals and H2O2 about twice as
much compared to normal erythrocytes. Higher level of this free radicals can
lead to oxidative stress.
Oxidative
stress, termed as an imbalance between production and elimination of reactive
oxygen species (ROS) leading to plural oxidative modifications of basic and
regulatory processes, can be caused in different ways. Increased steady-state
ROS levels can be promoted by drug metabolism, over-expression of ROS-producing
enzymes, or ionizing radiation, as well as due to deficiency of antioxidant
enzymes. The consequence of oxidative stress once it is high, it can cause
damage to the brain, metabolic disorders affecting electron transport chain.
Reactive oxygen species (ROS), generated by endogenous and exogenous sources,
cause significant damage to macromolecules, including DNA (Salmon et al., 2004).
Furthermore,
Spermatozoa are highly vulnerable to oxidative attack because they lack
significant antioxidant protection due to the limited volume and restricted
distribution of cytoplasmic space in which to house an appropriate armoury of
defensive enzymes. In particular, sperm membrane lipids are susceptible to
oxidative stress because they abound in significant amounts of polyunsaturated
fatty acids. Susceptibility to oxidative attack is further exacerbated by the
fact that these cells actively generate reactive oxygen species (ROS) in order
to drive the increase in tyrosine phosphorylation associated with sperm
capacitation. However, this positive role for ROS is reversed when spermatozoa
are stressed. Under these conditions, they default to an intrinsic apoptotic pathway
characterised by mitochondrial ROS generation, loss of mitochondrial membrane
potential, caspase activation, phosphatidylserine exposure and oxidative DNA
damage. In responding to oxidative stress, spermatozoa only possess the first
enzyme in the base excision repair pathway, 8-oxoguanine DNA glycosylase. This
enzyme catalyses the formation of abasic sites, thereby destabilising the DNA
backbone and generating strand breaks. Because oxidative damage to sperm DNA is
associated with both miscarriage and developmental abnormalities in the
offspring, strategies for the amelioration of such stress, including the
development of effective antioxidant formulations, are becoming increasingly
urgent (Aitken et at., 2016).
The
process of lipid peroxidation involves a complex chain reaction utilizing the
interaction of oxygen-derived species with polyunsaturated fatty acids (e.g.
docosahexaenoic acid, linoleic acid and arachidonic acid), resulting in highly
reactive electrophilic aldehydes and free radicals (Esterbauer et al., 1991). This process is extremely
detrimental to cellular functions as it disrupts membrane integrity, fluidity
and function (Esterbauer et al.,
1991). Lipid peroxidation is a self-propagating process involving initiation
and propagation steps which continue through an ongoing free radical chain
reaction until termination occurs. The retina is particularly prone to lipid
peroxidation since it is highly enriched in polyunsaturated fatty acids (PUFAs)
(Catalase). The predominant PUFA in photoreceptor outer segments is
docosahexanoic acid which is the most unsaturated fatty acid in the body. Lifelong accumulation of chronic oxidative
damage will lead to dysfunction in retinal cells and increase their susceptibility
to exogenous and endogenous insults eventually culminating in loss of visual
function and cell death (Esterbauer et al.,1991).
Malaria infection has been found to be associated with lipid peroxidation
accompanying reduction in antioxidant capacity of the infected patients
especially Plasmodium falciparum infection. Instantaneous reduction in
antioxidant potency in tandem with increased lipid peroxidation is also
observed to be equally accountable for development of oxidative stress in
malaria patients (Das
and Nanda, 1999; Upadhyay
et al., 2011; Egwunyenga
et al., 2004). Any infection,
including malaria, activates the immune system of body thereby causing release of reactive oxygen species
as an antimicrobial action (Kulkarni
et al., 2003). In addition to
host’s immune system, malaria parasite also stimulates certain cells in
production of reactive oxygen species
thereby resulting in hemoglobin degradation (Loria
et al., 1999; Pradines
et al., 2005). One of the major
reasons for development of malarial anemia seems to be oxidative stress (Das
and Nanda, 1999; Kremsner
et al., 2000) while changes in
micronutrient metabolism alter disease progression and severity (Singotamu
et al., 2006).
Proteins
are the largest constituent of the cellular milieu and are frequent targets of
oxidative damage (Stadtman, 2004). Protein oxidation can involve direct
reaction with amino acids, cleavage of the polypeptide chain, and conversion of
the protein to derivatives that are highly sensitive to proteolytic
degradation. It has also been established that all of these protein
modifications can be mediated by metal-catalyzed oxidation systems. All amino
acid residues of proteins are potential targets for oxidation by HO· or by H2O2
in the presence of metal ions. For example, oxidation of tyrosine residues is
damaging to the red blood cells, as this amino acid is converted to a
3,4-dihydroxyphenylanine derivative, which itself can undergo redox cycling to
generate further ROS (Sugiura and Ichinose, 2011).
Antioxidants
are molecules that inhibit or quench free radical reactions and delay or
inhibit cellular damage (Young et al.,
2001). Though the antioxidant defenses are different from species to species,
the presence of the antioxidant defense is universal. Antioxidants exists both
in enzymatic and non-enzymatic forms in the intracellular and extracellular
environment. . Enzymatic antioxidants work by breaking down and removing free
radicals. The antioxidant enzymes convert dangerous oxidative products to
hydrogen peroxide (H2O2) and then to water, in a
multi-step process in presence of cofactors such as copper, zinc, manganese,
and iron. Non-enzymatic antioxidants work by interrupting free radical chain
reactions (Young et al.,2001).
The
antioxidants can also be categorized according to their size, the
small-molecule antioxidants and large-molecule antioxidants. The small-molecule
antioxidants neutralize the ROS in a process called radical scavenging and
carry them away.
Glucose-6-phosphate
dehydrogenase (G6PD) deficiency is the most common enzymopathological disease
in humans. This disease is described as a widespread, heritable, X-chromosome
linked abnormality (Reclose et al.,
2000). It is estimated that it affects approximately 400 million people
worldwide (Daloii et al., 2004). This
disease is seen most frequently in approximately all of Africa, Asia, and the
countries near the mediterranean Sea (Frank, 2005). G6PD enzyme was
demonstrated to play an active role in survival of erythrocytes. It is known
that in the pentose phosphate pathway of erythrocytes, glucose-6 phosphate
dehydrogenase (G6PD) enzyme provides the production of NADPH and Glutathione (GSH). GSH, produced by pentose
phosphate pathway can react with H2O2 and reduce it to H2O. This prevents the generation of oxidative
stress within red blood cells; oxidative stress can be induced in erythrocytes
whose G6PD enzymes are deficient. In this situation, GSH is not produced and H2O2
is not reduced to H2O, leading to oxidative stress and hemolysis.
1.2 Justification /
Need for study
The
need for this study is to carefully ascertain whether G6pD deficiency has an
impact on oxidative stress and malaria infection. To known if G6pD can promote
malaria infection in patient. This study is equally designed to give
information about the prevalence of G6PD and malaria infection among malaria
patient in Enugu metropolis.
1.3 Statement of the
problem
So
many reasons have been attributed to susceptibility of G-6-PD deficient
patients to malaria infection. Recall that the pentose phosphate pathway is
essential in producing enough NADPH capable of reducing oxidized glutathione
but when there is a default in the production of enough NADPH from PPP as the
case may be in G-6-PD deficient patients, leading to a decrease in the level of
reduced glutathione which leads to an increased in oxidative stress – a
possible risk factor in the development of malaria infection will be
complicated.
1.4 Aim of the study
This
study was undertaken to evaluate the relationship between oxidative stress among
malaria patients visiting Enugu State University Teaching Hospital, Parklane,
Enugu Nigeria.
1.5 Objective of the study
1.
To screen patients for malaria infection.
2.
To assess anaemia in malaria patients.
3.
To assess G6PD deficiency in malaria patients.
4.
To assess oxidative stress indices (lipid peroxidation and protein oxidation)
in malaria patients.
1.6 Limitations of the
study
Recruitment
of participants into the study.
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