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Research Article
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Oxidative Stress and Micronutrient Therapy in Malaria: An In vivo Study in Plasmodium berghei Infected Mice |
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O.I. Iribhogbe,
E.O. Agbaje,
I.A. Oreagba,
O.O. Aina
and
A.D. Ota
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ABSTRACT
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Free radical production from oxidative stress induced by malaria infection plays a major role in the pathogenesis of malaria. However, the use of agents with antioxidant activity may interfere with malaria progression. The study involves an in vivo evaluation of the role of some antioxidant micronutrients in the modulation of malaria infection. Rodent malaria model using Plasmodium berghei NK-65 strain (chloroquine sensitive) was used for the study. Fourty five mice of either sex weighing 20.05±0.02 g were procured for the study. Fourty mice were inoculated intraperitoneally with 1x107 million Plasmodium berghei infected erythrocyte and were administered with 0.2 mL of distilled water, 0.2 mL of vehicle; Tween 80 (control and vehicle group), chloroquine 25 mg kg-1 and artesunate 4 mg kg-1 (standard drug group), vitamin A 60 mg kg-1, vitamin E 100 mg kg-1, selenium 1 mg kg-1, zinc 100 mg kg-1 (test group F, G, H and I, respectively) 72 hours post inoculation. Antioxidant micronutrients demonstrated significant (p<0.05) schizonticidal activity when compared with negative control during the 4 day curative test. Erythrocyte membrane distability was most markedly elevated in the tween 80 group (426.15%), followed closely by the chloroquine (373.85%) treated group and artesunate group (329.23%) and least in the zinc treated group (32.31%). There was no significant (p>0.05) difference in MCFI values (0.115±0.002; 0.114±0.002 g dL-1) between vitamin A treated group and selenium treated group respectively. However, this was significant (p<0.05) between the micronutrient treated groups and the control (negative, positive and vehicle). Conclusively, antioxidant micronutrients have antimalarial activity which may be due potentiation of erythrocyte membrane stabilization.
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Received: December 07, 2012;
Accepted: February 12, 2013;
Published: March 16, 2013
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INTRODUCTION
Micronutrients are known to be an integral part of endogenous antioxidants
and are known to influence malaria progression in man. A randomized trial in
Papua New Guinea has shown that periodic supplementation with vitamin A reduced
the incidence of febrile episodes and parasitaemia due to Plasmodium falciparum
(Hussey and Clements, 1996; Shankar
et al, 2000). Also, vitamin A is essential for normal immune function
and has been shown to influence both antibody response and cell-mediated immunity
(Semba, 1998). Other studies have documented that antioxidants
such as carotenoids, vitamins C and E could provide protection against oxidative
stress induced by malaria infection (Adelekan et al.,
1997). Zinc deficiency has been observed to decreases the ability of the
body to respond to infection, affecting both cell mediated immune responses
as well as humoral immune responses (Okochi and Okpuzor,
2005). In another study, the morbidity and outcome of avian malaria infection
with Plasmodium spartani was more severe in ducklings fed with vitamin
E and selenium deficient diets than in ducklings fed with vitamin E and selenium
adequate diets (Yarrington et al., 1973). Free
radicals produced from oxidative stress are aggravated in malarial infection
which leads to decrease in the antioxidant defense system. Consequently, oxidative
stress in malaria infection can result in the development of malarial anemia
(Kremsner et al., 2000; Clark
and Hunt, 1983). Hence, there is need to establish the mechanism that underlies
the antimalarial activity of antioxidant micronutrients as well as determine
its role in malarial induced oxidative stress.
MATERIALS AND METHODS Materials: Chemicals and equipments: Heparinised capillary tubes, Light Microscope (Olympus, Japan), EDTA bottles, Feeding trochars, Syringes (1 mL, 5 mL), Cotton wool, Microscopic slides (Olympus, China), Hand gloves, Giemsa stain (Sigma), 98% Methanol (Sigma) and Tween 80 (sigma). Drugs: Vitamin A (Clarion Medical Pharmaceuticals, Nigeria), Vitamin E (Clarion Medical Pharmaceuticals, Nigeria), Zinc gluconate (Mason Vitamins Incorporated USA), Selenium-organic (Mason Vitamins Incorporated USA), Chloroquine (Emzor Pharmaceuticals, Nigeria) and Artesunate (Emzor Pharmaceuticals, Nigeria).
Preparation of animals: Fourty five in bred and pure Swiss albino mice
of either sex weighing between 18- 25 g was used for the study. They were obtained
from the animal house of the Nigerian Institute of Medical Research, Yaba Lagos
State and housed in stainless steel cages with wire screen top. The animals
were about 7-8 weeks old and were maintained on commercial feeds (Vital feeds,
Jos) and tap water ad libitum for the entire duration of the study. The
mice were allowed to acclimatize for 1 week in the laboratory environment under
a controlled temperature of 20°C and at optimum humidity before being subjected
to the experiment (Obernier and Baldwin, 2007). Good
hygiene was maintained by constant cleaning and removal of faeces and spilled
feeds from the cages daily.
Preparation of Inoculum of Chloroquine Sensitive Strain of Plasmodium
berghei: Plasmodium berghei NK 65 strain maintained in the laboratory
of Nigerian Institute of Medical Research, Yaba by serial blood passage from
mouse to mouse was used for the study. Donor mouse with a rising parasitaemia
of 20-30% confirmed by thin and thick blood film microscopy was used. Blood
(0.2 mL) was collected in a heparinized tube from the auxiliary plexus of veins
in the donor mouse using heparinized capillary tubes. The blood was diluted
with 5 mL of Phosphate Buffer Solution (PBS) pH 7.2 so that each 0.2 mL contained
approximately 1x107 infected red cells (Peter
et al., 1975; Fidock et al., 2004).
Each animal received inocula of about 10 million parasites per kilogram body
weight, which is expected to produce a steadily rising infection in mice.
Preparation of drugs
Chloroquine: Fifty milligram of powdered chloroquine sulphate were
dissolved in 20 mL of distilled water. So that 1 mL will contain 2.5 mg of chloroquine
sulphate. Dosage administered to the animals in the standard drug group (A)
was 25 mg kg-1. Hence the 0.2 mL of solution administered contained
0.5 mg of chloroquine sulphate.
Artesunate: Four milligram of artesunate powder was dissolved in 10 mL of distilled water. Hence, 1 mL of distilled water contained 0.4 mg of artesunate. Dosage administered was 4 mg kg-1 equivalent to 0.2 mL of solution. Vitamin A: Two hundred thousand International Units of vitamin A caplet, which is equivalent to 60 mg of vitamin A, was used to prepare the dose administered (60 mg kg-1). The drug was dissolved in 0.2 mL of Tween 80 used as a vehicle and distilled water in a ratio of 0.2:0.2:9.6 to make up a total volume of 10 mL. The final volume of drug administered was 0.2 mL, which is equivalent to 0.495 mg of vitamin A. Vitamin E: One hundred milligram of vitamin E caplet was dissolved in 0.2 mL of Tween 80 and distilled water in a ratio of 0.2:0.2:9.6 making up a total volume of 10 mL. The dose administered to the animal was 100 mg kg-1. Hence, the final volume of drug administered to the animal was 0.2 mL, which is equivalent to 1.6 mg of vitamin E. Selenium: One milligram of selenium was dissolved in 10 mL of distilled water in its powdered form. A dose of 1 mg kg-1 b.wt. was administered to the animals. The final volume of drug administered was 0.2 mL equivalent to 0.0145 mg of selenium. Zinc: The dose of zinc administered was 100 mg kg-1. 100 mg of zinc was dissolved in 10 mL of distilled water in its powdered form. 0.2 mL of the solution was administered which is equivalent to 1.91 mg of zinc.
Drugs/micronutrient administration: A 4-day curative test was performed
using the methods of Peters, (1965). Peter
et al. (1975) and Fidock et al. (2004).
Mice were grouped into nine groups of 5 and drug/micronutrient administration
was done daily for 4 days as shown in Table 1. Antioxidant
micronutrients were administered orally using doses based on LD50
values as reported by Schrauzer (2000), Oncu
et al. (2002) and Oreagba and Ashorobi (2006),
while the standard dose of chloroquine and artesunate were used.
At the end of the 4 day curative treatment (day 5 post treatment) blood samples
were collected via the auxiliary vein into EDTA specimen bottles for laboratory
analysis.
Laboratory analysis
Osmotic fragility test: Erythrocyte osmotic fragility was determined as
described by Azeez and Oyewale (2010); Alanazi
(2010). 0.02 mL of blood was added to tubes containing increasing concentration
of phosphate-buffered sodium chloride (NaCl) solution at pH 7.4 (0, 0.1, 0.3,
0.5, 0.7, 0.8 and 0.9%). The tubes were mixed and incubated at room temperature
(29°C) for 30 min. The content of each tube was then centrifuged at 3500
rev min-1 for 10 min. The relative amount of hemoglobin released
into the supernatant was determined spectrophotometrically at a maximum wave
length of 540 nm. The quotient of absorbance of the content of individual test
tubes that caused 50% lyses of red blood cells was the Mean Corpuscular Fragility
Index (MCFI) (Chikezie, 2007). This was extrapolated
from the Osmotic Fragility Curve (OFT) obtained by plotting the percentage lysis
against saline concentrations. The relative capacity of the antimalarials and
antioxidant micronutrients to stabilize or destabilize red blood cell membrane
was evaluated as percentage of the quotient of the difference between the MCFI
values of the test and control samples (Chikezie, 2007).
Thus:
Table 1: |
Drug administration (per os) in animals |
 |
Data analysis: Statistical analyses of the data were performed using statistical soft ware package SPSS version 17.0. Students t-test and one way ANOVA were used to compare the mean of laboratory data between groups. The statistical significance level was set at 95% confidence interval and p-value<0.05 was considered significant. RESULTS
Table 2 revealed the mean % hemolysis in the treated groups
at varying saline concentration (0.0-0.9 g dL-1). This was used to
plot the osmotic fragility curves as shown in Fig. 1a-i.
The osmotic fragility curve showed a similar sigmoidal pattern from which the
MCFI was extrapolated after a 4 day curative treatment of established Plasmodium
berghei infection in mice. The general hemolytic trend observed was a decrease
in % hemolysis with increasing saline concentration in all the micronutrient
treated groups when compared to the negative and vehicle control groups.
As shown in Table 3, there was a significant difference in the Mean Corpuscular Fragility Index (MCFI) between groups (F = 2275.65; p<0.05). This determines the % erythrocyte membrane stability or disability of the treatment groups. However, there was no statistically significant difference (p>0.05) in MCFI values of vitamin A and selenium treated groups. Erythrocyte membrane disability (Fig. 2) was most marked in the tween 80 group (426.15%), followed closely by the chloroquine (373.85%) treated group and artesunate group (329.23%) and least in the zinc treated group (32.31%). DISCUSSION
The findings of the present study connote erythrocyte membrane protection from
oxidative stress during malaria infection.
Table 2: |
Percent hemolysis in different groups after 4 days curative
test in P. berghei parasitized mice |
 |
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Fig. 1(a-i): |
Osmotic fragility curve in, (a) Control group MCFI = 0.065,
(b) Dist. H20 Group MCFI = 0.239, (c) Tween 80 Group MCFI = 0.343, (d) Chloroquine
Group MCFI = 0.308, (e) Artesunate Group MCFI = 0.279, (f) Vitamin A Group
MCFI = 0.115, (g) Vitamin E Group MCFI = 0.129, (h) Selenium Group MCFI
= 0.114, (i) Zinc Group MCFI = 0.08 |
Table 3: |
Mean Corpuscular Fragility Index and % Membrane Disability
after 4 Day Curative Test in P. berghei Parasitized Mice (n=5mice each) |
 |
Values are expressed as Mean±SEM. df = 4, Mean difference
is significant at *p<0.05 when compared with control (negative, positive
and vehicle). aNo significant difference in MCFI value between
vitamin A and selenium treated groups |
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Fig. 2: |
Percent Membrane distability in different groups after 4 day
curative test in parasitized mice, Key: A: Control (no inoculation, no treatment),
B: Dist. H2O, C: Tween-80, D: Chloroquine, E: Artesunate, F:
Vit A, G: Vit E, H: Selenium, I: Zinc |
This proposed mechanism is further corroborated by the work of Stocker
et al. (1985) which revealed that erythrocyte membranes are better
protected by antioxidants than parasite membrane. According to Kraus
et al. (1997), vitamins C and E supplementation reduced erythrocyte
osmotic fragility and oxidative damage in rats. Similarly, exercise stress,
heat stress and other forms of oxidative stress have been associated with increased
erythrocyte osmotic fragility, concurrently with elevated levels of Thiobarbituric
Acid Reacting Substances (TBARS) and Malondialdehyde (MDA) which are products
of lipid peroxidation in the erythrocyte membrane (Kelle
et al., 1999; Ozturk and Gumuslu, 2004).
Damage to the erythrocyte membrane proteins and lipids due to oxidative stress
leads to hemolysis as a result of destruction of the spectrin bands which is
the base of the erythrocyte cytoskeleton (Reid and Mohandas,
2004). Also damaged by oxidative stress according to Reid
and Mohandas (2004) are band 3, glycophorin C and RhAG; the membrane proteins
that link the lipid bilayer to the spectrin cytoskeleton. These linkages play
a significant role in regulating cohesion between the lipid bilayer and the
cytoskeleton; the loss of which results in lipid loss, decreased membrane surface
area and loss of deformability of erythrocytes. Erythrocyte membrane appears
to be protected by the antioxidant micronutrients (vitamin A, E, Zinc and Selenium)
as evidenced by a significantly lower MCFI in the antioxidant treated groups
when compared to the infected control groups. This proposed mechanism is further
corroborated by the work of Stocker et al. (1985)
which revealed that erythrocyte membranes are better protected by antioxidants
than parasite membrane. According to Kraus et al.
(1997), vitamins C and E supplementation reduced erythrocyte osmotic fragility
and oxidative damage in rats. Similarly, exercise stress, heat stress and other
forms of oxidative stress have been associated with increased erythrocyte osmotic
fragility, concurrently with elevated levels of Thiobarbituric Acid Reacting
Substances (TBARS) and Malondialdehyde (MDA) which are products of lipid peroxidation
in the erythrocyte membrane (Kelle et al., 1999;
Ozturk and Gumuslu, 2004). Additionally, the % membrane
distablity was also significantly lower in the antioxidant groups when compared
to infected control. Also among the micronutrient treated groups, MCFI appears
to be lower in the zinc treated group when compared to other micronutrient groups.
This finding is supported by other studies which revealed that dietary zinc
deficiency in rats is associated with increased hemolysis of erythrocytes in
hypotonic saline (ODell et al., 1987;
Paterson and Bettger, 1985; Roth
and Kirchgessner, 1994) and in the presence of various detergents, alcohols
and toxins (Paterson and Bettger, 1985). Additionally,
alterations in the composition of the erythrocyte membrane have been detected
in zinc-deficient rats (Avery and Bettger, 1988, 1992;
Driscoll and Bettger, 1991; Johanning
and ODell, 1989; Paterson et al., 1987).
These findings corroborate the finding of the present study which revealed that
zinc had the lowest MCFI value among the antioxidants used for treatment of
malaria. In vitro addition of zinc to red blood cells is also protective
against hemolysins (Avigad and Bernheimer, 1976; Takeda
et al., 1977). Consequent upon these findings, it is pertinent to
state that with membrane stabilization, hemolysis is impaired, merozoite release
is reduced and progressive parasitization of uninfected RBC is also significantly
reduced. It has been revealed that the trace element zinc plays an important
role in the structure and function of biological membranes (Bettger
and ODell 1993) which also corroborates the present study. Oxidative
modifications of the membrane increases fragility of red blood cells (Stern
1986; Wagner et al., 1988). Because there
is some evidence for a physiological role of zinc as an antioxidant (Bray
and Bettger, 1990), greater oxidative damage in zinc deficiency could be
responsible for impaired stability of erythrocytes. In a previous study by Kraus
et al. (1997), enrichment of the diet with antioxidants in combination
(vitamin C, vitamin E and β-carotene) prevented the elevated osmotic fragility
of erythrocytes in zinc-deficient rats. Indeed, this suggested an important
role of oxidative damage in the impaired stability of erythrocytes in zinc deficiency.
Vitamin E on the other hand is a natural constituent of biological membranes;
it acts as an antioxidant by donating hydrogen atom at 6-hydroxyl group on the
chromatin ring and by scavenging singlet oxygen and other reactive species (Lee
et al., 2004; Powers and Jackson, 2008).
An association between Reactive Oxygen Species (ROS) generation and erythrocyte
loss has been observed in malarial infection, when such markers were measured
in the erythrocyte (Das and Nanda, 1999). The malarial
parasite is known to perturb the lipid composition of the host RBC (Sherman,
1979), with RBCs in in vitro culture showing an increase in total
lipid content and a decrease in percentage PUFA in the erythrocyte membrane
(Hsiao et al., 1991). The effect of ROS on erythrocytes
is probably a balance between the parasite and host response. The significant
reduction in membrane percentage PUFA and α-tocopherol concentration in
malarial subjects supports oxidative stress. Wo and Yang
(1986) observed that during the ageing of erythrocyte membrane, the spectrin
content, Na/K-ATPase activity as well as the lipid fluidity were obviously decreased.
However, supplementation of a trace amount of a selenium compound (Na2SeO3)
in the medium prevented the dissociation of spectrin from membrane and delayed
the changes of Na/K-ATPase activity and lipid fluidity. The effectiveness is
proportional to selenium concentration within the range of 0.1-1.0 ppm (Wo
and Yang, 1986). A similar effect of supplementation of selenium on the
intact erythrocytes during ageing has also been observed (Wo
and Yang 1986). This supports the potent membrane protective role of selenium
as evidenced in the present study which showed a significantly lower MCFI and
% membrane distability when compared to other treated groups. The protective
action of selenium on biomembranes is generally interpreted in terms of the
activity of selenium-containing Glutathione Peroxidase (GPx). Findings from
this present study has further reiterated the fact that antioxidant play a significant
role in membrane protection. This is further corroborated by the findings of
Wambi et al. (2008) which observed that dietary
antioxidant supplements increased survival when administered as a preventive
measure prior to radiation exposure as well as when given as treatment after
radiation exposure. The administration of dietary antioxidants prior to the
radiation exposure was associated with significant protective effects against
radiation-induced leukocyte depletion in peripheral blood and bone marrow, suggesting
that antioxidants may improve the survival of irradiated animals by attenuating
the deleterious effects of radiation on the host immune system. Earlier studies
have previously demonstrated the preventive effect of antioxidants against radiation-induced
oxidative stress in vitro and in vivo, which was measured by radiation-induced
reductions of serum or plasma total antioxidant status in animals (Guan
et al., 2004, 2006; Kennedy
et al., 2004; Wan et al., 2005, 2006).
CONCLUSION Consequent upon these findings, it is pertinent to state that the antimalarial activity exhibited by these micronutrients may be due to membrane stabilization, impaired hemolysis, impaired merozoite release and the inhibition of progressive parasitisation of uninfected RBC.
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