Malaria is a prevalent disease in tropical and subtropical areas of Africa. I t is estimated that 1 -3 million deaths occurs worldwide, mostly… [600270]

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CHARPTER ONE
INTRODUCTION
1.1 Background of Study
Malaria is a prevalent disease in tropical and subtropical areas of Africa. I t is
estimated that 1 -3 million deaths occurs worldwide, mostly involvin g children
under the age of 5years (Gouado et al ., 2007). Malaria is a major public health
challenge in Nigeria and it accounts for more cases and deaths than any other
country in the world ( Olasehinde et al ., 2010) . This disease is often linked to
changes in climate, poverty, malnutrition and the double re sistance of the malaria
parasite to usual anti -malaria drugs and insecticides (Müller and Garenne, 1999).
Infection by malaria can cause serious health problems and this often leads to death
especially in children (Gouado et al ., 2007). The dis ease is caus ed by malaria
parasites (Plasmodium species) which are transmitted by the female anopheles
mosquito (vector). There are today more than 25 named plasmodium species which
infect primates. Four of the species are human parasites; P. falciparum, P. vivax, P.
malaria and P. ovale (Trampuz et al ., 2003). Epidemiological studies have
demonstrated that P. falciparum is the most dangerous specie as it is responsible
for most of the deaths caused by malaria (Greenwood et al., 2005). The malaria
parasite is transmitt ed when an individual is bitten by infected female anopheles
mosquito (Ochei and Kolhatkar, 2008). The main symptom of uncomplicated

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malaria in children is fever. Older children may present with headache, backache,
chills, myalgia and fatigue ( Olasehinde et al., 2010). Severe anaemia may exist
alone or in combination with other complications particularly cerebral malaria and
respiratory distress in which it portends worse prognosis (WHO, 2004).
Consequences of severe malaria include coma and death if untrea ted, young
children are especially vulnerable (Anemana et al., 2004). Laboratory diagnosis of
malaria could be made by detection of parasite in blood ] or by serological
techniques (Ochei and Kolhatkar, 2008).
Micronutrients are trace elements that are requ ired in small quantities to ensure
normal metabolism, growth and physical well ‐being. Some studies relating
micronutrient status and malaria infection reported low plasma levels of certain
micronutrient in acute malaria infection (Alonso, 2004). Iron and b eta carotene
which are reported to have modulatory effect on the pathogen esis of malaria, have
been observed to be deficient in acute plasmodium falciparum infection (Lavender,
1993; Shankar and Prasad 1998 ;; Beard, 2001; Caulfield et al., 2004). The level s of
micronutrients in children are of particular interest since adequate intake is of great
importance for the well being, proper development, and functioning of the body
starting from fetal life and throughout childhood. Micronutrients com prise of
vitami ns and minerals. Examples of vitamins are vitamin A, pro -vitamin A

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(Beta‐carotene), vitamin B1, vitamin B2, vitamin B6, vitamin B 12, biotin, vitamin C,
vitamin E, vitamin D, vitamin K ,folic acid, niacin and pantothenic acid while
minerals include the trac e elements such as iron, copper, iodine, manganese,
selenium and zinc together with the macro elements calcium, magnesium,
potassium and sodium. ( Asaolu and Igbaakin, 2009; Crook, 2012). Micronutrients
have been implicated to play important roles in immuni ty and physiologic
functions. For instance, Calcium is an important nutrient that plays a major role in
bone and teeth formation, impulse transmission, catalytic activation among others
(Nordin, 1997). Iron plays an important role in the production of heam oglobin,
oxygenation of red blood cells and lymphocytes. It improves the function of
enzymes in protein metabolism and enhances the function of calcium and copper
(Asaolu and Igbaakin, 2009) . Vitamin B 12 is involved in the maturation of red
blood cells. The folic acid coenzymes are specifically concerned with metabolic
reactions involving the transfer and utilization of the one carbon moiety
(Crook, 2012). Micronutrients are found in small quantities within the body and
they are obtained from a wide v ariety of foods. No single food contains all of the
micronutrients we need and, therefore, a balanced and varied diet is necessary for
an adequate intake. Micronutrients deficiency is more frequent amongst children in
developing countries (Gibson and Ferg uson, 1998). These deficiencies may

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contribute to an increased risk of parasitic infection such as malaria (Mahomed,
2000).
1.2 Justification of Study
Malaria has been a major challenge to the world’s population especially in Africa
and indeed Nigeria. It has been implicated in increased rate of morbidity and
morta lity among children (Sachs and Malaney, 2002) . Research has shown that
malaria causes 0.5 – 3.0 million deaths each year and that 75% of these deaths
occur in African c hildren under the age of 5y ears (Greenwood et al ., 2005). The
increased clinica l state of malaria infection may be due to poor nutritional status
more especially as a resu lt of micronutrients deficiency (Gouado et al ., 2007) .
Micronutrients play vital role both in combating anaemia a nd other adverse effects
of malaria infection in humans and animals in developing resistance against the
disease. Micronutrients are not only necessary in the regeneration of heamolyzed
red cells during malaria infection, but also served as antioxidants he nce protecting
the red cells against damage by malaria toxins (Jain, 2006). It is therefore of
tremendous importance to assess micronutrients status of children with malaria .
1.3 Aim of Study
The aim of this study is to assess the levels of micronutr ients in children with
malaria infection in Paediatric Ward, Central Hospital, Benin city.

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1.4 Specific Objective
 To evaluate the levels of vitamin B 12, folic acid, iron, magnesium , and
calcium in malaria infected children .
 To correlate the micronutrient l evels with the severity of malaria infection.
 and to compare the results with the control group ( non-infected children ).
1.5 Research Design
This is a case study designed to assess micronutrients status of malaria infected
children and then compare findi ngs with non -infected children in Benin City.
1.6 Ethical consideration
Ethical approval was sought and approved by the ethical committee of Central
Hospital, Benin City, Edo State .
1.7 Research Hypothesis
H1: malaria parasites affec ts calcium , magnesium, iron ,vitamin B 12,folic acid in
children infected with malaria.
H0 malaria parasites does not affect calcium , magnesium, iron ,vitamin B 12,folic
acid in children infected with malaria.

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1.8 Questionnaire/informed consent
A questionnaire, written in Engl ish was administered to the parents of the subjects
to fill. An informed consent was given to the parents or guardian of the children
before sample was collected.
1.9 Sample size
The sample size (N) was ca lculated using prevalence from previous studies
The sample (Olasehinde et al., 2010). size for this study will be obtained using the
formula described by Dean et al., (1995).
N = Z2P (1 – P)
D2
N = required sample size
Z = confidence level at 95% (standard value of 1.96)
P = estimated prevalen ce of intestinal parasites of pupils (84.7 %)
D = margin of error at 5% (standard value = 0.05)
N=1.962 X 0.847(1 -0.847 )
0.052
3.8416 X 0.847(0.153 )
___________________
0.0025
N=199 minimum sample sizes

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Therefore a minimum of 200 test sa mples and 10 0 control will be used for this
research.

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CHARPTER TWO
2.0 LITERATURE REVIEW
2.1 Malaria
Malaria which is one of the most prevalent and dangerous disease known to man
most especially in tropical and subtropical climate regio ns (Sachs and Malaney,
2002 ), is the major cause of ill health in Africa, malaria is responsible for over
10% of the overall African disease burden (Salako, 2002). It is caused mainly by
protozoan parasites belonging to the genus Plasmodium . Today, there a re more
than 25 named plasmodium species which infect primates. Four of the species are
human parasites; P. falciparum, P. vivax, P. malaria and P. ovale . The most
serious forms of the disease is caused by P. falciparum and P. vivax (Trampuz et
al., 2003 ). The parasite is transmitted by female anopheles mosquitoes (Adefioye
et al., 2007).
2.2 Epidemiology
It is estimated that more than a third of the world’s population live in malaria –
endemic areas. Estimates suggest that one billion people are infected wit h the
parasite at any given time. P. falciparum is the most dangerous specie; it is
responsible for most of the deaths caused by malaria. 95% of the malaria cases are
caused by either p. falciparum or p. vivax . Malaria causes 0.5 – 3.0 million deaths
each year. 75% of these deaths occur in Africa n children under the age of 5years .

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20% of the tot al deaths in children under 5 years of age in Africa is due to malaria,
and malaria constitutes 10% of the total disease burden in Africa. It is estimated
200-450 mi llion cases of fever in children infected by malaria parasites each year
in Africa. The most affected regions in the world are Sub -Saharan Africa and
Southeast Asia (Greenwood et al., 2005).
Malaria is endemic throughout Nigeria. Malaria is a major public health problem in
Nigeria where it accounts for more cases and deaths than any other country in the
world. Malaria is a risk for 97% of Nigeria’s population. The remaining 3% of the
population live in the malaria free highlands. Malaria is also the reaso n for
outpatient hospital attendance in seven (7) out of every ten (10) patients seen in
Nigerian hospitals and occurs in younger ch ildren up to three 3 -4 times a year and
is responsible for 25% of infant mortality and 30% of childhood death in Nigeria
(Iloh et al ., 2013). Prevalence and management study of p. falciparium malaria
among infants and children was carried out in Ota, Ogun State, Nigeria. In the
study the prevalence of falciparum malaria in c hildren between 0 and 12 years wa s
80.5% with mean par asite density of 750. Prevalence rate of 84.7% was recorded
among children between zero and five years. It was observed that newborns and
infants between age 5 days and three months who presented with fever were found
to be positive for falciparum malaria (Olasehinde et al., 2010). According to the
study carried out by Okafor and Oko -Ose, (2012 ), the prevalence of malaria

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infections among children of ½ -11years olds in Edo State, Nigeria. Children aged
six months to five are the most vulnerable to malaria in fections and deaths.
Epidemiological study of malaria among children of 1 -15 years of age in Southern
Nigeria revealed that the use of insecticide treated bed net, curtains and mats with
lambdacyhalothrin can serve in controlling mosquito bite thus reducin g malaria
infection among the experimental area (Oko et al., 2014).

Figure 1 : Malaria cases (per 100,000) by Country Worldwide (WHO, 200 0)

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2.3 Malaria parasites
Genetic evidence suggests that the ancestor of the malaria parasite arise more than
500 m illion years ago. These “pre -malaria parasites” acquired at an early stage in
their evolution an asexual form of reproduction called schizogony. Schizogony is a
form of reproduction where the parasite forms a number of daughter cells within
one host cell. This “technique” greatly enhanced the growth rate of the parasite.
About 150 million years ago the “pre -malaria parasite” adapted to Diptera insects,
which is the ancesto r to today’s Anopheles mosquito ( Carter and Mendis, 2002).
There are today more than 2 5 named plasmodium species which infect primates.
Four of the species are human parasites; P. falciparum, P. vivax, P. malaria and P.
ovale. P. falciparum’s lineage separated from the other human malaria species
more than 130 million years ago. P.vivax, P. malaria and P. ovale diverged over
100 million years ago. P. ovale is known to solely infect humans. P. malaria on the
other hand has been found in an indistinguishable form to infect other primates,
and P. vivax has close related malaria parasites which infect monkeys in south –
southeast Asia. Evidence from human genetic data suggests that the origin of P.
falciparum is in West Africa within a thousand years ago, due to the agrarian
revolution (Trampuz et al., 2003).

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2.3.1 Life cycle of Malaria Parasite
The female Anopheles mosquito requires blood as nutrition for reproduction, and
this blood is drawn from people. When one mosquito bites a malaria -infected
person, the mosquito becomes a carrier for the parasite and is able to transmit the
disease to the ne xt person on the mosquito’s menu. Normally it takes a number of
bites from different carrier mosquitoes for a person to get infected, depending on
the mosquito and the immunity of the victim. The infected anopheles mosquito
feed on a human being. The para site is injected into the blood of the human host.
The infectious stage of the parasite is called sporozoites. The sporozoites go with
the blood to the liver where they enter the liver cells. They multiply inside the liver
cells and the daughter cells of the sporozoite are called merozoites. When the
infected cell ruptures, thousands of these merozoites will be released into the blood
stream where they multiply until the red blood cel l rupture and release more
meroz oites. After invading more red blood ce lls, the merozoites will then
differentiates into the sexual (male and female gametocytes) stage which is
eventually transmitted to a feeding mosquito. In the gut of the mosquito the
gametocytes goes through different stages to form an oocyst. Inside the o ocyst,
sporozoites develop and these sporozoites infect the s alivary glands of the
mosquito (Ochei and Kolhatkar, 2008).

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Figure 2: Life Cycle of Malaria Parasite (Ayodotun and Olugbenga, 2012).

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2.3.2 Signs and Symptoms of Malaria in Children
The ma in symptom of uncomplicated malaria in children is fever. The fever is
normally recurrent and irregular. Other symptoms in infants are irritability, poor
feeding, vomiting, jaundice and splenomegaly. Older children may present with
headache, backache, chil ls, myalgia and fatigue. Between attacks, the patient may
look quite well. In children anemia, convulsions, hypoglycemia, coma and
metabolic acidosis are the most common signs of severe malaria, in adults
jaundice, renal failure and pulmonary edema are mos t often present (Olasehinde et
al., 2010).
2.3.3 Consequences of Malaria in Children
Consequences of severe malaria include coma and death if untreated, young
children are especially vulnerable . In malaria infested zones , treatment is often less
satisfacto ry and the overall fatality rate for all cases of malaria can be as high as
one in ten. For reasons that are poorly understood, but which may be related to
high intracranial pressure, children with malaria frequently exhibit abnormal
posturing, a sign indi cating severe brain damage ( Anemana et al., 2004 ). Malaria
has been found to cause cognitive impairments, especially in children. Malaria
causes widespread anemia during a period of rapid brain development and also
direct brain damage and this neurologic d amage results from cerebral malaria to

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which children are more vulnerable ( Boivin, 2002 ). Over the longer term,
developmental impairments have been documented in children who have suffered
episodes of severe malaria (Trampuz et al., 2003).
2.3.4 Severe Mal aria Anaemia in Children
Severe malaria anaemia is defined as haemoglobin concentration less than 5g/dl
associated with Plasmodium falciparum parasitaemia. Other causes of anaemia
have to be excluded as asymptomatic falciparum parasitaemia is common in
endemic areas. Severe anaemia may exist alone or in combination with other
complications particularly cerebral malaria and respiratory distress in which it
portends worse prognosis (WHO, 2004). Study carried out by Ayodotun and
Olugbenga, (2012 ) has demonstra ted the clinical burden of severe malaria anaemia
as a common indication for admission of young children into emergency units
particularly children less than 24 months of age. Malaria infection has been a
major cause of morbidity and mortality among childr en below the age of five
years . Children below 3 years are predominantly affected with a mean age of 1.8
years (Krause, 2000).
2.3.5 Laboratory Diagnosis of Malaria
Laboratory diagnosis of malaria could be made by detection of parasite in blood or
by serol ogical techniques.

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1. Detection of Parasite in Blood
a. In thick and thin films : These blood films are prepare d and stained by
Field’s or Giemsa method. The thick and thin blood films are then examined
under the oil immersion lens. The thick film is meant to de tect the presence
of the parasite while thin film enables the identification of the various
species (Ochei and Kolhatkar, 2008).
b. By concentration using buffy coat preparation : In this method, the buffy
coat obtained from spun capillary tube containing blo od is used to make
thick and thin films The films are the n stained and examined
microscopically. The parasite present can be estimated (Ochei and
Kolhatkar, 2008).
2. Serology : Serological tests have a limited use in the diagnosis of malaria
because the antib ody tests are unable to differentiate between the past and
current infections . They are helpful epidemiological surveys for the control
of malaria . The tests used include enzyme linked immunosorbent assay
(ELISA), indirect fluorescent antibody test (IFAT) and radioimmunoassay
(RIA) (Ochei and Kolhatkar, 2008).
2.4 Micronutrients
Micronutrients comprise of vitamins and minerals which are required in small
quantities to ensure normal metabolism, growth and physical well ‐being. The

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levels of micronutrients quantitively in children are of particular interest since
adequate intake is of great importance for the well being, proper development, and
functioning of the body starting from fetal life and throughout childhood. Vitamins
as a class of micronutrients are essential organic nutrients, most of which are not
made in the body, or only in insufficient amounts, and are mainly obtained through
food. When their intake is inadequate, vitamin deficiency disorders are the
consequence. Although vitamins are only presen t and required in minute
quantities, compared to the macronutrients, they are as vital to health and need to
be considered when determining nutrition security. Each of the 13 vitamins known
today has specific functions in the body: vitamin A, provitamin A (Beta‐carotene),
vitamin B1, vitamin B2, vitamin B 12, vitamin B6, biotin, vitamin E , vitamin C,
vitamin D, folic acid, vitamin K , niacin and pantothenic acid. Minerals as another
class of micronutients are inorganic nutrients that also play a key role in ensur ing
health and well ‐being. They include the trace elements copper, iodine, iron,
manganese, selenium and zinc together with the macro elements calcium,
magnesium, potassium and sodium. Like vitamins, minerals are found in small
quantities within the body a nd they are obtaine d from a wide variety of foods
(Asaolu and Igbaakin, 2009; Crook, 2012).

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2.4.1 Functions of Micronutrients
Micronutrients have been implicated to play important roles in immunity and
physiologic functions . For example, Iron carries oxy gen to the cells and is
necessary for the production of energy, the synthesis of collagen, and the
functioning of the immune system and copper is required with iron for synthesis of
hemoglobin. It works with many enzymes such as those involved in protein
metabolism and hormone synthesis (Gebremedihin, 1976; Gebre -Medhin and
Birgegård, 1981; Moreno et al., 1998). Calcium plays an important role in muscle
contraction and regulation of water balance in cells. Modification of plasma
calcium concentration leads to the alteration of blood pressure. Magnesium has
been known as an essential co -factor for many enzyme systems. It also plays an
important role in neurochemical transmission and peripheral vasodilation (Rude,
1996). Zinc is an integral part of more than 2 00 enzymes and has significant task in
nucleic acid metabolism, cell replication, tissue repair, and g rowth (Nicola et al.,
2002). The antioxidation functions of selenium in glutathione peroxidase are
essential in protecting the biological system from oxid ation caused by peroxides .
Superoxide dismutases, which usually contain copper and/or zinc, act as
antioxidants ag ainst superoxides (McKenzie et al., 1998).

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2.4.2 Micronutrients Deficiency in children
Micronutrients deficiency such as vitamin A, zinc, ir on, and calcium are more
frequent amongst children in developing countries (Gibson and Ferguson, 1998).
These deficiencies may contribute to an increased risk of parasite infection such as
malaria (Mahomed, 2000). It has been observed for instance, that th e deficiency of
calcium from food is responsible for serious diseases such as osteoporosis, cardio –
vascular diseases, diabetes, obesity and colon cancer (Miller, 1997). Women who
suffer from folate deficiency prior to and during pregnancy, have an increase d risk
of giving birth prematurely to children , with a lower birth weight and
malformations of the brain and spinal cord, together with other birth defects such
cleft lip and palate, certain heart defects and limb malformations. Also, iron
deficiency is th e most widespread health problem in the world, impairing normal
mental development in 40 ‐60% of infants in the developing world, debilitating the
health and energies of 500 million women, and leading to more than 115,000
deaths during childbirth a year. Research has also shown that Zinc deficiency
hampers the functioning of the immune system by lowering T – lymphocytes
response and the production of cytokines. This can eventually contribute to the
increase risk of infection (Steketee, 2003). About 5 million children under the age
of five worldwide are affected by xerophthalmia, a serious eye d isorder caused by

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vitamin A deficiency. These children are at risk of becoming blind and are more
likely to die of common childhood diseases (Gouado et al., 2007).
2.4.3 The Prevalence of Micronutrient Deficiency in Diet Plans
There have been several resea rches that have shown that micronutrient deficiency
is linked scientifically to higher risks of obesity, overweight, and other dangerous
diseases. Researchers have also shown that out of the two -third of the U.S obese
and overweight population, one -third of that population is on a diet at any given
time. Therefore there arise a need to determine whether the current popular diet
plan could aviate micronutrient deficiencies by providing minimum levels of 27
micronutients, as determined by the U.S. Food and Dr ug Administration’s (FDA),
Reference Daily Intake (RDI) guidelines.
The daily menus suggested by Atkins for life diets, South Beach Diet, and Dash
Diet plans, where evaluated micronutrients and calorie content of each ingredient
in each meal were determin ed using the data from the food composition of the U.S.
Department of Agriculture Nutrient Database for Standard References. From the
results gotten, the diet plans did not meet 100% sufficiency by the RDI guideline.
It was discovered that individuals who follow the popular diet plans (with food
alone) will likely have micronutrient deficiency which may eventually lead to
increased susceptibility to having dangerous health conditions and diseases
(Calton, 2010).

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2.5 Vitamin B 12 (Cobalamin )
Vitamin B 12 or co balamin (CbI) is essential for the normal maturation of red blood
cells. Cobalamin is synthesized by bacteria and is found in soil and in
contaminated water. Foods of animal origin (meat, eggs and milk) are the primary
dietary sources. The amount of CbI in the average western diet (5 -15µg/ d) is more
than sufficient to meet the recommended dietary allowable of 2µg/d. Therefore,
except in strict vegetarians, the presence of CbI deficiency implies the presence of
an absorptive problem. The body stores a large amount of CbI (2 -5mg) relative to
daily requirements (Snow, 1999).
2.5.1 Structure of Vitamin B 12
Central portion of the molecule consists of four reduced and extensively
substituted pyrrole rings , surrounding a single cobalt atom (Co) . This central
structure is called Corrin Ring system. The above system is similar to porphyrins,
but differs in that two of pyrrole rings; r ings I and IV are joined directly. Below
the corrin ring system, is DBI ring –5, 6- dimethyl b enzimidaz ole riboside which is
connect ed at one end to central cobalt atom and at the other end from the riboside
moiety to the ring IV of corrin ring system. One phosphate ( PO 4) group connects
ribose moiety to aminopropanol (esterified) , which in turn is attached to propionic
acid side chain of ring IV. A cyanide group is coordinately bound to the cobalt
atom and then is called cyanocobalamine (Chatterjea and Shinde, 2012).

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Figure 3 : Structure of Vitamin B 12 (Chatterjea and Shinde, 2012).
2.5.2 Metabolism of Vitamin B 12; Absorption
Vitamin B 12 is absorbed from ileum of the small intestine. For its proper absorption
it requires the presence of hydrochloric acid (HCl) secreted by the mucosa of the
stomach and intrinsic factor (IF) of c astle, a constituent of normal gastric juice. It
is a glycoprotein secreted by parietal cells. It is a constituent of gastric
mucoproteins. In addition to amino acids, contain hexoses, hexosamines and sialic

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acid. It is non-dialyzable and thermolabile , destroyed by heating at 70° to 80°C for
½ hour. It can b e inactivated by prolonged digestion with pepsin or trypsin. Found
in ‘Cardiac’ end and fundus of stomach, but not in the pylorus . Atrophy of fundus
of stomach and a lack of free HCl (achlorhydria) is usually associated with
pernicious anaemia, caused by B 12 deficiency (Arpita et al., 2012; Chatterjea and
Shinde, 2012).
2.5.3 Mechanism of Absorption of Vitamin B 12
Recently, it has been shown that two binding proteins are required for absorption
of vit. B12. They are cobalophilin (binding protein secreted i n the saliva ) and
intrinsic factor ( a glycoprotein secreted by parietal cells of gastric mucosa ). Gastric
acid (HCl) and pepsin release the vit . B12 from protein binding in food and make it
available to bind to salivary protein, cobalophilin. In the duode num, cobalophilin
is hydrolyzed releasing the vitamin for binding to “ intrinsic factor” (IF). Vitamin
B12 is absorbed from the distal third of the ileum via specific binding site
(receptors) that binds the “B 12-IF complex”. The removal of B 12 from ‘ intrins ic
factor’ (IF) in presence of calcium ions (Ca++) and a releasing factor (RF) secreted
by duodenum take place and B 12 enters the ileal mucosal cells for absorption into
the circulation (Arpita et al., 2012; Chatterjea and Shinde, 2012) .

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2.5.4 Transportat ion, Storage and Excretion of Vitamin B 12
Vitamin B12 is transported in blood in association with specific proteins named
Transcobalamine I and Transcobalamine II and III. Physiologically
Transcobalamine II is more important. Transcobalamines have α 2 to β mobility.
Normal serum level varies from 0.008 to 0.42 μg/dl. (Average = 0.02 μg/dl). Main
storage site is Liver. A man on normal non -vegetarian diet may store several
milligrams (= 4 mg). As storage is high, development of deficiency state takes long
time. Normally there is practically no urinary excretion. But following parenteral
administration there is urinary excretion up to 0.3 μ g/day (Chatterjea and Shinde,
2012).

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Figure 4: Mechanism of Absorption of Vit. B 12 (Chatterjea and Shinde,
2012).

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2.5.5 Functions of Vitamin B 12
Biologically active forms are cobamide coenzyme which acts as coenzyme with
various enzymes .In human it is required in adenosylcobalamin, coenzyme to L –
methyl malonyl -CoA mutase in the conversion of L -methylmalonyl CoA to
succin yl CoA. It is also required in methyl cobalamin, coenzyme to methionine
synthase in the conversion of homocysteine to methionine. Congenital defects of
the mutase synthesis or inability to synthesize adenosylcobalamin (adenosyl -CbI)
result in life -threaten ing methylmalonic aciduria and metabolic ketoacidosis.
Congenital defects in methionine synthase or the synthesis of methyl -CbI result in
severe h yperhomocysteinemia (Tietz, 2008).
2.5.6 Deficiency of Vitamin B 12
Deficiency of vitamin B 12 in human is assoc iated with megaloblastic anaemia and
neuropathy. The most common cause of vitamin B 12 deficiency is pernicious
anaemia, an autoimmune disease in which chronic atrophic gastritis results from
antibodies to gastric parietal cells and intrinsic factor (IF), directed against gastric
parietal cell H+/K+-ATPase. Pernicious anaemia may also occur in children
because of either failure of IF secretion or secretion of biologically inactive IF
(Weir and Scott, 1995; Prachi et al., 2010).

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2.5.7 Laboratory Assessment s tatus of Vitamin B 12
Both direct and indirect functional tests are available for assessing vitamin B 12.
Microbiological, competitive protein binding (CPB) and immunoassays have been
used for the direct quantification of serum vitamin B12. The indirect test s include
assays for urinary and serum concentration of methylmalonic acid, plasma
homocysteine, the deoxyuridine suppression test and the vitamin B 12 absorption
test ( Gregory, 1995; Tietz, 2008).
2.6 Folic Acid (Folate)
The designation “folic acid” is app lied to a number of compounds which contain a
pteridine nucleus (pyrimidine and pyrazine rings ), Para -aminobenzoic acid
(PABA) and Glutamic acid. Folates are synthesized by microorganisms and by
plants and are widely distributed in the diet. Vegetables, fr uits, dairy products and
cereals are the most important sources . Unlike CbI, the body stores of folate (5 –
10mg) are small relative to daily requirements (Snow, 1999 ; Chatterjea and
Shinde, 2012 ).
2.6.1 Structure of Folic acid
Structure of folic acid is chemically called pteroyl glutamic acid (PGA). There are
at least three chemically related compounds of nutritional importance which occur
in natural products, all may be termed pteroyl glutamates. These three compounds

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differ only in the number of glutamic a cid residues attached to pteridine PABA
complex (pteroic acid). The first is monoglutamate which has one glutamic acid
and second is triglutamate having three glutamic acid residues while the third is
known as heptaglutamate having seven glutamate residues (Chatterjea and Shinde,
2012 ).

Figure 5 : Structure of Folacin (Chatterjea and Shinde, 2012 ).

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2.6.2 Formation of Folic acid (F.H 4)
Active “coenzyme” form of the vitamin is the reduced tetrahydroderivative,
tetrahydrofolate (F.H 4) obtained by addition of four hydrogen atoms to the
pteridine moiety at 5, 6, 7 and 8 positions. Because of their l iability, these occur
naturally only in small quantities, being present mainly in the form of N5-formyl or
N5-methyl derivatives (Quinlivan et al., 2002). Folic a cid, before functioning as a
coenzyme, must be reduced first to 7, 8-dihydrofolic acid (F.H 2) and then to 5, 6, 7,
8 tetrahydrofolate (F.H 4). Both reactions are catalyzed by Folic acid reductases
enzyme, which use NADPH as hydrogen donor. Also requires vit amin C (ascorbic
acid) as cofactor. The steps of the reactions are as follows:

Figure 6: Steps involved in the Formation of F.H 4 (Snow, 1999) .

30

Figure 7: Structure of F.H 4 (Chatterjea and Shinde, 2012 ).

2.6.3 Biosynthesis of Folic acid
Many microorganisms including those inhabiting the intestinal tract can synthesise
folic acid. Some of them cannot synthesise PABA, which has to be supplied. In
presence of adenosine Triphosphate (ATP) and coenzyme A -Sulphuryl group
(CoA -SH), PABA reacts with glutamic acid to form “p-amino -benzoyl glutamic
acid”. The latter then reacts with a “Pterin” to produce “pteroyl monoglutamic
acid, PGA” (folic acid); pterin moiety is probably derived from guanosine. Higher
animals including human be ings cannot synthesize folic acid and it has to be
supplied in diet. In human beings, intestinal bacteria can synthesize and is a good
source (Gregory, 198 9; Chatterjea and Shinde, 2012 ).

31
2.6.4 Functions of Folic acid
The folic acid coenzymes are specifi cally concerned with metabolic reactions
involving the transfer and utilisation of the one carbon moiety (C1). One carbon
moiety (C1) may be either methyl (–CH3), formyl ( –CHO), formate (H.COOH),
formimino group ( –CH=NH) or hydroxymethyl ( –CH2OH). Most of them are
metabolically “interconvertible” and catalysed by nicotinamide adenosine
dinucleotide phosphate (NADP) -dependant hydroxymethyl dehydrogenases The
“one -carbon” moiety can be derived from several sources and can be utilised to
form several compound s (Tietz, 2008).
2.6.5 Deficiency of Folic acid
Deficiency of folate may from the absence of intestinal microorganisms (gut
sterilization) , poor intestinal absorption (for example after surgical resection or in
celiac disease or sprue), insufficient dietar y intake (including alcoholism),
excessive demands (as in pregnancy, liver disease and malignancies),
administration of anti -folate drugs (methotrexane) and anticonvulsant therapy (that
increases folate requirements, especially during pregnancy). Megalobla stic
anaemia is the major clinical manifestation of folate deficiency, although sensory
loss and neuropsychiatric changes may also occur (Tietz, 2008).

32
2.6.6 Laboratory Assessment Status of Folic acid
Folate status may be reliably assessed by direct meas urement of serum and
erythrocyte or whole blood concentrations and its metabolic function as coenzyme
assessed by metabolite concentrations such as plasma homocysteine. Serum folate
concentrations are considered indicative of recent intake and not of tissu e stores,
but serial measurement s have been used to confirm adequate intake. Whole blood
or erythrocyte folate concentrations are more indicative of tissue stores and have
been shown to have a moderate correlation with liver folate concentrations. CPB
assays have now largely replaced microbiological procedures for the measurement
of serum, whole blood or erythrocyte folate ( Gregory, 1989; Tietz, 2008).
2.7 Iron (Fe)
Iron is one of the most essential trace elements in the body. In spite of the fact that
iron is the fourth most abundant element in the earth’s crust, iron deficiency is one
of the most important prevalent nutritional deficiencies in some parts of the world,
for example in India. Much of the iron in the body is contained in haemoglobin.
Haemogl obin is the component of red blood cells that enables them to carry
oxygen and deliver it to the body's tissues. Iron is also an important component of
muscle cells and is necessary for the formation of many enzymes in the body. Total
iron content in a hum an of 70 kg body weight varies approximately from 2.3 gm to
3.8 gm. Average iron content of adult males is about 3.8 gm and of females about

33
2.3 gm . There are two broad categories that are used to describe iron in the body.
They are essential (or functiona l) iron and storage iron (Domellof, 2007).
2.7.1 Essential Iron
Essential or functional iron is one which is involved in the normal metabolism of
the cells in the body. The essential iron is divided into three which are haem
protein, iron present in cytoch rome and iron requiring enzymes. Examples of haem
proteins are haemoglobin, myoglobin, catalases and peroxidases. Xanthine
oxidase, cytochrome C reductase, acyl -CoA dehydrogenase and NADH reductase
are examples of iron requiring enzymes (Rangarajan, 2007).
2.7.2 Storage Iron
Storage iron is present in two major compounds. They are ferritin and
haemosiderin. Free iron is toxic and catalyses the conversion of O-
2 to hydroxy
OH- oxy radicals. Iron bound to ferritin is nontoxic. It is the storage protein of ir on
and found in blood, liver, spleen, bone marrow and intestine (mucosal cells).
Evidence suggests that haemosiderin is derived from ferritin and is usually
described as ferritin with partially stripped shell. Haemosiderin contains a larger
fraction of its mass as Fe2+ than does ferritin and exists as microscopically visible
Fe-staining particles. Haemosiderin is usually seen in states of iron overload or
when Fe is in excess, when the synthesis of apoferritin and its uptake of Fe are
maximum. Haemosiderin is rather insoluble. Fe in haemosiderin is available for

34
formation of haemoglobin, but mobiliz ation of iron is much slower from
haemosiderin than ferritin ( Rangarajan and D’Souza, 2007 ).
2.7.3 Absorption of Iron and Factors Regulating Absorption
Around 10 to 20 mg of Fe is taken in the diet and only about 10% is absorbed. The
greatest need of iron is during infancy and adolescence. The only mechanism by
which total body stores of iron are regulated is at the level of absorption. Garnick
proposed a “mucosal block theory” for iron absorption . According to the theory
soluble inorganic salts of iron are easily absorbed from the small intestine.
Hydro chloric acid (HCl) present in gastric juice liberates free Fe 3+ from non -haem
proteins. Vitamin C and glutathion e in diet reduce Fe 3+ to Fe 2+, which is less
polymerisable and more soluble form of iron. Vitamin C and amino acids can form
iron-ascorbate and iron -amino acid chelates which are readily absorbed. Haem is
absorbed as such. Gastroferrin, a glycoprotein in g astric juice is believed to bind
iron and facilitate its uptake in duodenum and jejunum. The absorption of iron
from intestinal lumen into mucosal cells takes place as Fe 2+.Enterocytes in the
proximal duodenum are responsible for absorption of iron. Incom ing iron in the
Fe3+ state is reduced to Fe 2+ by an enzyme “ferrireductase” present on the surface
of enterocytes, it is helped by vitamin C present in the foods. The transfer of iron
(Fe 2+) from the apical surfaces of enterocytes into their interiors is p erformed by a
proton -coupled divalent metal transporter (DMT1). This protein is not specific for

35
iron as it can transport a wide variety of divalent cations. Once it is inside, it can
either be stored as “ferritin” or it can be transferred across the busol ateral
membrane into the plasma where it is carried bound to transferrin . Passage of Fe 2+
across the basolateral membrane is carried out by another protein called iron
regulatory protein 1 (IREG 1) . Most of Fe 2+ required to be absorbed is transferred
to plasma by a Fe2+ transporter (FP). Fe2+ in the enterocytes also comes from
haem by the action of “haem oxidase” enzyme on haem. IREG1 may interact with
the copper containing protein called “hephaestin” , a protein similar to
caeruloplasmin. Hephaestin is t hought to have a ‘ferroxidase’ activity which is
important in the release of iron from cells as Fe 3+, the form in which it is
transport ed in the plasma by transferrin. Overall regulation of iron absorption is
complex and not well understood mechanistically . It is exerted at the level of the
enterocyte where further absorption of iron is blocked if sufficient amount taken
up, for body need. This is known as dietary regulation exerted by “mucosal block”
explained according to Garnick’s hypothesis (Chatterjea and Shinde, 2012 ).

36

Figure 8: Absorption of Iron (Chatterjea and Shinde, 2012 ).
2.7.4 Iron Transport and Utilization
Transport of Fe throughout the body is accomplished with a specific protein called
transferrin. Transferrin transports Fe from th e gastrointestinal tract (GIT) to the
bone marrow for haemoglobin synthesis and to all other cells as required.
Transferrin can transport a maximum of two atoms of iron as Fe3+ per molecule.
Normally, in plasma or serum transferrin is about 33% saturated w ith Fe. Cell

37
surface specific receptors are available for the iron -transferrin complex. Tissues
having high uptake, for example, liver, have a larger number of receptors present.
The number of receptors decreases when a person is replete with iron and incr eases
with depletion. Fe2+ is incorporated in protoporphyrin IX with the help of the
enzyme “ferrochelatase” for the formation haem. Iron is also transported into cells
where it is used for both oxidative phosphorylation and as an enzyme cofactor .
Plasma “transferrin iron pool” is in equilibrium with the iron in sto rage forms, that
is, ferritin and haemosid erin ( Sherman, 1990; Crook, 2012; Chatterjea and Shinde,
2012).
2.7.5 Iron Requirements
Requirement of iron varies according to age, sex, weight and st ate of health . An
adult male requires approximately 10 mg/day and adult female 20 mg/day.
Pregnancy and lactation demands more; pregnant women require 10 mg/day and
lactating mothers 25 to 30 mg/day. Children require 10 to 15 mg/day (Oski, 1993).
2.7.6 Iron Deficiency (I.D)
Iron deficiency is one of the most common mineral deficiencies in the world.
Young children, and especially low birth weight infants, are at high risk of I.D
since their rapid growth leads to high iron requirements . Iron deficiency us ually
results from loss of blood in adults (including bleeding from menstrual periods)
but, in children and pregnant women, may result from an inadequate diet. . I.D

38
causes anaemia in men, women, and children. Globally, about 25% of pre -school
children are estimated to have I.D anemia (I.D.A), the most severe form of I.D.
Anaemia developed due to I.D make people appear pale and feel weak and tired.
Iron supplements, usually taken by mouth, are often needed . In addition to
anaemia, iron deficiency may cause pica (a craving for non foods such as ice, dirt,
or pure starch), spoon nails (thin, concave fingernails), and restless legs syndrome
(Beard, 2003; Hider and Kong, 2013 ).
2.7.7 Diagnosis of I ron deficiency
Iron deficiency is diagnosed based on symptoms an d blood test results. Results
include a low level of haemoglobin (which contains iron), a low haematocrit (the
percentage of red blood cells relative to the total blood volume ), and a low number
of red blood cells. These results indicate anaemia. In iron d eficiency anaemia, red
blood cells are abnormally small. Blood tests also include measurement of
transferrin , protein that carries iron in blood when iron is not inside red blood cells.
If the percentage of iron in transferrin is less than 10%, iron defic iency is likely.
Ferritin level is also estimated. Feritin is a protein that stores iron. Iron deficiency
is confirm ed if the Ferritin level is low. However, the ferritin level may be normal
or high when iron deficiency is present if people have inflammati on, an infection,
cancer, or liver damage ( Hider and Kong, 2013).

39
2.7.8 Iron toxicity
Excess iron can accumulate in the body. Causes include repeated blood
transfusions, iron therapy given in excessive amounts or for too long, chronic
alcoholism, an overdo se of iron or hereditary disorder called hemochromatosis.
Excess iron consumed all at once causes vomiting, diarrhoea, and damage to the
intestine and other organs. Excess iron consumed over a period of time may
damage coronary arteries (Oski, 1993).
2.8 Calcium (Ca)
Calcium is an important mineral mainly found in bone and teeth. Most of the
body’s calcium (over 99%) is located in the bones and teeth. The remaining 1% is
which is present on other tissues including the extracellular fluids, blood, play the
role of vascular contraction mediation, muscle contraction, vasodilation, glandular
secretion, and nerve transmission. In the bone, calcium is primarily in the form of
hydroxyapatite (Ca 10 (PO 4)6 OH 2) and the bone mineral makes up about 40% the
total bone weight. The osteoclastic and osteoblastic activity in the bone varies
between the different age group where osteoblastic activities is more in children as
against the high osteoclastic activities in adults and even more in aging adults
(Crook, 2012).

40
2.8.1 Dietary sources and Absorption of Ca
It is widely distributed in food substances such as milk, cheese, egg -yolk, beans,
lentils, nuts, figs, cabbage. Calcium is taken in the diet principally as calcium
phosphate, carbonate and tartarate. Unlike sodium (Na) and potassium (K) which
are readily absorbed, the absorption of Ca is rather incomplete. About 40% of
average daily dietary intake of Ca is absorbed from the gut. Calcium is absorbed
mainly from the duodenum and first half of jejunum against electri cal and
concentration gradients ( Ritchie and King, 2000; Chatterjea and Shinde, 2012 ).
2.8.2 Mechanism of Ca Absorption
Calcium is mainly absorbed in the intestine by both active transport and passive
diffusion into the enterocytes and out on the serosal side of the enterocytes
respectively. This transport is mediated by 25 -dihydroxycholecalciferol ( the active
form of vitamin D) in the intestinal mucosa (Ritchie and King, 2000; Chatterjea
and Shinde, 2012 ).
2.8.3 Factors Affecting Ca Absorption
Variou s factors which influence the absorption of calcium are discussed as follows:
a. pH of intestinal milieu : An acidic pH favours calcium absorption
because the Ca -salts, particularly phosphates and carbonates are quite
soluble in acid solutions. In an alkaline medium, the absorption of

41
calcium is lowered due to the formation of insoluble tricalcium phosphate
(Pettifor, 1994).
b. Composition of the diet : A high protein diet favours absorption, 15% of
dietary Ca is absorbed. If the protein content is low, only 5$ may be
absorbed. This is because amino acids increase the solubility of Ca -salts
and thus its absorption. Lysine and arginine obtained from basic proteins
cause maximal absorption of Ca. In malabsorption syndrome, fatty acids
are not absorbed properly. Fatty acids produce insoluble calcium soaps
which are excreted in faeces, thus, decreasing the Ca absorption. Also, an
organic acid produced by microbial fermentation of sugars in the gut
increase the solubility of Ca -salts and therefore increases their
absorpti on. Citric acid also may increase the absorption of calcium.
Cereals which contain phytic acid (inositol hexaphosphate) form
insoluble Ca -salts and decrease the absorption of Ca. Furthermore,
oxalates present in vegetable like cabbage and spinach forms ins oluble
calcium oxalates which are excreted in the faeces, thus lowering the
calcium absorption. Excess of phosphates and high content of magnesium
in the die lower calcium decreases absorption of calcium (Pettifor, 1994;
Chatterjea and Shinde, 2012 ).

42
c. State of health of the individual and aging : A healthy adult absorbs
about 40% of dietary calcium. Above the age of 60 years, there is a
gradual decline in the intestinal absorption of Ca. In sprue syndrome, the
intestinal absorption of calcium suffers due to formation of Ca -soap with
fatty aci ds which are excreted in faeces ( Chatterjea and Shinde, 2012).
d. Hormonal : Parathormone ( PTH) directly cannot increase the calcium
absorption. But PTH stimulates “1, α hydroxylase” enzyme in the kidney
and increases the synthesis of 1, 25 -(OH)2-D3 (calcitriol) which enhances
calcium absorption . Calcitonin directly cannot also affect Ca absorption.
Increased calcitonin level inhibits “ 1-α- hydroxylase ” enzyme, thus
decreasing synthesis of calcitriol and Ca -absorption. Glucoc orticoids also
diminish intestinal transport of calcium (Pettifor, 1994).
2.8.4 Regulation of Ca Concentration
Kidneys filter about 250 mmol of Ca++ very day, about 95% of which is reabsorbed
by the tubules. The major portion of this filtered Ca+2 is take n up by proximal
tubule without hormonal regulation. A fine adjustment to the amount reabsorbed
occurs in distal tubules under the influence of PTH (PTH -uptake). Plasma level of
ionized calcium concentration is the principal regulator of PTH secretion by a
simple negative feedback mechanism. A threshold level of magnesium is required
for PTH release. Hypermagnesaemia inhibits PTH secretion. PTH secretion is also

43
subject to negative feedback by the vitamin D metabolite 1,25 (OH) 2D3. PTH
rapidly stimulates os teoclast activity, the increased bone resorption causing an
increase in plasma Ca+2 and PO 4; Vitamin D3 plays a permissive role for this effect.
PTH stimulates more slowly (days) osteoblast activity. PTH via cyclic adenosine
monophosphate ( c-AMP ) increases the distal nephron reabsorption of calcium and
decreases that of PO 4 in the proximal tubule. In doing so, PTH increases the
tubular synthesis and excretion of c -AMP. PTH also stimulates the enzyme
complex that converts 25, OH D 3 to 1, 25 (OH) 2 D3, thereb y increasing calcium
uptake from the gut (Gazarini et al., 2003; Crook, 2012).
2.8.5 Functions of Ca
Calcium is needed for calcification of bones and teeth. Calcification is the process
involved in the formation of bones and teeth . Osteoblasts secrete an enzyme;
alkaline phosphatase which can hydrolyse certain phosphoric esters. Calcium plays
a role in blood coagulation by producing substances for thromboplastic activity of
blood. It also plays a role in neuromuscular transmission. Calcium ions are needed
for excitability of nerves and for muscle contraction. Normal excitability of heart is
Ca ion dependent. Calcium also functions as a secondary or tertiary messenger in
hormone action and in permeability of gap junctions (Kon et al., 2003).

44
2.8.6 Hyperca lcaemia and Hypocalcaemia
When the serum calcium level exceeds 11.0 mg/dl, it is called as hypercalcaemia
(Normal serum calcium level is 9 to 11 mg/dl). Hypercalcaemia may be caused by
primary hyperparathyroidism, malignancy, hyperthyroidism, hypothyroidis m,
acromegaly, acute adrenal insufficiency, granulomatous diseases (such as
Tuberculosis, Sarcoidosis, Berylliosis, Coccidioidomycosis ), Vitamin A
intoxication, hypervitaminosis D or drug -induced ( such as Thiazide diuretics,
Spironolactone, Milk -alkali syn drome ).
Hypocalcaemia is said to exist when serum calcium is less than 8.5 mg/dl as
determined by a standard method. The commonest cause of hypocalcaemia is
hypoalbuminaemia, closely followed by renal disease or renal failure. The other
most common cause o f hypocalcaemia is surgically -induced hypoparathyroidism
(Pettifor, 1994; Crook, 2012)
2.8.7 Measurement of total Ca
Spectrophotomet ric, ion specific electrodes (ISEs) and occasionally atomic
absorption methods are routinely used for the measurement of Ca . According to the
College of American Pathologists Comprehensive Chemistry Survey, in 2007
approximately 75% of participating clinical laborator ies used spectrophotometric
methods. Spectrophotometric methods use metallochromic indicators that change
colou r when they bind calcium. Although less accurate than atomic absorption

45
spectrometry, they have been easier to automate. With ISEs the specimen is
acidified to convert protein -bound and complexed Ca to free Ca before
measurement of free Ca (Tietz, 2008).
2.9 MAGNESIUM
Magnesium, the major intracellular divalent cation is one of the most crucial
micronutrients needed for normal physiological functions of the body.
(Swaminathan, 2003). The two important properties of magnesium which enable it
play these roles are:
(a) Its ability to form chelates with most important intracellular ligands that are
anionic in nature e.g. ATP.
(b) It is able to undergo competitive binding with calcium for binding sites on
membranes and proteins (Ryan, 1991).
Magnes ium play s a major rol e in the synthesis of proteins and nucleic acids and
act as a co -factor for most enzymes and membrane transporters. Also, when
combined with ATP, magnesium help to maintain proper cardiovascular system
functioning as well as other biological processes such as cell replication, protein
synthesis, and in energy metabolism (Noronha and Matuschak, 2002).
Magnesium enhances enzyme activities by its ability to:
(i) Bind to ligands such requiring enzymes.
(ii) Bind to the active sites of enzymes.

46
(iii) Cause conformational chan ge especially during catalytic processes, e.g. Na+,
K+-ATPase,
(iv) Promote aggregation of multi -enzymes complexes e.g. aldehyde
dehydrogenase (Ryan 1991 ).
2.9.1 CHEMISTRY
Magnesium belongs to group 2 of the periodic table, along with Beryllium,
Calcium, Str ontium and Barium. The element has an atomic number of 12, an
atomic mass of 24, one main oxidation state (+2) and three naturally occurring
isotopes (24Mg, 25Mg and 26Mg), of which 24Mg is the major isotope at 79% of the
total mass. Magnesium is the sev enth most abundant element in the Earth’s crust
with a quoted average of 2.76% (Fyfe 1999), and the Mg2+ ion is the second most
abundant cation in sea water, after Na+. Its chemistry is intermediate between that
of Beryllium and the heavier alkali earth e lements. Magnesium is a lithophile
metallic element and a major constituent of many mineral groups, including
silicates, carbonates, sulphates, phosphates and borates. It forms several important
minerals, including magnesite MgCO 3, dolomite CaMg(CO 3)2, pyrope garnet
Mg 2Al2(SiO 4)3 and kieserite MgSO 4.H2O.

47
2.9.2 DIETARY SOURCES
In nature, magnesium is abundant in mostly green vegetables such as spinach,
broccoli (which have magnesium -rich chlorophyll), nuts, banana, legumes, cereal ,
and grain. Animal produ cts such as meat, fish, and chocolates contain fewer
amounts compared to those mentioned above, while dairy products contain very
low amount (Saris et al., 2000). Magnesium is also present in drinking water
especially in hard water which contains up to ab out 30mg/L . processes such as
food processing, heating (boiling) of magnesium rich food can lead to magnesium
depletion up to 85% reduction (Fawcett et al., 1999 ). This may therefore explain
the reason for the apparently high magnesium deficiency in many p opulations.

48
Table 1: Food sources of Magnesium (Saris et al., 2000)
Food Magnesium (mg/100 g)
Milk, cow’s whole 11
Cheese, cheddar 25
Eggs 12
Meat 21 – 24
Fish 25 – 50
Cabbage 4
Potatoes 14
Peanuts, dry roasted 190
Bread, white 24
Bread, w holemeal 76
Chapatti 37
2.9.3 Absorption, Transport, Metabolism a nd Excretion of Magnesium
An apparently healthy adult human have approximately 1000mmol of magnesium
(Saris et al., 2000). Most of the absorption (about 30-40%) takes place in the
jejunum a nd ileum in the small intestine where the fractional intestinal absorption
is inversely proportional to the intake levels (i.e. 65% at a low intake and 11% at a
high intake) . Normally, absorption is passive with a saturable demonstration at a
low intake (Kayne1993). Although factors affecting magnesium absorption is
idiopathic, studies have suggested that the parathyroid hormone play a vital role in
this regard. Vitamin D (1, 25 -dihydroxycholecalciferol) has been known to

49
promote stimulate its absorption ef ficiency. However, the role of vitamin D and its
active metabolite 1, 25 (OH) 2 D is controversial (Swaminathan 2003).
Results from studies on magnesium distribution in the body revealed that about
60% is present in the bone where about 30% is exchangeable and acts as a body’s
reservoir for magnesium to stabilize serum concentration. Other magnesium
storage organs include skeletal muscles (about 20%), other soft tissues (about
19%), liver (about 7 -9mmol/kg) and 1% in the extracellular fluid (Swaminathan
2003 ).
Table 2: Distribution of magnesium in the adult human (Saris et al., 2000)
Tissue Weight (Kg
wet wt) Concentration
(mmol/Kg wet wt) Content
(mmol) % of total body
magnesium
Serum 3.0 0.85 2.6 0.3
Red blood cells 2.0 2.5 5.0 0.5
Soft tissue 22.7 8.5 193.0 19.3
Muscle 30.0 9.0 270.0 27.0
Bone 12.3 43.2 530.1 52.9
TOTAL 70.0 1000.7 100

50
The normal range for magnesium in adults is between 0.70 -1.10mmol/L where
about 20% is protein -bound, 65% is in the ionized form and others are complexed
with variou s anions such as citrates and (Saris et al., 2000). About 60 -70% of the
protein -bound are bound to albumin while others to the globulins (Kroll and
Elin, 1985).
The kidney is the major organ that plays a key role in magnesium homeostasis in
the body where urinary magnesium excretion coincides with its absorption in the
intestine at approx. 4mmol/ day/. In maintaining balance in plasma magnesium
levels, 84mmol is filtered daily and about 95% of this value is reabsorbed leaving
about 3 -5mmol in the urine, wher e about 20% is reabsorbed in the proximal
tubules, 65 -75% in the thick ascending limb of the loop of Henle, and the
remaining 5 -10% in the distal convoluted tubule (Quamme and Rabkin, 1990 ). In
the proximal tubule depending on sodium/water re -absorption an d the luminal
magnesium concentration, magnesium movement tends to be unidirectional
passive process (de Rouffignac and Quamme 1994 ). Approximately 25% of the
filtered NaCl is reabsorbed in the In thick ascending limb via an active
transcellular transport (NaCl -K2 transport) ( Konrad and Weber, 2003 ). Thereby
creating a favorable lumin al positive potential at the thick ascending limb where
most of the magnesium is reabsorbed (Dai et al., 2001 ).

51
2.9.4 FUNCTIONS OF MAGNESIUM
Magnesium is a cofactor in more th an 300 enzyme systems that regulate diverse
biochemical reactions in the body, including protein synthesis, muscle and nerve
function, blood glucose control, and blood pressure regulation. Magnesium is
required for energy production, oxidative phosphorylat ion, and glycolysis. It
contributes to the structural development of bone and is required for the synthesis
of DNA, RNA, and the antioxidant glutathione. Magnesium also plays a role in the
active transport of calcium and potassium ions across cell membrane s, a process
that is important to nerve impulse conduction, muscle contraction, and normal
heart rhythm (Noronha and Matuschak, 2002 ).
2.9.5 HYPERMAGNESAEMIA
Hypermagnesaemia has a prevalence rate that varies from 5.7% to 9.3% (Whang
and Ryder 1990 ; Huey et al., 1995). It has been reported that the highest serum
magnesium levels so far is 18mmol/L in a premature 33 week old baby and
13.4mmol/L in a 78 year old woman who was reported to have swallowed water
from the Dead Sea (Huey et al., 1995) , and severe hypermagnesaemia have been a
feature of those who have drown in the Dead Sea (Oren et al., 1987 )

52
2.9.5.1 CAUSES OF HYPERMAGNESAEMIA
Most often, Hypermagnesaemia is due to excessive administration of medications
with high magnesium salt or magnesium -contain ing contents mostly in patients
with impaired renal (Swaminathan, 1998 ). These magnesium containing drugs are
commonly used as antacids, rectal enemas and laxatives. Also drugs used as
treatment for drug overdose usually contain magnesium (magnesium -contai ning
cathartics) which may also induce hypermagnesaemia in these persons (Jaing et al.,
2002 ). However, 75% of those patients did not develop hypermagnesaemia due to
the fact that the ingested dose did not exceed the normal dose but developed bowel
disorde rs which may enhance the absorption (Clark and Browns, 1992 ) hence, the
need to maintain the serum magnesium levels in these patients is paramount. There
have been reports that revealed a high serum magnesium levels of 9.5mmol/L after
an overdose of magnes ium-containing cathartics which resulted in coma
(Swaminathan 1998 ), fatal hypermagnesaemia observed following magnesium
sulphate gargles (Birrer et al., 2002), and also urethral irrigation with hemiacidrin
was reported by Swaminathan , (1998 ).

53
2.9.6 HYPO MAGNESAEMIA
Hypomagnesaemia which is the reduction of serum magnesium may be cause by
any of the following factors:
a) Reduce intake
b) Reduced intestinal absorption
c) Redistribution
d) Increased gastrointestinal and renal loss
Most commonly, hypomagnesaemia caused by a magnesium shift from the
extracellular fluid into the cells or to the bone tissue have been seen in those with
re-feeding syndrome in starved patients, treatment of metabolic acidosis, hungry
bone syndrome (seen in post -parathyroidectomy or in diffuse osteoblastic
metastasis). Hypomagnesaemia have been observed in patients with acute
pancreatitis (about 20%) probably due to magnesium deposition in the areas with
necrosis (Ryzen and Rude 1990) . The intracellular shift caused by the high
concentrations o f catacholamines among those after cardiac surgery and those with
congestive heart failure may contribute to the hypomagnesaemia seen in these
patients (Satur et al., 1995).

54
2.10 Effects of Malaria parasite on Micronutrients in Children
Malaria parasite, a pathogenic agent of malaria remain s a major cause of morbidity
and mortality in children below the age of five in many developing countries
(Jeffrey and Pia, 2002). Micronutrients deficiency such as vitamins, Magnesium,
iron, and calcium are more frequent amongst children in developing countries
(Gibson and Ferguson, 1998). These deficiencies are often associated to increased
susceptibility to infections such as malaria (Shankar and Prasad, 1998). Some
authors have associated malaria acquisition and its se verity (or Plasmodium
virulence) to the concentrations of micronutrients in children (Nyakeriga et al.,
2004; Wander et al., 2009).
2.11 Iron Levels in Malaria parasite Infection
Plasmodium falciparum has a damageable effect on serum iron and significantly
decrease its level (Onyesom et al., 2012). A study conducted by M’boh and
colleagues (2010) showed a low value of serum Iron levels among children
infected with malaria parasite. Iron is an essential constituent of haemoglobin
which transports and stocks oxygen in organism, playing a functional role in
oxidative metabolism, cellular proliferation and others physiological processes
(Oppenheimer, 2001). This micronutrient is important for all pathogenic

55
organisms; in free state it may encourage their virulen ce and as such, its rapid
metabolism will result in consequent serum decrease.
2.12 Calcium Levels in Malaria Parasite Infection
Malaria parasite significantly decreases calcium levels. Gouado et al ., (2007)
reported a significant decrease in calcium level in children infected with malaria
parasite. This translates to the negative effect of malaria on the levels of calcium.
Reduction in calcium observed in malaria cases is caused by the clinical
manifestation of malaria: fever, increase in pulse rate, sweat , shivering (Golvan,
1983) which affects neuromuscular excitability, nerve conduction and muscular
contraction. These physiological phenomena require calcium for their functioning
(Nestec, 1989). Thus half of the cases of tetany, neonatal convulsion, and l ow birth
weight are due to hypocalcemia (Pettifor, 1994). Gazarini et al. (2003) also
reported that trophozoites concentrate calcium in their internal compartment for
metabolism. Decrease in calcium can also be caused by losses during digestive and
renal p roblems following malaria parasite infection.
2.13 Magnesium Levels in Malaria Parasite Infections
Malaria parasite infection significantly reduced serum Magnesium levels among
children infected with malaria parasite (Onyesom et al., 2012). This micronutri ent
is further reduced as the degree of malarial infection increases (Onyesom et al.,

56
2012). The malarial causative agent – P. falciparum , is capable of sticking to blood
vessels by a process known as cytoadherence. This sticking red cells leads to
obstruct ion of microcirculation which results in dysfunction of multiple organs and
breakdown in the body’s immune system. These resultant effects of P. Falciparum
cytoadherence could modify micronutrient levels including Mg and other essential
trace elements in s erum of infected patients. Therefore, the activities of P.
falciparum reduces the amounts of red blood cells (Nyakeriga et al., 2004) and its
replenishment is interrupted by the depleted levels of iron which is needed to
produce red blood cells (Wander et al., 2009). The associated reduction in
magnesium levels will also hamper magnesium -dependent functions in the body.
2.14 Folate Levels in Malaria Parasite Infection
In children with malaria, folate concentrations have been reported as either normal
or rai sed. In a holoendemic area in Kenya, P. falciparum infection had little
influence on cord plasma or red cell folate activity in newborns (Brabin, 1985).
Raised red cell folate in malaria was first reported from Nigeria, where the mean
red cell folate level in 198 children exposed to malaria was significantly higher
than in 185 age -matched control children (Bradley -Moore et al ., 1985). Among
106 children in the Gambia with malaria and anemia, normal or increased levels of
red cell folate were found in 75 at presentation and in 15 at 1 to 2 weeks after
treatment with antimalarials alone. In a further study in the Gambia, only 8% of

57
600 children with falciparum malaria had low red cell folate (Herrmann et al.,
2003). Red cell folate concentrations in Malawian c hildren with P. falciparum
malaria were within the ranges reported for other populations. Serum folate was
normal in all the children with malarial anemia in whom it was measured in a
recent study in Zambia (Helman, 1987). In New Guinea, where there is a h igh rate
of malaria transmission, red cell folate was higher in infants with malaria
parasitemia, both with P. falciparum and with P. vivax (Carmel, 1995). The
significance of the raised red cell folate concentrations reported in malarial
infection is crit ical in the assessment of a possible relationship of folate nutritional
status to the risk of malaria, and various hypotheses have been considered. The
raised levels could be due to de novo parasite folate synthesis, as in vitro studies
have shown an incre ase in folate in animal cells infected with malaria (Carmel et
al., 1999).
2.15 Vitamin B 12 Levels in Malaria Parasite Infection
Vitamin B12 absorption is a complex process, involving a series of steps that can
be affected adversely by intestinal disease, infections, and medications. Plasma
vitamin B12 concentrations were not different in malaria parasite infected children
(Allen, 2008). However, evidence suggests that Vitamin B12 been fundamental for

58
erythropoietic process, its absorption may be compromise d in P. falciparum
malaria (Areekul et al., 1972).

59
CHAPTER THREE
3.0 MATERIAL AND METHODS
3.1 Study Area
This study was carried out in Central Hospital, Benin city, Edo state .
3.2 Study Populatio n
The Study group consists of children attending paediatric clinic in Central
Hospital, Benin City.
3.3 Inclusion criteria: children within the age of 1 -10years
3.4 Exclusion criteria : children who are
i. Receiving anti -malaria
ii. Currently receiving vitamin su pplement s
iii. Children above 10years
3.5 Control group : children above ten years without malaria
3.6 Sample Collection
5ml of blood was collected (with minimum of stasis) into a plastic tube containing
1ml of aqueous tri -sodium citrate without delay ,the sampl e was centrifuged at
1200g for 15minutes.The pl asma was separated into plain plastic tube and kept
frozen till the day of analysis. 2ml of blood sample was collected into EDTA for
malaria parasites test.

60
3.7 LABORATORY ANALYSIS
3.7.1 PROCEDURE FOR MALARI A PARASITE TEST
Thick Film
Using a grease free microscope slide, a large drop of blood about 15mm, was
placed on the slide. Without delay, the end of a plastic bulb pipette was used to
spread the drop of blood to make the thick smear which co vers an area o f about 15
× 15mm . The blood films were then allowed to dry.
Staining of thick film
1. The slide was placed in a staining rack and was flooded with I:10 dilution of
giemsa stain and was allowed to act for 30minutes.
2. The slides wer e rinsed and allowed to air d ry.
3. A drop of immersion oil was added to the smear and examined on the
microscope using
100 objective
4. Parasites was counted and reported in +
3.7.2 ASSAY OF SERUM CALCIUM CONCENTRATION
Method: Calcium Arsenazo III
Principle: At midly acidic p H, metallo -chromogen Arsenazo III combines with
calcium to form a coloured complex which absorbance measured at 650nm (640 –
660) is proportional to the amount of calcium in the sample.
Reagent composition:
Arsenazo III reagent

61
Imidazol buffer pH 6.8 at 25oc >90mmol/L
Arsenazo III >0.18mmol/L
Surfactant preservative 0.1%
Standard
calcium conc. 10mg/L (2.5mmol/L)
Table 3: Procedure/protocol table for calcium estimation.
No of tubes Blank Standard Sample
Reagent 1mL 1mL 1mL
Distilled water 20µL – –
Standard – 20µl –
Sample – – 20µL

Mix well and allow to stand for 1 minute at room temperature.
Read absorbance at 650nm against a reagent blank.
The colour is stable for 1 hour away from light.

62
CALCULATION
Calcium Conc. = _________________ ___

Reference Range:
8.6 – 10.0mg/dL
3.8 QUALITY CONTROL
In other to ensure accuracy and precision, quality control sera supplied by bio -labo
with reference number 95010 was included in the assay .
3.9 ANALYSIS FOR VITAMIM B12 AND FOLIC ACID
3.9.1 Electro luminescence (ECL) For Measurement of Vitamin B12
Principle
Electroluminescence (ECL) is a process in which reaction of highly reactive
molecules are generated from stable state electrochemically by an electron flow
cell forming highly reacted species on a surface of a platenium electrode producing
light. This method uses ruthenium (II) -tris (bipyridyl) [Ru (bpy)3 ]2+ complex and
triproplamine (TPA) and react them with each other to emit light. The applied
voltage creates an electrical field that causes the reaction of all materials. Abs of sample
Abs of standard X 10µg/dL

63
Tripropylamine (TPA) oxidized at the surface of the electrode, releases an electron
and forms an intermediate which may further react by releasing a proton. In turn
the ruthenium complex releases an electron at the surface of th e electrode forming
an oxidized form of Ru(bpy)33+ cation, which is the second reaction component
for the chemiluminescent reaction. Then this cation will reduce and form Ru(bpy)3
2+ and an exited state via energy transfer which is unstable and decays with
emission of photon at 620 nm to its original state (Mathew et al., 2005). The
florescence emitted by Ru(bpy)32+ is detected by standard photomultiplier, and
the results are expressed as ECL intensity, which is the measurement of the whole
luminescence emi tted from the sample. This method employs various test
principles (such as competitive principle, sandwich and bridging) for the
measurement. The most important one in measuring vitamin B12 concentration is
the competitive principle.
3.9.2 Methodology/Step s
1. Antibodies (intrinsic factor) for vitamin B12 is labeled with ruthenium
complex.
2. The antibodies were incubated with the sample
3. biotinylated vitamin B12 and streptavidin which is coated with paramagnetic
miroparticles are added to the mixture.
4. The free binding sites of the labeled antibody become occupied with the

64
formation of an antigen -hapten complex.
5. The entire complex is bonded to biotin and streptavidin.
6. After incubation, the reaction mixture is transported into the measuring cell
where the immun e complexes are magnetically entrapped on the working
electrode and the excess unbound reagent and sample are washed away.
7. The reaction is stimulated electrically to produce light which is indirectly
proportional to the amount of vitamin B12 that is measu red.
8. The sample duration time is 27 minutes, and the test is very sensitive with
detection limit of 22 pmol/L (30 pg/ml).

Figure 9: Electroluminescence method, competitive principle for measuring
vitamin B12.

65
3.9.3 Enzyme -linked Immunosorbent Assay Kit F or Folic Acid (FA)
Principle
This assay employs the competitive inhibition enzyme immunoassay technique. A
monoclonal antibody specific to folic acid has been pre -coated onto a microplate.
A competitive inhibition reaction is launched between biotin labele d folic acid and
unlabeled folic acid (Standards or samples) with the pre -coated antibody specific to
folic acid. After incubation the unbound conjugate is washed off. Next, avidin
conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and
incubated. The amount of bound HRP conjugate is reverse proportional to the
concentration of folic acid in the sample. After addition of the substrate solution,
the intensity of color developed is reverse proportional to the concentration of folic
acid in the sample.
3.9.4 Reagents and Quantity
I. Pre-coated, ready to use 96 -well strip plate
II. 1 Plate sealer for 96 wells 4
III. Standard 2
IV. Standard Diluent 1×20mL
V. Detection Reagent A 1×120μL
VI. Assay Diluent A 1×12mL
VII. Detection Reagent B 1×120μL

66
VIII. Assay Diluent B 1×12mL
IX. TMB Substrate 1×9mL
X. Stop Solution 1×6mL
XI. Wash Buffer (30 × concentrate) 1×20mL
3.9.5 Methodology/Steps
1. wells for standard, sample and blank were pre pared and 50μL each of dilutions
of standard, blank and samples were added into the appropriate wells, respectively
and 50μL of detection Reagent A were added to each well immediately. The plate
was shaked gently and cover with a Plate sealer and Incubated for 1 hour at 37oC.
2. The solution was aspirated and washed with 350μL of 1X Wash Solution to
each well using a squirt bottle, multi -channel pipette, manifold dispenser or
autowasher, and allowed to stand for 1 -2 minutes. The remaining liquid was
remov ed from all wells completely by snapping the plate onto absorbent paper and
this was repeated
3. 100μL of Detection Reagent B working solution was added to each well and
incubated for 30 minutes at 37oC after covering it with the Plate sealer.
4. The aspi ration/wash process was repeated for total of 5 times as conducted in
step 2.

67
5. 90μL of Substrate Solution was added to each well and covered with a new Plate
sealer and incubated for 15 – 25 minutes at 37oC.
6. 50μL of Stop Solution was added to each well. The liquid will turn yellow by
the addition of Stop solution. The liquid was mixed by tapping the side of the plate.
7. The microplate read was measured at 450nm immediately.s

Calculation of Results
This assay employs the competitive inhibitio n enzyme immunoassay technique, so
there is an inverse correlation between folic acid concentration in the sample and
the assay signal intensity. Average the duplicate readings for each standard,
control, and samples. Create a standard curve on log -log or semi-log graph paper,
with the log of folic acid concentration on the y -axis and absorbance on the x -axis.
Draw the best fit straight line through the standard points and it can be determined

68
by regression analysis. Using some plot software, for instance, curve expert 1.30, is
also recommended. If samples have been diluted, the concentration read from the
standard curve must be multiplied by the dilution factor.
3.9.6 ASSAY OF SERUM MAGNESIUM CONCENTRATION
Method : Xylidyl blue
Principle: Magnesium reacts wi th xylidyl Blue to form a coloured compound in
alkaline solution. The intensity of the colour formed is proportional to the
magnesium in the sample.
Reagent composition:
Xylidyl Blue 110mmol/L
Ethanolamine (pH 11.0) 1mol/L
GEDTA 60mmol/L
Standard
Magnesium conc. 2mg/dL

69
Table 4: Procedure/Protocol table for magnesium estimation
No of test tube Standard Sample Blank
Reagent 1000µ l 1000µ l 1000µ l
Serum – 10µl –
Standard 10µl – –
Mix and incubate for 5minutes at 37oc. The absorbance of sample and standard was
measured against reagent blank at 546nm.
CALCULATION

Reference Range:
Serum: 1.8 – 2.6mg/dL
3.10 PROCEDURE FOR SERUM IRON
Principle
Iron III reacts with chromoazural b (CAB) &cetyltrimethyl ammonium bromide
(CTMA ) to form a coloured ternary complex with an absorbance maximum at 623
nm. The intensity of the colour produced is directly proportional to the
concentration of iron in the sample.

70
Procedure
1. Fresh test tubes were set up together with a test tube labelled standard and
blank
2. 50μl of sera (test) and standard were pipetted and dispensed into the test tubes
and standard test ubes respectively.
3. 50μl of distil water was pipetted into the tube labeled blank
4. 1000μL of reagent was pipetted into all tubes
5. The tubes were mixed properly and incubated in room temperature for 15 mins.
6. The absorbance of all tubes were read against reagent blank at 623nm
7. The concentration of iron in each sample was calculated using Beer -Lambert’s
Law.
Calculation of Iron concentration
From Beer -Lambert’s Law (First principle)
Tab x conc. STD
Sab 1
Where T ab = absorbance of sample
Sab = absorbance of standard, = 100μg/dl
Δ Ab Sample x 10
Conc of sample (μg/dl.) = Ab STD
This method is linear up to an iron concentration of 500μg/dl or 89.5μmol/l.
Reference value
Male: 59 -148 μg/dl or 10.6 -28.3μ mol/l.

71
Female: 37 -145μg/dl or 6.6μ mol/l.
3.11 STATISTICAL ANALYSIS
Values were represented as mean±SEM. The values were analyzed using the
Duncan’s multiple ranged analysis of variance of the SPSS (version 17.0). The
means were also represented as bar -charts using the excel Microsoft word spread
sheet (version 7).

72
CHAPTER FOUR
4.0 RESULTS
Table 1 : Age, sex, and malaria load of study and control subjects

Table 1 above shows that a total sample of 100 tests and 50 control subjects
recruited for this st udy were males while 100 test and 50 control subjects were
females. 60 tests and 40 control subjects fell under ages 6months -4 years, 71tests
and 40 controls fell under ages 5 -10 years, and 69 tests and 20 controls group fell
under ages 10 years and above. 50, 80, and 70 tests subjects were observed to give
a malaria load of one plus ( +), two pluses (++), and three pluses (+++) respectively.

Variables Study group (n = 200) Controls (n = 10 0)
Age brackets 6mnths -4 years 60 40
5-10 yrs 71 40
>10 yrs 69 20
Sex Males 100 50
females 100 50
Malaria load + 50 –
++ 80 –
+++ 70 –

73

Figure 1 : showing the ages of the study and control groups

Figure 2 : showing the sex of the study and contro l groups

74
Figure 1 and 2 above are graphical representation that shows that a total sample of
100 tests and 50 control subjects recruited for this study were males while 100 test
and 50 control subjects were females. 60 tests and 40 control subjects fell under
ages 6months -4 years, 71tests and 40 controls fell under ages 5 -10 years, and 69
tests and 20 controls group fell under ages 10 years and above.

75
Table 2 : Comparison between the PCV, RBC, HgB conc., red cell indices, total
and differential leucocyte counts, NLR, and PLR of the study and control
groups.
Values are represented as mean ± SEM for six determinations. Means with ** are
significantly different (p<0.05) by independent t- test
Variables Study subjects mean ± SEM
(n = 200) Controls mean ± SEM
(n = 100) P-value
PCV (%) 31.59±0.10* 36.85±0.10** 0.032
RBC (×103 /ul) 4.21±0.12* 8.22±0.03** 0.023
Hb. conc. (g/dl) 9.08±0.20* 13.65±0.09** 0.021
MCV 80.31±0.92* 79.20±0.40* 0.980
MCH 20.25±0.40* 23.20±0.30* 0.872
MCHC 33.84±0.30* 32.26±0.20* 0.074
PLT 267.21±2.00* 263.33±2.00* 0.653
TWBC 9.34±0.62* 1.04±0.03** 0.023
LYMC 41.43±2.00* 9.07±0.20** 0.037
NEUTROPHIL 55.86±2.00* 47.00±4.00* 0.614
EOSINOPHIL 9.19±1.00* 10.20±1.00* 0.067
NLR 1.46± 0.01 6.36± 1.20 0.021
PLR 8.15 ± 1.02 26.45± 2.01 0.021

76
Table 2 shows that there was a significant decrease in PCV, RBC, and
haemoglobin concentration, increase total white blood cell count with high
lymphocyte count, decreased neutrophil -lymphocyte ratio (NLR) and platelet –
lymphocyte ratio (PLR) in children tested positive for malaria when compared to
those of the control group at P<0.05. All other variables showed no significant
change (P>0.05).

77

Figure 3 : Comparison between the PCV, RBC, HgB conc., and red cell
indices, of the study and control groups.
Figure 3 above is a graphical representation that shows that there was a significant
decrease in PCV, RBC, and haemoglobin concentration while changes in red cell
indices show no significance in children sero -positive for malaria.

78
Table 3 : Comparing the calcium, magnesium, iron, vit. B12, and folic acid
levels between the malaria Positive subjects and Malaria negative subject s
Values are represented as mean ± SEM for six determinations. Means with ** are
significantly different (p<0.05) by independent t- test
Table 3 showed a significant decrease in serum calcium, iron, vitamin B12 and
folic acid in children infected with malaria when compared to those of the control
group at P<0.005. There was no significant change in serum magnesium
(P>0.005).

Variables Malaria positive subjects
mean ± SEM (n = 200) Malaria negative subjects
mean ± SEM (n = 100) P-value
Calcium (mg/dl) 8.45±0.20* 12.26±0.30** 0.048
Magnesium (mg/dl) 2.13±0.07* 5.85±0.30* 0.776
Iron (mg/dl) 102.12±1.96* 167.2 6±3.00** 0.035
Vitamin B12 435.53±43.00* 772.31±57.12** 0.030
Folic acid 7.28±0.20* 17.67±0.50** 0.024

79

Figure 4 : Comparing the calcium, magnesium, iron, Vit. B12, and folic acid
levels between the malaria Positive subjects and Malaria negative subjects
Figure 4 above is a graphical representation of the significant decrease in serum
calcium, iron, vitamin B12 and folic acid in children infected with malaria when
compared to those of the control group. There was no significant change in serum
magnesium.

80
CHAPTER FIVE
DISCUSSION
This research was aimed at establishing the effect of malaria parasite on
neutrophil -lymphocyte ratio, platelet lymphocyte ratio and some essential plasma
electrolytes (calcium, magnesium and iron) as well a s vitamins (vitamin B12 and
folate). These trace metals and vitamins are important for the formation of RBC,
and by extension, well being and longevity. A total of 300 subjects were used for
the study; 200 tests (malaria patient s) within the ages of 1 -5yea rs and 100 controls,
aged between the age of 10 years and above.
PCV decreased significantly compa red to the control group p>0.05 . This decrease
is supported by the significant decrease in haemoglobin concentration. However,
RBC decreased significantly for the test group compared to the control group. The
noticed decreased in PCV level in the malaria subjects is basically attributed to the
increased lysis of RBC by the plasmodium parasites as well as, membrane damage
by the generated reactive oxygen speci es. This is in agreement with previous work
done by (Oppenheimer, 2001).
White blood cell increased (p<0.05) compared to the control, as well as
lymphocytes (40%) compared to the control (10%). The malaria parasites are
capable of inducing immune reaction th rough antigen -antibody interaction and
secretion of toxins which initiate the release of inflammatory pyrogens that act on

81
the thermo -regulatory apparatus of the hypothalamus, thus resetting the body
temperature from the physiologic 37oc to higher than 40oc (Fever) seen in malaria
patients. The significant increase in lymphocytes is a defence response against the
malaria plasmodium parasites. This is in agreement with research done by (Gouado
et al (2007) who also reported increase in total white blood cell count in persons
with malaria.
Plasma calcium, magnesium and iron decreased significantly p >0.05 indicating a
possible loss through vomiting and/or diarrhoea that may be associated with the
feverish condition of the patients. Iron is an important co -factor required for the
formation of the heme component of RBC haemoglobin. The significant decrease
in the element might be a contributory factor to the low haemoglobin concentration
(10g/dl) compared to the control group (13g/dl) observed. The decrease in these
elements is in consonant with previous work done by Onyesom et al., (2012) who
reported that Malaria parasite infection significantly reduced serum Magnesium
levels among children i nfected with malaria parasite. Gouado et al ., (2007)
reported a signif icant decrease in calcium level in children infected with malaria
parasite. Plasmodium falciparum has a damageable effect on serum iron and
significantly decrease its level (Onyesom et al., 2012).Calcium and magnesium are
important components of the bone m arrow which is an important site RBC
generation making the decrease of these metals a possible reason for the decreased

82
RBC. Apart from loses of these metals through vomiting and stooling, the decline
in appetite and lack of feeding in these patients could also be possible reasons for
the decrease in RBC count.
Vitamin B12 and folate are important co -enzymes that are needed for the
maintenance and proper formation of the RBC structure and membrane integrity.
The unavailability of these vitamins results in t he formation of poorly structured
and lysis -prone RBC so that the primary function is compromised. There was
observed significant decrease in these two vitamins in the malaria patients
attributable to poor feeding in the patients . The low values gotten fo r vitamin B 12
is in consonant with work done by (Areekul et al ., 1972) who repo rted that
Vitamin B12 being fundamental for erythropoietic process, its absorption may be
compromised in P. falciparum malaria . The loss or decrease in folate values in
this re search does not agree with previous work who reported that in children with
malaria, folate concentrations have been reported as either normal or raised. In a
holoendemic area in Kenya, P. falciparum infection had little influence on cord
plasma or red cel l folate activity in newborns (Brabin, 1985 ).

83
5.1 CONCLUSION AND RECOMMENDATION
The increased clinical state of malaria infection may be due to poor nutritional
status more especially as a resu lt of micronutrients deficiency . Micronutrients play
vital role both in combating anaemia and other adverse effects of malaria infection
in humans and animals in developing resistance against the disease. Micronutrients
are not only necessary in the regeneration of heamolyzed red cells during malaria
infection, bu t also served as antioxidants hence protecting the red cells against
damage by malaria toxins. It is therefore of tremendous importance to assess
micronutrients status of children with malaria .
.

84
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