Case study of megaloblastic aneamia

Dissertation Submitted to the

Dr. Babasaheb Ambedkar Technological University,
Lonere,Maharashtra

In Partial fulfillment of the requirements for the degree of

BACHELOR OF PHARMACY
By
Mr. Ravi madhukar Bhombe

Under the guidance of

Miss. Priti kale
Assistant Professor

Department of Pharmaceutics

SSJP’s, Ojas College of Pharmacy, Revgaon road,

Rohanwadi, Jalna- 431 203 (M.S.)
2021 -2022
 CERTIFICATE BY THE
GUIDE

This is to certify that, this dissertation entitled“Case study of Aneamia”is a bonafied

and genuine practice school work carried out [Link] madhukar Bhombe under
my guidanceat, SSJP’s, Ojas College of Pharmacy, Revgaon road, Rohanwadi,
Jalna,in partial fulfillment of the requirements for the degree of Bachelorof Pharmacy.

Place: Jalna Signature

Date: Miss. Priti kale
Assistant professor
Department of
Pharmaceutics2(MS.)
 DECLARATION BY THE
CANDIDATE

I hereby declare that, this dissertation entitled“Case study of Asthmais a bonafied

and genuine practice school work carried outunder the guidance of Miss. Priti kale,
Assistant Professor, Department of Pharmaceutics,SSJP’s, Ojas College of Pharmacy,
Revgaon road, Rohanwadi, Jalna.

Place:Jalna Signature of Candidate

Date: Mr. Ravi madhukar Bhombe

SSJP’s, Ojas College of Pharmacy, Revgaon road,

Rohanwadi, Jalna- 431 203 (M.S.)
 ENDORSEMENT BY THE
PRINCIPAL

This is to certify that, this dissertation entitled“Case study of Asthma”is a bonafied

and practice school work carried out byMr. RushikeshGopal Gawhaleunder the
guidance ofMiss. Priti kale,Assistant Professor, Department of
Pharmaceutics,SSJP’s, Ojas College of Pharmacy, Revgaon road, Rohanwadi, Jalna.

Seal and sign of Principal

Dr. V. G. Kuchake
Principal,

SSJP’s, Ojas College of Pharmacy, Revgaon road,

Rohanwadi, Jalna- 431 203 (M.S.)
 EXAMINER’S CERTIFICATE

It is to certify that,Mr Ravi madhukar Bhombe, (Exam seat Number-

20258920181382310047), studying in the Seventh Semester of Bachelor of
Pharmacy, has been attended the viva-voce and defended the dissertation/project
entitled“Case study of Aneamia” on (Date:) at SSJP’s, Ojas College of Pharmacy,
Revgaon road, Rohanwadi, Jalna.

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Place: Place:
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 Case study of megaloblastic aneamia

 Introduction:
Anaemia is defined as a reduction in haemoglobin (Hb) concentration, haematocrit,
or a number of red blood cells per litter below the reference interval for healthy
individuals of similar age, sex, and race, under similar environmental conditions .
According to the World Health Organization (WHO), for under five years children,
the threshold Hb level for being anaemic is less than 11.0g/dl Anaemia is a
common clinical condition characterized by decreased Hb levels which are
insufficient for the body’s demand Anaemia, has been shown to be a public health
problem that affects low, middle and high income countries at different degrees and
the prevalence varies with socioeconomic state.
Anaemia resulting from iron deficiency has been reported to increases morbidity
and mortality in preschool-aged children and pregnant women . Several factors
contribute to the occurrence of anaemia and nearly half of (43%) the anaemia cases
in childhood are due to iron deficiency. The deficiency may result from inadequate
dietary intake of iron, malabsorption of iron, an increased iron demand during rapid
growth in children and chronic blood loss. Other causes of anaemia include folate,
vitamin B12 deficiencies, malaria, intestinal helminth, viral infections, etc.
The development of anaemia is multifactorial and could stem from a variety of
factors. Nutritional deficiencies especially iron, but also folate, vitaminB12, and
protein appear to be the biggest factors Anaemia during childhood remains a major
public health challenge in Previous studies on the prevalence of iron deficiency
anaemia among children have led to discrepant results.
Anaemia is used as an indicator of nutritional and health status in children because
It is not only a risk factor for impaired physical activity performance and school
participation in children, but it may also persist into adulthood. Anaemia in
adulthood may lead tobecause it has been shown to result in low productivity due
to reduced work capacity.

Haemoglobin:

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Haemoglobin is a tetramer composed of two pairs of identical Alpha (α) like and
Beta (β) subunits. Zeta (ζ) is α like globin and Epsilon (ɛ), Gamma (γ) and Delta
(δ) are β like globins. The four subunits have the same tertiary folding, and they
each contain eight α helices, which are identified as A through H. Regarding the
number of amino acids; α chain has 141 amino acids, and the β chain has 146
amino acids. Hb functions to transport oxygen from the lungs to the tissues and
Co2 from the tissues to the lungs .

after, when production of the first enucleated definitive RBC commences from
stem and progenitor cells in the fetal liver, the predominant β-like globin molecule
produced is gamma globin .. This molecule is encoded by two duplicated globin
genes found within the β-globin gene cluster. The γ- globin chains combine with
adult α-globin chains into a stable tetramer forming HbF. This remains the
predominant Hb for much of gestation.

Heme synthesis:
Heme biosynthesis involves 8 enzymes, 4 of which are cytoplasmic and 4 of which
are mitochondrial The first step occurs in the mitochondria and involves the
condensation of succinyl coenzyme A (CoA) and glycine to form 5-aminolevulinic
acid (ALA), catalysed by ALA synthase (ALA-S). The next 4 biosynthetic steps
take place in the cytosol. ALA dehydratase (ALA-D) converts 2 molecules of ALA
to a monopyrrole porphobilinogen (PBG). Two subsequent enzymatic steps convert
4 molecules of PBG into the cyclic tetra pyrrole uroporphyrinogen III, which is
then decarboxylated to form coproporphyrinogen III. The final 3 steps of the
biosynthetic pathway, including the insertion of ferrous iron into protoporphyrin IX
by Ferro chelatase occur in the mitochondria. Physiological reasons or possible
advantages for the mitochondrial localization of the initial and last 3 enzymes, as
opposed to the cytoplasmic location of the remaining enzymes, are not known.

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Fig: Heme biosynthesis pathway

Reason of anaemia:Thehaemoglobin level at which symptoms and signs of

anaemia develop depends upon 4 main factors:

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1. The speed of onset of anaemia: Rapidly progressive anaemia causes more

symptoms than anaemia of slow-onset as there is less time for physiologic
adaptation.

2. The severity of anaemia: Mild anaemia produces no symptoms or signs but a

rapidly developing severe anaemia (haemoglobin below 6.0 g/dl) may produce
significant clinical features.

3. The age of the patient: The young patients due to good cardiovascular
compensation tolerate anaemia quite well as compared to the elderly. The elderly
patients develop cardiac and cerebral symptoms more prominently due to
associated cardiovascular disease.

4. The haemoglobin dissociation curve: In anaemia, the affinity of haemoglobin for

oxygen is depressed as 2,3-BPG in the red cells increases. As a result,
oxyhaemoglobin is dissociated more readily to release free oxygen for cellular use,
causing a shift of the oxyhaemoglobin dissociation curve to the right.

In symptomatic cases of anaemia, the presenting features are: tiredness, easy

fatiguability, generalised muscular weakness, lethargy and headache. In older
patients, there may be symptoms of cardiac failure, angina pectoris, intermittent
claudication, confusion and visual disturbances.

1. Pallor. Pallor is the most common and characteristic sign which may be seen in
the mucous membranes, conjunctivae and skin.

2. Cardiovascular system. A hyper dynamic circulation may be present with

tachycardia, collapsing pulse, cardiomegaly, mid systolic flow murmur, dyspnoea
on exertion, and in the case of elderly, congestive heart failure.

3. Central nervous system. The older patients may develop symptoms referable to
the CNS such as attacks of faintness, giddiness, headache, tinnitus, drowsiness,
numbness and tingling sensations of the hands and feet.

4. Ocular manifestations. Retinal haemorrhages may occur if there is associated

vascular disease or bleeding diathesis.

5. Reproductive system. Menstrual disturbances some of the manifestations

involving the reproductive system in anaemic subjects.

6. Renal system. Mild proteinuria and impaired concentrating capacity of the

kidney may occur in severe anaemia.

7. Gastrointestinal system. Anorexia, flatulence, nausea, constipation and weight

loss may occur. In addition to the general features, specific signs may be associated

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with particular types of anaemia which aredescribed later together with discussion
of Specific type of anaemia

Symptoms:

Symptoms of anaemia may include shortness of breath, particularly on exertion,

weakness, lethargy, palpitation and headache. Signs of anaemia may be divided
into general and specific. General signs include pallor of mucous membranes or
nail beds, which occurs if the haemoglobin level is less than 9 g/dL. A hyper
dynamic circulation may be present with tachycardia, cardiomegaly and a systolic
flow murmur especially at the apex. Specific signs are associated with particular
types of anaemia Iron deficiency anaemia increases the risk of having infectious
diseases, reduced physical ability, reduced concentration, and reduced learning
ability

Classification of anaemia:
Maxwell Win Trobe was an Austrian born physician who was a 20th-century
authority in the medical field ofhaematology. He proposed 80 years ago the
anaemia classification based on the mean cell volume (MCV) obtained by
haematocrit (Hct) /RBC ratio from the measurement of spun Hct and manual RBC
count and the mean corpuscular haemoglobin concentration (MCHC), calculated as
Hb/Hct ratio, where Hb was also based on manual measure.

Of these two indices, which allowed to classify the anaemia as microcytic,

normocytic, and macrocytic based on MCV value and hypochromic, normochromic
or hyper chromic based on MCHC, only MCV has survived as key parameter for
the classification of anaemia with auto-mated haematology analysers .

Anaemia is usually classified based on the size of RBCs, as measured by the mean
corpuscular volume (MCV) into three types:

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Microcytic anaemia:

The microcytic anaemias are those characterized by the production of red cells that
are smaller than normal. The small size of these cells is due to decreased
production of haemoglobin
The causes of microcytic anaemia include
a) iron deficiency
b) decreased globin chain synthesis (thalassemia)
c) anaemia of chronic diseases
d) Defects in the synthesis of the heme group (sideroblastic anaemia).

a) Iron Deficiency Anaemia:

Iron deficiency is the most common mineral deficiency world-wide and is also the
leading cause of anaemia . Lack of sufficient iron stores leads to a failure of
haemoglobin production and anaemia with production of hypochromic microcytic
red cells.
Incidence peaks in the early toddler years and again in teenage girls after the onset
of menstruation. There are several risk factors for developing iron deficiency
anaemia in children. Among these are prematurity, exclusive breastfeeding without
regular intake of iron-fortified foods .

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Figure 1.4 Hypochromic microcytic red cells [53].

b) Thalassemia:
Thalassemia are inherited diseases that disrupts the structure of haemoglobin,
leading to anaemia. Haemoglobin is made of 2-α and 2-β chains. The α chains rely
on four genes and the β chains rely on two genes. Mutations of these genes lead to
the different types of thalassemia..Thalassemia is one of the most common genetic
disorders worldwide, with about 5% of the global population carrying at least one
gene mutation [54]. It is seen most commonly in people of Mediterranean and
Southeast Asian descent .
α-thalassemia has four different types. One gene mutation presents as a silent
carrier and mutation of two genes results in a mild microcytic anaemia. These two
types do not require intervention. Mutation of three genes is known as haemoglobin
H disease and results in chronic hemolysis and often the need for transfusion.
Mutations of all 4- α chain genes is not compatible with life .

c) Anaemia of chronic disease:

This type of anaemia occurs in patients with acute or chronic immune activation.
The condition has thus been termed anaemia of inflammation [5658].This type of

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anaemia may be caused by renal failure, various infectious, inflammatory

(including rheumatological diseases) and neoplastic diseases The cause of this
anaemia is twofold . First, renal production of erythropoietin is suppressed by
inflammatory cytokines, resulting in decreased red cell production. Second, lack of
iron availability for developing red cells can lead to microcytosis. The lack of iron
is largely due to the protein hepcidin, an acute-phase reactant that leads to both
reduced iron absorption and reduced release of iron from body stores . The protein
ferroportin mediates cellular efflux of iron. Hepcidin binds to and down regulates
ferroportin, thereby blocking iron absorbed by enterocytes from entering the
circulation and also preventing the release of iron from its body stores to
developing red cells.

d) Sideroblastic anaemia:
The sideroblastic anaemias are a heterogeneous group of disorders that have in
common the presence of ring sideroblasts in more than 10% to 15% of nucleated
red cells .
After Prussian blue staining of iron deposited in individual erythroblasts (Perls
stain), ring sideroblasts are identified as immature red cells in which 10 or more
blue granules representing iron loaded mitochondria form a ring around the
nucleus. The sideroblastic anaemias display remarkable clinical and hematologic
heterogeneity, but share this mitochondrial iron loading . Despite recent advances
in our understanding of iron metabolism, the abnormal mitochondrial iron
metabolism that characterizes these conditions is poorly understood .

Normocytic anaemia:

Normocytic anaemias are due to either RBC underproduction or increased red

blood cell loss or red cell destruction.

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Anaemia due to underproduction of haemoglobin:

This group includes anaemia of chronic diseases, the aplastic anaemias (congenital
or acquired), bone marrow infiltration (leukemia, lymphoma, disseminated
malignant solid tumors, etc.), myelofibrosis, and osteopetrosis. In addition to the
clinical picture, which may suggest the diagnosis in some cases, these anaemias are
characterized by a low reticulocyte count. Figure shows a blood film with
normochromic red cells .

Figure 1.5 Normochromic red cells [61].

Macrocytic anaemia:

Macrocytic anaemia is characterized by the presence of red cells larger than normal
(high MCV), figure . While megaloblastic anaemia is the most common cause of
macrocytosis, not every macrocytosis is megaloblastic anaemia.

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 Case study of megaloblastic aneamia

Figure 1.6
Oval macrocytes in a case of megaloblastic anaemia .

a) Megaloblastic anaemias:
Megaloblastic anaemia results from impaired DNA synthesis resulting in large
RBCs. Bone marrow RBC precursors are dysplastic with asynchronous maturation
of the nucleus and cytoplasm [44]. The most common causes of megaloblastic
anaemia are folate deficiency and vitamin B12 deficiency. Further studies are
needed to confirm the diagnosis of megaloblastic anaemia and to identify an
etiology (e.g. antiparietal cell antibodies and anti intrinsic factor antibody).

Several drugs interfere with DNA synthesis and cause megaloblastic changes in the
RBC precursors with or without anaemia. These drugs include anticonvulsants
(phenytoin, phenobarbital), chemotherapeutic agents (methotrexate, 6-
mercaptopurine, hydroxyurea), and trimethoprim sulfamethoxazole. Other causes
of megaloblastic anaemia include inborn errors of metabolism such as orotic
aciduria, homocystinuria and methylmalonic aciduria. Thiamine-responsive
anaemia is usually associated with sideroblastc anaemia. The diagnosis is
suggested by the exclusion of other causes of megaloblastosis and response to
thiamine therapy

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 Case Study : Aneamia

Patients Name: Mr. Ravi madhukar Bhombe

Age:19
Gender: Male
Patient ID:9101
Received:20/9/2020
Reported:20/9/2020
Referred physicians: Dr. Kishor tepale
Main physicians: Dr. Aadityanath patil
after obtaining the full medical history pertaining to different general and specific
signs and symptoms, the patient is examined for evidence of anaemia. Special
emphasis is placed no colour of the skin, conjunctivae, scleral and nails. Changes in
the retina, atrophy of the papillae of the tongue, rectal examination for evidence of
bleeding, and presence of hepatomegaly, splenomegaly, lymphadenopathy and
bony tenderness are looked for. In order to confirm or deny the presence of
anaemia, its type and its cause, the following plan of investigations is generally
followed, of which complete blood counts (CBC) with reticulocyte count is the
basic test.
A. HAEMOGLOBIN ESTIMATION. The first and foremost investigation in any
suspected case of anaemia is to carry out a haemoglobin estimation. Several
methods are available but most reliable and accurate is the cyan methaemoglobin
(HiCN) method employing Drabkin’s solution and a spectrophotometer. If the
haemoglobin value is below the lower limit of the normal range for particular age
and sex, the patient is said to be anaemic. In pregnancy, there is haemodilution and,
therefore, the lower limit in normal pregnant women is less (10.5 g/dl) than in the
non-pregnant state.
B. PERIPHERALBLOOD FILM EXAMINATION. It is specially for
microcytic Aneamia by using haemoglobin estimation is invariably followed by
examination of a peripheral blood film for morphologic features after staining it
with The blood smear is evaluated in an area where there is neither Rouleaux
formation nor so thin as to cause red cell distortion. Such an area can usually be
found at junction of the body with the tail of the film, but not actually at the tail.
The following abnormalities in erythroid series of cells are particularly looked for
in a blood smear:
1. Variation in size : Normally, there is slight variation in diameter of the red cells
from 6.7-7.7 μm (meanvalue 7.2 μm). Increased variation in size of the red cell is

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 Case study of megaloblastic aneamia

termed anisocytosis. Anisocytosis may be due to the presence of cells larger than
normal (macrocytosis) or cells smaller than normal (microcytosis). Sometimes both
microcytosis and macrocytosis are present .
Macrocytes are classically found in megaloblastic anaemia; other causes are
aplastic anaemia, other dyserythropoietic anaemias, chronic liver disease and in
conditions with increased erythropoiesis. Microcytes are present in iron deficiency
anaemia, thalassemia and spherocytosis. They may also result from fragmentation
of erythrocytes such as in haemolytic anaemia.
2. Variation in shape :Increased variation in shape of the red cells is termed
poikilocytosis. The nature of the abnormal shape determines the cause of anaemia.
Poikilocytes are produced in various types of abnormal erythropoiesis e.g. in
megaloblastic anaemia, iron deficiency anaemia, thalassemia, myelosclerosis and
Microangiopathic haemolytic anaemia.
3. Inadequate haemoglobin formation :.lIt may develop either from lowered
haemoglobin content (e.g. in iron deficiency anaemia, ), or due to thinness of the
red cells (e.g. in thalassemia, ). Unusually the red cells due to increased
haemoglobin concentration is termed hyperchromasia and may be found in
megaloblastic

4) Polychromasia is defined as the red cells having more than one type of colour.
Polychromatic red cells are slightly larger.

C. RED CELL INDICES:An alternative method to diagnose and detect the

severity of anaemia is by measuring the red cell indices:
In iron deficiency and thalassaemia, MCV (mean cell volume) normal range ( 84-
98 ), reduce value ( 112.30) MCH ( mean corpuscular haemoglobin ) normal range
(27-34) reduce value ( 30.60)and MCHC ( mean corpuscular haemoglobin
concentration ) normal value (31-36 ) and reduced value ( 34.40)are reduced. In
anaemia due to acute blood loss and haemolytic anaemias, MCV, MCH and MCHC
are all within normal limits. In megaloblastic anaemias, MCV is raised above the
normal range.
[Link] AND PLATELET COUNT:Measurement of leucocyte and
platelet normal range ( 4000-11000 &15000-45000) and reduce value ( 3000&
11800)count helps to distinguish pure anaemia from pancytopenia in which red
cells, granulocytes and platelets are all reduced. In anaemias due to haemolysis or
haemorrhage, the neutrophil count and platelet counts are often elevated. In
infections and leukaemias, the leucocyte counts are high and immature leucocytes
appear in the blood.

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E. RETICULOCYTE COUNT:Reticulocyte count (normal 0.5-2.5%) and is

done in each case of anaemia to assess the marrow erythropoietic activity. In acute
haemorrhage and in haemolysis, the reticulocyte response is indicative of impaired
marrow function.
Hemegaloblastic anaemias are disorders caused by impaired DNA synthesis and
are characterised by a distinctive abnormality in the haematopoietic precursors in
the bone marrow in which the maturation of the nucleus is delayed relative to
that of the cytoplasm. Since cell division is slow but cytoplasmic development
progresses normally, the nucleated red cell precursors tend to be larger which
Ehrlich in 1880termed mega oblasts. Mega oblasts are both morphologically and
functionally abnormal with the result that the maturated cells formed from them
and released into the peripheral blood are also abnormal in shape and size, the
most prominent abnormality being macrocytosis. The underlying defect for the
asynchronous maturation of the nucleus is defective DNA synthesis due to
deficiency vitamin B12 (cobalamin) and/or folic acid (folate). Less common
causes are interference with DNA synthesis by congenital or acquired
abnormalities of vitamin B12 or folic acid metabolism. Before considering the
megaloblastic anaemia, an outline of vitamin B12 and folic acid metabolism is
given for a better understanding of the subject. The salient nutritional aspects and
metabolic functions of vitamin B12 and folic acid are summarized.

Vitamin B12 Metabolism

BIOCHEMISTRYSOURCES:The only dietary sources of vitamin B12 are

foods of animal protein origin such as kidney, liver, heart, muscle meats, fish,
eggs, cheese and milk. In contrast to folate, fruits and vegetables contain
practically no vitamin B12 unless contaminated with bacteria. Cooking has little
effect on its activity. Vitamin B12 is synthesized in the human large bowel by
microorganisms but is not absorbed from this site and, thus, the humans are
entirely dependent upon dietary sources. The average daily requirement for
vitamin B12 is2-4 μg.

ABSORPTION:After ingestion, vitamin B12 in food is released and forms a

stable complex with gastric R-binder .R-binder is a form of glycoprotein found
in various secretions. salivaphagocytes and plasma. On entering the duodenum,
the vitamin B12-R-bindercomplex is digested releasing vitamin B12 which then
binds to intrinsic factor (IF). The IF is a glycoprotein of molecular weight 50,000
produced by the parietal cells of the stomach and its secretion roughly parallels

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that of hydrochloric acid. The vitamin B12-IF complex, on reaching the distal
ileum, binds to the specific receptors on the mucosal brush border, thereby
enabling the vitamin to be absorbed. The IF, therefore, acts as cell-directed
carrier protein similar to transferrin. The receptor-bound vitamin B12-IF
complex is taken into the ileal mucosal cells where after several hours the IF is
destroyed, vitamin B12 released and is transferred to another transport protein,
Tran’s cobalamin (TC) II. The vitamin B12-TC II complex is finally secreted
into the portal circulation from where it is taken by the liver, bone marrow and
other cells. There are 2 major vitamin B12 binding—TC I and TC II, and a minor
protein TC III. TC I is not essential for vitamin B12 transport but functions
primarily as a storage protein while TC III is similar to TC II and bind a small
amount of vitamin B12

TISSUE STORES:Normally, the liver is the principal storage site of vitamin

B12 and stores about 2 mg of the vitamin, while other tissues like kidney, heart
and brain together store about2 mg. The body stores of vitamin B12 are adequate
for 2-4years. Major source of loss is via bile and shedding of intestinal epithelial
cells. A major part of the excreted vitaminB12 is reabsorbed in the ileum by the
IF resulting in enterohepatic circulation.

FUNCTIONS: Vitamin B12 plays an important role in general cell metabolism,

particularly essential for normal haematopoiesis and for maintenance of integrity
of the nervous system. Vitamin B12 acts as a co-enzyme for 2 main biochemical
reactions in the body: Firstly, as methyl cobalamin (methyl B12) in the
methylation of homocysteine to methionine by methyl tetra hydro folate (THF).
The homocysteine-methionine reaction is closely to folate metabolism. Methyl
B12 Homocysteine Methionine When this reaction is impaired, folate
metabolism is deranged and results in defective DNA synthesis responsible for
megaloblastic maturation. Secondly, as adenosyl cobalamin (adenosyl B12) in
propionate metabolism for the conversion of methyl malonyl co-enzyme A to
succinyl co-enzyme A:Adenosyl B12Propionyl CoA → Methyl malonyl CoA →
Succinyl CoA Lack of adenosyl B12 leads to large increase in the level of methyl
malonyl CoA and its precursor, propionyl CoA. This results in synthesis of
certain fatty acids which are incorporated into the neuronal lipids. This
biochemical abnormality may contribute to the neurologic complications of
vitamin B12 deficiency

VITAMIN B12 DEFICIENCY:In Western countries, deficiency of vitamin

B12 is more commonly due to pernicious (Addison an) anaemia. True

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vegetarians like traditional Indian Hindus and breast-fed infants have dietary lack
of vitamin B12. Gastrectomy by lack of intrinsic factor, and small intestinal
lesions involving distal ileum where absorption of vitamin B12 occurs, may
cause deficiency of the vitamin. Deficiency of vitamin B12 takes at least 2 years
to develop when the body stores are totally depleted.

VITAMIN B12 DEFICIENCY

A. Inadequate dietary intake e.g. strict vegetarians, breast-fed infants.

[Link]

1. Gastric causes: pernicious anaemia, gastrectomy, congenital lack of intrinsic

factor.

2. Intestinal causes: tropical sprue, ileal resection, Crohn’s disease, intestinal

blind loop syndrome, fish-tapeworm infestation.

TESTS FOR VITAMIN B12 DEFICIENCY. The normal range of vitamin

B12 in serum is 280-1000 pg/ml. Valueless than 100 pg/ml indicate clinically
deficient stage. Traditional tests employed to establish vitamin B1deficiency are
serum vitamin B12 assay

1. SERUM VITAMIN B12 ASSAY. Assay of vitamin B12 in blood can be

done by 2 methods—microbiological assay and radio assay.

i) Microbiological assay. In this test, the serum sample to be assayed is added to

a medium containing all other essential growth factors required for a
vitaminB12-dependent microorganism. The medium along with microorganism
is incubated and the amount of vitaminB12 is determined turbimetrically which
is then compared with the growth produced by a known amount of vitaminB12.
Several organisms have been used for this test such as Euglena gracilise,
Lactobacillus leichmannii, Escherichia coli and Ochromonas malhamensis. E.

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 Case study of megaloblastic aneamia

gracilise is, however, considered more sensitive and accurate. The addition of
anti-biotics to the test interferes with the growth and yields false low result.

ii) Radio assay. Assays of serum B12 by radioisotope dilution (RID) and
radioimmunoassay (RIA) have been developed. These tests are more sensitive
and have the advantage over microbiologic assays in that they are simpler and
more rapid, and the results are unaffected by antibiotics and other drugs which
may affect the living organisms.

Folate Metabolism

BIOCHEMISTRY. Folate or folic acid, a yellow compound, is a member of

water-soluble B complex vitamins with the chemical name of pteroyl glutamic
acid (PGA). Folic acid does not exist as such in nature but exists as folates in
poly glutamate form (conjugated folates). For its metabolic action as co-enzyme,
poly glutamates must be reduced to di hydro- and tetrahydrofolate forms.

SOURCES. Folate exists in different plants, bacteria and animal tissues. Its main
dietary sources are fresh green leafy vegetables, fruits, liver, kidney, and to a
lesser extent, muscle meats, cereals and milk. Folate is labile and is largely
destroyed by cooking and canning. Some amount of folate synthesised by
bacteria in the human large bowel is not available to the body because its
absorption takes place in the small intestine. Thus, humans are mainly dependent
upon diet for its supply. The average daily requirement is100-200 μg

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Fig : contrasting pathway of absorption and transport of iron, vitaminB12, folic acid

ABSORPTION AND TRANSPORT. Folate is normally absorbed from the

duodenum and upper jejunum and to a lesser extent, from the lower jejunum and
ileum. However, absorption depends upon the form of folate in the diet.
Polyglutamate form in the foodstuffs is first cleaved by the enzyme, folate
conjugates, in the mucosal cells to mono- and di glutamates which are readily
assimilated. Synthetic folic acid preparations in Polyglutamate form are also
absorbed as rapidly as mono- and diglutamate form because of the absence of
natural inhibitors. Mono- and diglutamate undergo further reduction in the mucosal
cells to form tetrahydrofolate (THF), a mono glutamate. THF circulates in the
plasma as methylated compound, methyl THF, bound to a protein. Once methyl
THF is transported into the cell by a carrier protein, it is reconverted to
Polyglutamate

TISSUE STORES. The liver and red cells are the main storage sites of folate,
largely as methyl THF Polyglutamate form. The total body stores of folate are about
10-12 mg enough for about 4 months. Normally, folate is lost from the sweat,
saliva, urine and faeces.

FUNCTIONS. Folate plays an essential role in cellular metabolism. It acts as a co-

enzyme for 2 important bio-chemical reactions involving transfer of 1-carbon units
([Link] and formyl groups) to various other compounds. These reactions are as
under: Thymidylate synthetase reaction. Formation of deoxy thymidylate

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monophosphate (dTMP) from its precursor, deoxy uridylate monophosphate

(dUMP).Methylation of homocysteine to methionine. This reaction is linked to
vitamin B12 metabolism (Fig. 2).These biochemical reactions are considered in
detail below together with biochemical basis of the megaloblastic anaemia.

2. FOLATE DEFICIENCY. Folate deficiency is more often due to poor dietary

intakeInadequate dietary intake e.g. in alcoholics, teenagers, infants, old age, pover,
Malabsorption e.g. in tropical sprue, coeliac disease, partial gastrectomy, jejunal
resection, Crohn’s disease.

Excess demand

[Link]: pregnancy, lactation, infancy.

2. Pathological: malignancy, increased Haematopoiesis, chronic exfoliative skin

disorders, tuberculosis, and rheumatoid [Link] urinary folate loss e.g. in
active liver disease, congestive heart failure.

TESTS FOR FOLATE DEFICIENCY. The normal range of serum folate is 6-18
ng/ml. Values of 4 ng/ml or less are generally considered to be diagnostic of folate
deficiency. Measurement of formiminoglutamic acid (FIGLU) urinary excretion
after histidine load was used formerly for assessing folate status but it is less
specific and less sensitive than the serum assays. Currently, there are 3 tests used to
detect folate deficiency—urinary excretion of FIGLU, and red cell folate assay.

1. URINARY EXCRETION OF FIGLU. Folic acid is required for conversion of

form iminoglutamic acid (FIGLU) to Glutamic acid in the catabolism of histidine.
Thus, on oral administration of histidine, urinary excretion of FIGLU is increased if
folate deficiency is present

Treatment: Most cases of megaloblastic anaemia need therapy with appropriate

vitamin. This includes: hydroxycobalamine as intramuscular injection 1000 μg for 3
weeks and oral Folic acid 5 mg tablets daily for 4 months. Severely-anaemic

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patients in whom a definite deficiency of either vitamin cannot be established with

certainty are treated with both vitamins concurrently. Blood transfusion should be
avoided since it may cause circulatory overload. Packed cells may, however, be
infused slowly. Treatment of megaloblastic anaemia is quite gratifying. The marrow
begins to revert back to normal morphology within a few hours of initiating
treatment and becomes normoblastic within 48 hours of start of treatment. Reticulo-
cytosis appears within 4-5 days after therapy is started and peaks at day 7.
Haemoglobin should rise by 2-3 g/dl each for the night. The peripheral neuropathy
may show some improvement but subacute combined degeneration of the spinal
cord is irreversible.

Patients name: Mr. Ravi Bhombe

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Age:19

Gender:male

Physician name: Dr. Aadityanath patil

Sr. No Dosage form Brand name Time

1 Tab Zincovit 0-0-1

2 Tab Foalite 1-0-0

3 Tab Sway 1-0-0

4 Tab Bizfex xt 0-0-1

5 Tab Combiflam 1-0-1

6 Inj. Vitcofol

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Summary:19old Ravi Bhombe has megaloblastic aneamia symptoms and get

hospitalized and then following observation of case report of megaloblastic anaemia are
observed

i) Haemoglobin. Haemoglobin estimation reveals values below the normal range. The fall
in haemoglobin concentration may be of a variable degree.

ii) Red cells. Red blood cell morphology in a blood film shows the characteristic
Macrocytosis. However, Macrocytosis can also be seen in several other disorders such as:
haemolysis, liver disease.

iii) Reticulocyte count. The reticulocyte count is generally low to normal in untreated
cases file

iv) Absolute values. The red cell indices reveal an elevated MCV (above 120 fl)
proportionate to the severity Macrocytosis, elevated MCH (above 50 pg) and normal or
reduced MCHC.

v) Leucocytes. The total white blood cell count may be reduced. Presence of
characteristic hyper segmented Neutrophils (having more than 5 nuclear lobes) in
megaloblastic anaemia. An occasional myocyte may also be seen.

vi) Platelets. Platelet count may be moderately reduced in severely anaemic patients.
Bizarre forms of platelets may be seen.

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Reference:

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