BLOOD BIOCHEMISTRY

THE ROLE OF IRON,FOLIC

ACID, CYANOCOBALAMIN IN
THE FORMATION OF
HEMOGLOBINE & RBC
BY
Liniyanti.D.Oswari, M.D.; MNS.MSc
For
Medical student,Sriwijaya University
Block 8

Chapt.
44
Biochemistry of Erythrocytes
Student Learning Outcomes:
Describe the structure/ function of blood cell types:
Erythrocytes, leukocytes, thrombocytes
Explain the metabolism of the red blood cell
Explain basics of hematopoiesis from bone marrow
Describe some errors of hemoglobin function,
anemias, hemoglobin switching
Describe the structure/ function of blood group
antigens

Learning Objective
Understand of the Hematopoesis
Understand the Metabolism of folic acid
Cyanocobalamine in Erythropoesis.
Understand the etiology and the
management of Anemia.

Blood - Functions
Respiratory
Transport O2 from lungs to tissues
Transport CO2 from tissues to lungs

Nutrition
Transport food from gut to tissues (cells)

Excretory
Transport waste from tissues to kidney (urea, uric acid, water)

Regulatory
Water Content of Tissues
Water exchanged through vessel walls to tissue (interstitial fluid)

Body Temperature
Water- high heat capacity, thermal conductivity, heat of vaporization
Typical heat generation is 3000 kcal/day

Protective
Antibodies, antitoxins, white blood cells (WBC)

Blood Composition
Blood composition
5-6 L in an adult
70 mL/kg of body weight
Suspension of cells in a carrier fluid (plasma)
Cells - 45% by volume
Plasma - 55% by volume

Cells
Red cells (erythrocytes)
5x106/L

White cells (leukocytes)

7x103/L

Platelets (thrombocytes)
3x105/L

Blood composition
Suspension of cells in plasma (carrier fluid)
45% Cells
55% Plasma

Cells
Red cells (erythrocytes)
5x106/mL
White cells (leukocytes)
7x103/mL
(thrombocytes)
3x105/mL

99%
< 1%
Platelets

Cells
of
blood

Blood composition

Composition of Blood
The three main cellular elements in blood are:
1. Erythrocytes: are formed in bone marrow
are very specialized cells whose main function
is to carry O2 to cells and CO2 away from them
have a half-life of about 60-120 days
are removed by the liver and spleen and
destroyed
2. Leukocytes (white blood cells)
are formed in the bone marrow
most of the different leukocytes destroy
invading bacteria or other foreign substances
by phagocytosis

Normal Blood
Red cell
Monocyte
Platelets
Lymphocyte

Neutrophil

Normal Blood

Reticulocyte

Blood cells

Table 1 Blood cells (cells/mm3):

Erythrocytes
carry oxygen

5.2 x 106 men

4.6 x 106 women

Neutrophils
4300
granules; phagocytic, O2 burst kills
Lymphocytes
2700
immune response, B- and T-cells, NK
Monocytes
500
macrophages for bacteria, damage
Eosinophils
230
granules destroy parasites (worms)
Basophils
40

granules hypersensitivity, allergic

histamine, proteases,

Composition of Blood, cont

3. Platelets or thrombocytes
are formed in the bone marrow and spleen
control bleeding when there is a cut or injury

Plasma: is 90% water

the fluid remaining after all cellular elements
have been removed from whole blood by
centrifugation
the dissolved solids are mainly proteins (7%)
the remaining 1% contains glucose, lipids,
enzymes, vitamins, hormones, and waste
products such as urea and CO2

Composition of Blood
if plasma is allowed to stand, it forms a
clot, a gel-like substance
serum: the clear liquid that can be
extracted from blood plasma
serum contains all the components of
plasma but lacks fibrinogen that makes
blood clot

Male versus female

Hematocrit (% volume that is red cells)
40-50% in males
35-45% in females

Blood Components:Plasma Transports Solutes

Blood Plasma
Straw colored clear liquid
Contains 90% water
7% plasma proteins
created in liver
confined to bloodstream

Albumin
maintain blood osmotic pressure

Immunoglobulins
antibodies bind to foreign
substances called antigens
form antigen-antibody complexes

Fibrinogen
for clotting

2% other substances
Nutrients, electrolytes, gases, hormones, waste products

Hematocrits
Plasma
White cells
Red cells

Normal, Hemorrhage, IDA, Leukemia, Hemolysis, B12, P Vera

Plasma Protein
More than 200
Most abundant
Albumin - 4-5 g/100 mL
-globulins - ~1 g/100 mL
fibrinogen - 0.2-0.4g/100 mL

Original classification by zone

electrophoresis at pH 8.6
Separation by pI with several molecular
weight species within each group

Blood Components:
"Blood Count" % of Each Component

RBC - Reversible Shape Changes

Surfactants result in cells becoming more
spherical
Mechanical stress - deformation in capillaries
to allow for passage of cells
Disease eg. Sickle Cell Anemia
Hemolysis - release of Hb from the cell
Osmotic swelling
Surface collisions with artificial organs

White Blood Cells (Leukocytes)

Total count - approximately 7000/L
Various types
Neutrophils 62% (50-70%)
Eosinophils 2.3% (2-4%)
Basophils 0.4% (0-1%)
Monocytes 5.3% (2-8%)
Lymphocytes 30% (25-40%)
Plasma cells (mainly in the lymph)

Monocytes in tissue become macrophages

Functions of eosinophils
Secrete lethal substances at the time of exposure to
foreign proteins/parasites
1. Function protection from microorganisms,
allergic reactions
2. Eosinophill peroxidase detroy worms, bacteria
and tumor cells.
3. Eosinophill cationic protein (ECP)- destroys
helminths.
4. Eosinophill derived neurotoxin destroys nerve
fibres (myelinated nerve fibres)

Functions of basophils
Function allergic reactions, blood clotting
Energy mainly from oxidative phosphorylation
Basophill granules release some important substances like
1. Histamine Acute hypersensitivity reaction- vascular
changes, increase capillary permeability
2. Heparin prevents intravascular blood clotting
3. Hyaluronic acid necessary for deposition of ground
substances in basement membrane
4. Proteases exaggerate inflammation

Basophill have IgE receptor hypersensitivity reaction

Functions of neutrophils
1. First line of defence against invading micro-organisms.
2. Main source of energy: Glucose in glycolysis (small
amount of mitochondria)
Hundreds of granules (contain hydrolyses,
peroxidases, phosphatases, lysocim....)
3. Powerful and effective killer machine contains enzymes
like protease, elastase, metalloproteinase, NADPH
oxidase; antibody like substances called defensins.
Defensins antimicrobial peptides active against bacteria and fungi .

4.Secrete Platelet Aggregation Factor (PAF) accelerates

the aggregation of platelet during injury to the blood
vessels

Function
Defense against foreign invaders
bacteria
viruses
foreign materials (including biomaterials)

Phagocytosis
Neutrophils, macrophages
Move to foreign particle by chemtaxis
Chemicals induce migration
Toxins, products of inflamed tissues, complement
reaction products, blot clotting products
Response is extremely rapid (approx 1 h)

Lymphocytes
B cells - responsible for humoral immunity
T cells - responsible for cell mediated
immunity

B cells responsible for production of

antibodies
Receptor matches antigen
Cells multiply
Antibodies

Abs are just immunoglobulins

discussed earlier

T cells
Cytotoxic T cells (Killer T cells)
Bind to cytotoxic cells (eg infected by virus)
Swell
Release toxins into cytoplasm

Helper T cells
Most numerous
Activate B cells, killer T cells
Stimulate activity by secretion of IL2
Stimulate macrophages

Suppressor T cells
Regulate activities of other cell types

AIDS
HIV - attacks many cell types
epithelial cells
macrophages
neurons
lymphocytes (helper T)

Infected helper T cells when stimulated,

produces viral proteins which kill the cell
Helper T cell population disappears

Monocytes
Function phagocytosis, exit into tissues
tissue macrophages
4-8 % of all leucocytes
Are accumulated in the
place of inflammation
A lot of lysosomal
hydrolases
Aerobic pathway of
energy obtaining prevails

Functions of Platelets
1. Blood clotting
2. Clot retraction
3. Defence mechanism
4. Homeostasis
5. Repair and rupture of blood vessel

Platelets
Non-nucleated disk shaped cells
3-4 m diameter
Volume 10 x 10-9 mm3
250 000 cells/L
10 day circulation time
Surface contains membrane bound receptors
(GP Ib and IIb/IIIa)
mediate surface adhesion reactions, aggregation
reactions
interact with coagulation proteins

Blood Components: Platelets

Coagulate, form plug, prevent blood loss
Formed by fragmentation from megakaryoctyes

Figure 16-10c: Megakaryocytes and platelets

Contain muscle proteins actin and myosin which

contract when platelet is activated
Also granules, dense granules, lysosomal
granules
Platelets activated by minimal stimulation
Become sticky
Shape change
Release of cell contents

Stimulate other platelets

Function
Initially arrest bleeding through formation of platelet
plugs
Stabilize platelet plugs by catalyzing coagulation
reactions leading to formation of fibrin

Overview of Hemostasis:
Clot Formation & Vessel Repair

Figure 16-11: Overview of hemostasis and tissue repair

Hemostasis: Vasoconstriction & Plug Formation

Platelet Adhesion
Site of injury - exposure of connective
tissue elements (eg collagen)
Artificial surfaces through forming thrombi
(clots)

Platelet Aggregation
Caused by ADP, collagen, thrombin,
epinephrine, PAF, TXA2

Release of cell contents

Induced by ADP, collagen, thrombin, TXA2
and epinephrine

Coagulation
Maintenance of hemostasis (prevention of
blood loss)
At least 12 plasma proteins interact in series
of reactions
Cascade of reactions
Inactive factors become enzymatically active
following surface contact, proteolytic cleavage
by other enzymes
Amplification is rapid
Reactions are localized

Coagulation Cascade

39

Zone Electrophoresis of Plasma Proteins

+

globulins

pI

6.0

5.6

5.1

albumin

4.7

Functions of Plasma Proteins

Maintenance of:
Colloid osmotic pressure(COP) ()
pH
electrolyte balance

COP relates to blood volume

P =
Protein
soln

Water

If membrane present important

Isotonic - same osmotic pressure
Human blood - 300 milliOsmoles /L
Normal saline - 0.9% NaCl by weight
0.15 mol/L
0.30 mol/L of particles

At physiological temperature, two solutions

differing in pressure by 1 mOsm have an
osmotic pressure of 19.3 mm Hg between
them.

Solutions with same concentration of

solute particles will have same osmotic
pressure even if solute particles are
different.
Solution with higher concentration of
solute particles is hyperosmotic
Solution with lower concentration of
solute particles is hyposmotic

Colloid - large particle that cannot easily

cross a membrane
Stays in the compartment
In blood protein = 20-30 mmHg
Total ~ 5000 mmHg

Protein stays in the blood as is

maintained in the blood
Water content is therefore maintained

Hypotonic - lower than normal

Hemolysis of RBC
Hb
H2 O

Ghost Cells

Hypertonic higher than normal

Creation of cells
Hypertonic
1.5% NaCl

Crenated Cells
H 2O

Functions of Plasma Proteins (contd)

Transport of ions, fatty acids, steroids,
hormones etc.
Albumin (fatty acids), ceruloplasmin (Cu 2+),
transferrin (Fe), lipoproteins (LDL, HDL)

Nutritional source of amino acids for tissues

Hemostasis (coagulation proteins)
Prevention of thrombosis (anticoagulant
proteins)
Defense against infection (antibodies,
complement proteins)

Function and Properties of

Selected Plasma Proteins
Consider three abundant plasma
proteins
Structure, function
Coagulation, fibrinolysis, complement

Albumin
MW 66 000
Single chain, 580 amino acids, sequence
is known
Dimensions - Heart shaped molecule
50% helix [He and Carter, Nature, 358 209
(1992)]

Modeled as:

80
30

Synthesis
Mainly liver cells then exported
Assembly time on ribosome ~ 1-2 min
t0.5 in circulation - 19 days
14 g lost per day
0.4 mg synthesized per hour per g of
liver
Need liver of approximately 1.5 kg in
weight to maintain

Functions
Colloid osmotic pressure of blood is 80%
due to albumin
relatively low molecular weight
regulates water distribution

Transport of fatty acids

Liver to tissues, binding

Source of amino acids for tissue cells

(pinocytosis)
60% albumin in tissue (interstitial) fluid

-Globulins
20% of plasma proteins
refers to electrophoretic mobility
Represents a group of proteins of variable
structure
immunoglobulins

Main functional task is immunochemical

Antibodies - combine with specific antigens

Basic 4 chain structural unit

MW = 2x55000 +2x27000 = 160000

Variable region varies with respect to

primary, secondary and tertiary structures
Basis of specificity of antigen binding (106
average number)
5 classes of immunoglobulins
IgG, IgA, IgM, IgD, IgE
Different structures of constant regions of
heavy chains
Some are polymers (multiples of 4 chain unit IgA - dimer - MW 350 000, IgM - pentamer MW 900 000
See any immunology book for more details

Classes of Immunoglobulins
IgG Identifies microorganisms for engulfment or lysis
IgE Inhibits parasite invasion; involved in allergic
reactions
IgD Unknown
IgA Basis for passive immunity provided by breast
milk, agglutinates infectious agents in secretions
outside the body, present in tears, mucous
IgM Identifies microorganisms for engulfment or lysis

Functions
Primary function is antigen binding
(immune response)
Secondary function is complement binding
(after antigen)

Synthesis
In lymphocytes (T and B)
Made in response to presence of antigen
(foreign macromolecule, virus particle
etc.)

Fibrinogen
Coagulation
Structure
MW 340 000
Sequence of amino acids is known (3000)
4y, 3y structure
6 polypeptide chains, 2 (67,000), 2 (56,000),
2 (47,000)

disulfide
Triple dumbell model (EM)
450
90
D

s, s and s are intertwined

Function
Blood coagulation (clotting)
Fibrinogen

Fibrin

Thrombin

Plasmin

Fibrin

Degradation (FDP)

Plasmin is end product of fibrinolytic system

Clot needs to be removed
Not needed forever
Could embolize to lungs, brain

Importance of Protein Structure Sickle Cell Anemia

Occurs because of a minor variation in one
amino acid in the chain of Hb
Results in Hb that, when exposed to low O2
concentrations precipitates into long
crystals
Elongate cell
Damage cell membrane
Decrease in amount of RBC

Cellular
Elements of Blood
Red cells
40 - 50% of blood volume
5 x 106 cells /L
bag of hemoglobin
non-nucleated
no proliferation
cell membrane in excess so that deformation
does not rupture

Shape
Biconcave disc
8 m in diameter, 2.7 m thick, volume ~ 90
m3, area ~ 160 m2

Scanning Electron Micrograph of Red Blood Cells

Why this shape?

Area to volume ratio is high (maximal?)
Facilitates diffusion of O2 and CO2
minimal distance of contents from surface
Originates in bone marrow (hematopoiesis)

Molecular explanation based on the

properties of the proteins in the cell
membrane is found in Elgsaeter et al.
Science, 234, 1217 (1986)

Oxygen Binding of Hb
Blood must carry 600 L of O2 from lungs
to tissues each day
Very little carried in plasma since O2 only
sparingly soluble
Nearly all bound and transported by Hb of
RBC
Possible for Hb to carry four O2 molecules,
one on each chain, one on each chain

O2 depleted Hb solution placed in

contact with O2(g)
Equilibrium reaction
Fraction (s) of Hb converted to
oxyhemoglobin

Binding of O2 to 4 heme sites given by:

Hb O2 HbO2
HbO2 O2 Hb(O2 ) 2
Hb(O2 ) 2 O2 Hb(O2 ) 3
Hb(O2 ) 3 O2 Hb(O2 ) 4
Equilibrium constants for different reactions
different
Binding of first O2 relatively low affinity
2nd, 3rd and 4th - much higher affinity
Cooperative effect

Compare with binding curve for myoglobin

Myoglobin - oxygen reaction

k1

Mb O2 MbO2
At equilibrium

k 1

k1C MbCO2 k 1C MbO2

s

C MbO2
C MbO2 C Mb
k1
C MbCO2
k 1

k1
C MbCO2 CMb
k 1

KCO2
1 KCO2

Acid Effect - O2 Dissociation

HHb O2 HbO2 H

O2 binding causes release of H+

pH decreases, [H+] increases then the
equilibrium moves to left
% saturation decreases, more dissociation
for a given pO2
Tissues are at a lower pH than the lungs
due to CO2 which facilitates release of O2
to tissues

Hb versus Mb
Hb carry O2 to tissues where it is
released
Releases quickly in tissues where pO2 is
lower

Mb store O2 in the muscle, make

available to cells
Releases very little in tissues
Reference: Science 255 54 (1992)

Extrinsic system
Blood comes in contact with traumatized
vascular wall or extravascular tissues

Intrinsic system
Initiated by surface contact (often
negatively charged surface)

Most reactions are Ca++ dependent

Chelaters of Ca++ effective
anticoagulants

Fibrinolysis
Results in dissolution of fibrin clot

Conversion of plasminogen to plasmin

Plasminogen activators synthesized by and
released from endothelial cells
TPA - tissue plasminogen activator

Erythrocyte
metabolism
Erythrocyte metabolism:
Only glycolysis
ATP for Na+/K+, Ca2+
HMP shunt makes NADPH
G6PD is 1st enzyme
Lifetime rbc by G6PD activity
2,3-BPG modulates O2 binding
Need Fe2+ Hb bind O2;
If ROS made Fe3+, NADH can reduce

Fig. 1

Hematopoiesis:
Hematopoiesis
Stem cells in bone
marrow (1/105)
Proliferate, differentiate,
mature
by growth factors,
hormones
signal transduction paths
Myeloid, lymphoid lines
Leukemias: immature cells
keep proliferating;
defined by cell type

Fig. 15

Hematopoiesis
Factors affecting erythropoiesis:A)-Oxygen supply of tissues:
Decreased oxygen supply (hypoxia) to tissues stimulates
secretion of erythropoietin (EP) hormone.
Hypoxia stimulates kidney to release renal erythropoietic
factor (REF).
Hypoxia stimulates liver to produce a special type of globulin.
Both REF & globulin unite in plasma and form EP.
EP then stimulates bone marrow to produce RBCs.
Erythropoietin accelerates nearly all stages of RBCs
formation,
i.e. it stimulates proliferation & differentiation of progenitor
stem cells to produce mature RBCs.

Hematopoiesis
Factors affecting erythropoiesis:B) Dietary factors:
i-Proteins: Proteins of high biological value are needed in the
formation of RBCs.
ii-Metal ions:

Iron Fe: is essential for RBCs formation because it enters

in the formation of the hem part.

Copper Cu: It is carried & transported by plasma protein

ceruloplasmin. It catalyses the oxidation of Fe++ to Fe+++,
a reaction that must occur before transferrin can combine
and transport iron.

Cobalt Co: It stimulates EP release from kidney. So, excess

Co may produce polycythaemia.

Hematopoiesis
Factors affecting erythropoiesis:B) Dietary factors:
iii-Vitamins:

Both vitamins B12 & folic acid are essential for final
maturation of RBCs because they are needed in DNA
synthesis.

Deficiency of either B12 or folic acid results in failure of

nuclear maturation and causing maturation failure
anemia.

Vitamin C is a strong reducing agent which is important

in reducing the ferric form of iron to ferrous to facilitate
its absorption and transport.

Hematopoiesi
s erythropoiesis:Factors affecting
C) Hormonal factors:
i-Androgens: increase erythropoiesis by stimulating
the production of erythropoietin from kidney.
ii-Thyroid hormones:
Stimulate the metabolism of all body cells including
the bone marrow cells, thus, increasing
erythropoiesis.
Hypothyroidism is associated with anemia while
hyperthyroidism is associated with polycythaemia.

Hematopoiesis
Factors affecting erythropoiesis:C) Hormonal factors:
iii-Glucocorticoids:

Stimulate the general metabolism and also stimulate the

bone marrow to produce more RBCs.

In Addisons disease (hypofunction of adrenal cortex)

anemia present, while in Cushings disease (hyperfunction
of adrenal cortex) polycythaemia present.

Hematopoiesis
Factors affecting erythropoiesis:C) Hormonal factors:
iv-Pituitary gland: Affects erythropoiesis both
directly and indirectly through the action of
several hormones.
v- Haematopoietic growth factors: Are secreted
by lymphocytes, monocytes & macrophages
to regulate the proliferation and differentiation
of proginator stem cells to produce blood
cells.

Hematopoiesis
Factors affecting erythropoiesis:D)-State of liver & bone marrow:
i-Liver: Healthy liver is essential for normal
erythropoiesis because the liver is the
main site for storage of vitamin B 12 , folic
acid, iron & copper. In chronic liver
disease anemia occurs.
ii-Bone marrow: When bone marrow is
destroyed by ionizing irradiation or drugs,
aplastic anemia occurs.

Anemia
Anemia means a decrease in
hemoglobin content,
or RBCs count,
or both of them below the normal
range.
Anemia leads to a decrease in
blood ability to transport oxygen to
tissue cells.

Anemias: hemoglobin concentration is low:

Anemia
Normal Hb g/dL: men 13.5-17.5; women 11.5-15.5
Anemias classified by red blood cell morphology:
Rbc morphology
Microcytic,
hypochromic

functional deficit
impaired Hb
synthesis

Macrocytic
normochromic

impaired DNA
synthesis

Normocytic
normochromic

red cell loss

possible cause
thalassemia, lead,
iron deficiency

vit B12 or folic acid

deficient, erythroleukemia
acute bleeding,
sickle cell defects

Anemia
Types & causes of anemia:
I-Blood loss anemia:
A-Acute blood loss anemia:
Due to severe hemorrhage.
Plasma volume is replaced rapidly by the fluids
present in tissue spaces.
This leads to marked dilution of the blood.
RBCs are replaced within 2-3 weeks.
Sufficient iron gives normocytic cells but
insufficient iron will produce microcytic RBCs.

Anemia
Types & causes of anemia:
I-Blood loss anemia:
B-Chronic blood loss anemia:
Due to repeated loss of small amounts of
blood over a long period e.g.:
-Gastrointestinal bleeding (peptic ulcer)
-Excessive menstruation.
-Hemorrhagic diseases.
Due to depletion in iron stores the newly
formed RBCS are microcytic.

Anemia
Types & causes of anemia:
II-Aplastic anemia:
It results from destructione of bone marrow.
It may result from:
1-Excessive exposure to x-rays or gamma rays.
2-Chemical toxins e.g. cancer therapy & prolonged
exposure to insecticides or benzene.
3-Invasion of bone marrow by cancer cells.
4-Following infection by hepatitis.
Damaged bone marrow dont produce any RBCs, so in
aplastic anemia RBCS are normocytic.
It is associated with decrease in WBCs & platelets.

Anemia
Types & causes of anemia:
III-Hemolytic anemia:
It results from increased rate of destruction of RBCs inside
the cardiovascular system.
Causes of hemolytic anemia:
A-Hereditary:
1-Membrane abnormalities.
2-Enzyme deficiency e.g. G-6-P Dehydrogenase.
3-Hemoglobin abnormalities.
B-Acquired:
1-Incompatible blood transfusion.
2-Parasitic infection e.g. malaria.
3-Toxic agents e.g. snake venom & insect poisons.
4-Thermal e.g. several burns.

Bone Marrow (BM) Biopsy

Normal

Aplastic

Anemia
Types & causes of anemia:
IV-Dyshemopoietic anemia: Which may be due to:
1-Iron deficiency anemia.
2-Maturation failure (megaloblastic) anemia:a-Vitamin B12 deficiency.
b-Folic acid deficiency.
3-Anemia of endocrine disorders.
4-Nutritional anemia.
5-Anemia of renal failure.

Classification of Anemia
Based on cell size (MCV)
Macrocytic (large) MCV 100+ fl
(femtoliters)
Normocytic (normal) MCV 80-99 fl
Microcytic (small) MCV<80 fl
Based on hemoglobin content (MCH)
Hypochromic (pale color)
Normochromic (normal color)

MEGALOBLASTIC
(Macrocytic) ANEMIA
High MCV
High MCH
Normal MCHC
Macrocytic Anemia
Megaloblastic : defective DNA synthesis
Non-megaloblastic : numerous
mechanisms

Nutritional Requirements for

Hematopoiesis
Metals : iron copper cobalt
B12 and Folate
Other vitamins: B6, A, E, C
Riboflavin, Niacin

Megaloblastic Anemias
A form of anemia characterized by the
presence of large, immature, abnormal red
blood cell progenitors in the bone marrow
95% of cases are attributable to folic acid or
vitamin B12 deficiency

CH3
CH3

H2NCOCH2CH2

CH2CONH2

H2NCOCH2
N CN

H3 C
H3 C
H

corin nucleus

Co
N

cobalt coordinated
N

H2NCOCH2
H2C

CH2CH2CONH2

CH3

CH3

CH3
CH3
CH2 CH2CONH2

N
O

H3C
O

P
O

OH

CH3

benzylimidazole
N

HO
VITAMIN B 12

CH3

Vitamin B12
Source : food of animal origin
- liver
- muscle
- eggs
- cheese and milk
- Not in plants
- Made by bacteria

B12 Absorption
1. Release from food sources gastric
proteases and acids
2. Binding by salivary cobalophilins
3. Digestion of cobalophilin-B12
complex by pancreatic enzymes
4. Binding to intrinsic factor (IF)
IF is secreted by gastric parietal cells
5. Attachment of B12-IF to receptors
6. Endocytosis and binding to
transcobalamin II

B12 Dependant Reactions

1. Synthesis of methionine from
homocysteine requires : B12 and
folate
2. Synthesis of succinyl CoA from
methyonyl CoA requires :
methylmalonyl CoA mutase

FOLIC ACID
Sources : synthesized by plants
and microorganisms
Vegetables, fruits, dairy products
Polyglutamated
Thermo labile

Folic Acid Absorption-Transport

1. Polyglutamates converted to monoglutamates Intestinal carboxypeptidase
2. Binding of monoglutamates to brush
border receptor
3. Conversion to methyltetrahydrofolate
during absorption
4. Bind to serum protein
5. Receptor mediated cell uptake
6. Polyglutamated in cytoplasm

Pernicious Anemia
An Autoimmune Disease
Antibodies against :parietal cells
intrinsic factor (IF)
Thyroid - myxedema
Melanosomes - vitiligo

Pernicious Anemia
Hematologic features :
anemia
pancytopenia
megaloblastic hematopoiesis
cellular bone marrow
ineffective hematopoiesis

Folate Deficiency
Hematologic features : same as
Pernicious Anemia.
Clinical Picture : no neurologic
findings

Folate Deficiency :Diagnosis

Dietary history
Clinical conditions:
pregnancy
malabsorption (sprue)
hemolytic anemia
drugs
Laboratory:
serum or red cell folate levels

Pernicious Anemia
Presenting Complaint
Symptoms of anemia : 58%
Sensory paresthesis :13%
GI complaints :11%
Sore tongue or mouth : 7%
Weight loss : 5%
Difficulty walking : 3%
Other :3%

Pernicious Anemia - Diagnosis

History and Physical
glossitis
pallor
neurologic exam
Laboratory
blood smear
antibody assays
B12 level
Other
Schilling test

Schilling Test
First stage :
1. Inject B12 IM (1,000 ug) to saturatetranscobalamin II
2. Administer oral B12 - radiolabeled
3. Collect 24 h urine
4. Measure radioactivity in urine
Second stage :
1. Inject B12 IM (1,000 ug) to saturate transcobalamin II
(Same as 1st stage)
2. Administer oral B12 radiolabeled plus intrinsic
factor (HOG)
3. Collect 24 h urine, (Same as 1st stage)
4. Measure radioactivity in urine,(Same as 1st stage)

Static Test for Folate/B12 Status

Folate
Measured in whole blood (plasma and
cells) and then in the serum alone
Difference is used to calculate the red
blood cell folate concentration (may
better reflect the whole folate pool)
Can also test serum in fasting patient
B12
Measured in serum

Functional Tests for

Macrocytic Anemias
Homocysteine: Folate and B12 are
needed to convert homocysteine to
methionine; high homocysteine may
mean deficiencies of folate, B12 or B6
Methylmalonic acid measurements
can be used along with homocysteine
to distinguish between B12 and folate
deficiencies ( in B12 deficiency)
Schilling test: radiolabeled cobalamin
is used to test for B12 malabsorption

Pernicious Anemia
A macrocytic, megaloblastic anemia caused by a deficiency of vitamin B 12.
Usually secondary to lack of intrinsic factor (IF)
May be caused by strict vegan diet
Also can be caused by gastric acid secretion, gastric atrophy, H-pylori, gastrectomy, disorders
of the small intestine (celiac disease, regional enteritis, resections), drugs that inhibit B12
absorption including neomycin, alcohol, colchicine, metformin, pancreatic disease

Symptoms of Pernicious Anemia

Paresthesia (especially numbness
and tingling in hands and feet)
Poor muscular coordination
Impaired memory and hallucinations
Damage can be permanent

Vitamin B12 Depletion

Stage Iearly negative vitamin B12 balance
Stage IIvitamin B12 depletion
Stage IIIdamaged metabolism: vitamin B12
deficient erythropoiesis
Stage IVclinical damage including vitamin
B12 anemia
Pernicious anemianumbness in hands and
feet; poor muscular coordination; poor
memory; hallucinations

Causes of Vitamin B12 Deficiency

Inadequate ingestion
Inadequate absorption
Inadequate utilization
Increased requirement
Increased excretion
Increased destruction by antioxidants

Treatment of B12 Deficiency

Before 1926 was incurable; until 1948 was
treated with liver extract
Now treatment consists of injection of 100
mcg of vitamin B12 once per week until
resolved, then as often as necessary
Also can use very large oral doses or nasal
gel
MNT: high protein diet (1.5 g/kg) with meat,
liver, eggs, milk, milk products, green leafy
vegetables
B12 by IM injection; Frequent at first
Monthly thereafter life long

Folic Acid Deficiency

Tropical sprue; pregnancy; infants born to
deficient mothers
Alcoholics
People taking medications chronically that affect
folic acid absorption
Malabsorption syndromes

Causes of Folate Deficiency

Inadequate ingestion
Inadequate absorption
Inadequate utilization
Increased requirement
Increased excretion
Increased destruction
Vitamin B12 deficiency can cause folate
deficiency due to the methylfolate trap

Methylfolate Trap
In the absence of
B12, folate in the
body exists as 5methyltetrahydrofolate (an inactive
form)
B12 allows the
removal of the 5methyl group to form
THFA

Stages of Folate Depletion and

Deficiency
Stage Iearly negative folate balance
(serum depletion)
Stage IInegative folate balance (cell
depletion)
Stage IIIdamaged folate metabolism
with folate-deficient erythropoiesis
Stage IVclinical folate deficiency
anemia

Diagnosis of Folate Deficiency

Folate stores are depleted after 2-4
months on deficient diet
Megaloblastic anemia, low leukocytes
and platelets
To differentiate from B12, measure
serum folate, RBC folate (more
reflective of body stores) serum B12
High formiminoglutamic acid (FIGLU)
in the urine also diagnostic

Hemolytic Anemia
Oxidative damage to cellslysis occurs
Vitamin E is an antioxidant that seems to be
protective.
This anemia can occur in newborns, especially
preemies.

Nonnutritional Anemias
Sports anemia (hypochromic
microcytic transient anemia)
Anemia of pregnancy: dilutional
Anemia of inflammation, infection, or
malignancy (anemia of chronic
disease)
Sickle cell anemia
Thalassemias

Sports Anemia
Transientusually in athletes who are runners;
from compression of RBCs in feet until they
burst, releasing hemoglobin
Check lab values
Counsel about a proper diet

References
First Known Heart Attack Associated With Beta- thalassemia Major
Reported." Heart Disease Weekly February 22, 2004: 10.
Bowden, Vicky R., Susan B. Dickey, and Cindy Smith Greenberg.
Children and Their Families: The continuum of care . Philadelphia:
W.B. Saunders Company, 1998.
"Thalassemias." In Principles and Practice of Medical Genetics ,
Volume 2, edited by Alan E.H. Emery, MD, PhD, and David L. Rimoin,
MD, PhD. New York: Churchill Livingstone, 1983.
Thompson, M.W., R. R. McInnus, and H. F. Willard. Thompson and
Thompson Genetics in Medicine , Fifth Edition. Philadelphia: W.B.
Saunders Company, 1991.
Olivieri, N. F. "The Beta Thalassemias." The New England Journal of
Medicine 341 (1999): 99-109.