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

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/mL

White cells (leukocytes)

7x103/mL

Platelets (thrombocytes)
3x105/mL

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 Reticulocyte

Platelets Lymphocyte

Neutrophil

Normal Blood

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

Blood Components:Plasma Transports Solutes

Plasma composition
Water - 90% of plasma volume Proteins - 7% of plasma volume Inorganic - 1% of plasma volume
Na+, K+, Mg2+, Ca2+, PO43-

Organic - 2% of plasma volume

urea, fats, cholesterol, glucose ...

Male versus female

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

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 g-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/mL Various types
Neutrophils 62% Eosinophils 2.3% Basophils 0.4% Monocytes 5.3% Lymphocytes 30% Plasma cells (mainly in the lymph)

Monocytes in tissue become macrophages

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

Platelets
Non-nucleated disk shaped cells 3-4 mm diameter Volume 10 x 10-9 mm3 250 000 cells/mL 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 a 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

Zone Electrophoresis of Plasma Proteins

globulins
g b a1 a2

+ albumin

pI

6.0

5.6

5.1

4.7

Functions of Plasma Proteins

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

COP relates to blood volume

DP = p
Protein soln Water

If membrane present p 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 pprotein = 20-30 mmHg

Total ~ 5000 mmHg

Protein stays in the blood as p is maintained in the blood Water content is therefore maintained

Hypotonic - lower p than normal

Hemolysis of RBC
Hb H2O

Ghost Cells

Hypertonic higher p than normal

Creation of cells
Hypertonic Crenated Cells H2O

1.5% NaCl

Functions of Plasma Proteins (contd)

Transport of ions, fatty acids, steroids, hormones etc.
Albumin (fatty acids), ceruloplasmin (Cu2+), 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% a 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

g-Globulins
20% of plasma proteins g 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, 2a (67,000), 2b (56,000), 2g (47,000)

a b g disulfide Triple dumbell model (EM) 450 90 D E D

as, bs and gs are intertwined

Function
Blood coagulation (clotting)
Fibrinogen Thrombin Fibrin

Fibrin

Plasmin

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 b 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

Red cells Cellular Elements of Blood

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

Shape
Biconcave disc 8 mm in diameter, 2.7 mm thick, volume ~ 90 mm3, area ~ 160 mm2

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 a chain, one on each b chain

O2 depleted Hb solution placed in contact with O2(g) Equilibrium reaction Fraction (s) of Hb converted to oxyhemoglobin

Described by empirical equation

K ( pO2 )

n n

1 K ( pO2 )

K depends on ionic strength and pH of Hb solution n generally given as 2.5 -2.6

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

Mb O2 MbO2
At equilibrium
k 1 k1

k1CMbCO2 k 1CMbO2 s CMbO2 CMbO2 CMb k1 CMbCO2 k 1 k1 CMbCO2 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.

Hematopoiesis
Factors affecting erythropoiesis: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 B12 , 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 Macrocytic normochromic functional deficit impaired Hb synthesis possible cause thalassemia, lead, iron deficiency

impaired DNA synthesis

vit B12 or folic acid deficient, erythroleukemia

Normocytic normochromic

red cell loss

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.