Blood Biochemistry

Tissue Chemistry & Biological Fluids

Biochemistry has passed from a state of descriptive to quantifiable science. As a biochemist, you should always be interested in things about metabolic

sequences:

The description of the enzymes & chemical changes that comprise the metabolic sequence

The rate at which material can be transformed by the sequence The amount of material utilized by the sequence among living

things The nature of the control mechanisms which adjust the amounts

of material utilized by the sequence

2

Wet weight (kg) Protein content (kg)

Skeletal muscle 30 6.6

Adipose tissue 13.2 0.92

Stomach & intestine 7.25 1.34

Liver 1.6 0.35

Brain 1.36 0.136

Kidneys 0.29 0.05

Heart 0.29 0.06

Adrenals 0.014 ?

Blood 6.4 1.02

Skin 4.9 ?

Bone 12.0 1.23

Roughly, the contributions of the different tissues to the body's metabolism are proportional to the weights of the tissue and the biological fluids

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From the following table…

It is not the sheer mass of tissue which determines its quantitative contribution to metabolic activity

Activity of tissue is determined by its enzyme content

4

The body can be crudely divided into two components:

Circulating Tissues Biological Tissues

Blood Cartilage

Water Bone

Lymph Skin

Interstitial fluid Muscles

Cerebrospinal fluid Liver5

Vertebrates have evolved 2 principal mechanisms for supplying their cells with a continuous & adequate flow of oxygen:

A circulating system that actively delivers oxygen to the cells

Acquisition of oxygenThe oxygen carriers in vertebrates are the

proteins hemoglobin & myoglobin

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Blood

In a normal weight, there is about 5-6 liters of blood (12%) or 85ml/kg

It circulates as a homogenous suspension of erythrocytes, leukocytes & platelets in a solution of proteins, inorganic ions, & low-molecular weight organic compounds

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Functions of the Blood

Transport of nutrients Exchange of respiratory gases Transport of waste products Distribution of hormones & other regulatory substances Protection against microorganisms Acid-base, electrolyte & water homeostasis Heat regulation Prevention of excessive

hemorrhage by coagulation8

General Composition

By volume, 40-45% of the blood consists of erythrocytes, leukocytes & platelets

1 mm3 of blood contains: 5 x 106 RBCs; 5-103 WBCs; 5-105 platelets

Male Adults Female Adults

RBC 4.5-5.9 x 1012

cells/L

4.0-5.2 x 1012

cells/L

WBC 3.9-10.6 x 109

cells/L

3.5-10.0 x 109

cells/L

Platelets 150-400 x 109 cells/L

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The Packed Cell Volume (PCV Hematocrit)

PCV Hematocrit = Volume of Red Cells/ Volume of whole blood x 100

Expressed as volume of erythrocyte per liter of whole

blood Normal adult males = 41-53; adult females = 36-

46 Hematocrit used to determine PCV Color of supernatant plasma gives rough idea of

bilirubin content & is often a useful clue about the nature of anemia:

White plasma ----- iron deficiency anemia Lemon yellow plasma ----- Hemolytic or

Megaloblastic anemia 10

Blood Volume & the Hematocrit

Rarely necessary to have an accurate blood volume

Hematocrit (HCT) is the volume percentage of erythrocytes in whole blood

HCT is obtained by centrifugation

The specific gravity of WBC's intermediate between plasma & RBC, thus forming "Buffy Coat"

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Errors in the Estimation of HCT

Usually up to 5% of the apparent RBC mass is plasma

HCT differs according to blood source:

- Some particles when centrifuged tend to accumulate in the center of the tube

- HCT value is affected by movements of fluid (hydrostatic pressure)

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Clinical value of HCT

HCT is important in the diagnosis of anemia

Rough estimation of blood loss after hemorrhage

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Continuation…

Whole Blood

Whole blood – formed elements = plasma

Plasma – Clotting factors = Serum

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Physical Characteristics

Arterial blood is crimson

Venous blood is darker red

Specific gravity = 1.035-1.090 & the viscosity is 5-6 times that of water

Specific gravity of plasma = 1.015-1.035

Ph = 7.3-7.5 15

Erythrocyte Sedimentation Rate

Rate of settling of RBCs after blood is drawnIn healthy men : 1-3mm/hr; 4-7 mm/hr in

young womenLow ESR in patients with anemiaFollows Stoke's Law (settling velocity) with an

equationWhere:Vs is the particles' settling velocity (m/s) (vertically downwards if ρp > ρf, upwards if ρp < ρf), r is the Stokes radius of the particle (m), g is the standard gravity (m/s2), ρp is the density of the particles (kg/m3), ρf is the density of the fluid (kg/m3), and η is the fluid viscosity (Pa s). 16

Continuation…

ESR greatly increased during menstruation & normal pregnancy

Increased rate also found in septicemia & pulmonary tuberculosis (increased globulin & fibrinogen content of plasma; also in elevated cholesterol & phospholipid levels

Inflammation of various types that cause cell necrosis will cause rate of RBC to fall, but the viscosity remains unchanged. 17

Continuation…

In alcoholic cirrhosis there is a rise in plasma bile acids & membrane cholesterol levels may rise by 55%. This has 2 effects:

Cholesterol to phospholipid ratio is increased, reducing membrane flexibility Increased cholesterol content also raises total lipid present, expanding its

surface area Increase by 8% in the total lipid is enough to cause formation of spherocytes

(removed from the circulation as a result of alteration in size, shape and flexibility)

Elevated levels of plasma bile acids (mainly cholic & deoxycholic acids) are observed in obstructive jaundice with similar consequences for the RBC membrane

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Plasma

Straw colored fluid with specific gravity from 1.015-1.035

Specific gravity of plasma is related to its protein content

Contains 90-92% water

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Continuation…

Blood owes much of its physiological importance to high water content:

Maintaining blood pressure Important for heart regulation& in osmotic exchange between

body fluid & compartments

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Plasma Composition

The solutes of the blood plasma constitute ≈ 10% of the volume

Protein ≈ 7%Inorganic salts ≈ 0.9%Other organic compounds

≈ the rest other than proteins.

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Separation of plasma proteins

Based on the different mobility in an electric field

Electrophoresis – widely used

Isoelectric focusing

Immunoelectrophoresis –

separates proteins on the basis of electrophoretic as well as immunologic properties

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Albumin

Alpha 1

Alpha 2 Beta

Gamma

Albumin & Globulins

Comprise most of the proteins in the blood plasma

Colloidal osmotic pressure (from the proteins of the plasma) is the force that opposes the hydrostatic pressure in the capillaries

Better terminology should be "potential osmotic pressure" or "osmotic tendency"

Albumin & Globulins

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Proteins move in electric field by the charge they carry…

Major fractions include:

Albumin (54-58%)α1 globulins (6-7%)

α2 globulins (8-9%)

β1- globulins (13-14%)Gamma globulins (11-12%)

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Enzymes of Plasma

Most plasma enzymes do not have metabolic roles in plasma with the exception of those involved in coagulation

Activity of certain plasma enzymes is useful as index of certain abnormal conditions:

- Serum amylase – elevated in acute pancreatitis

- Acid phosphatase – in cases of prostatic cancer

- Alkaline phosphatase – in hepatic obstruction and bone diseases

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Assay of tissue enzymes in plasma

When organs are damaged part of their enzyme complement in released into the plasma.

In a healthy persons, levels of intracellular enzymes are very low & a result of cellular turnover

Tissues contain 103-104 times higher content of soluble enzymes within their cells

Intracellular enzymes released into the plasma are inactivated & removed within days

Amount of enzyme released depends on the concentration of that enzyme & extent of tissue damage

Knowledge of cellular location of enzyme provides good clinical information

In practice, enzyme assays are most useful in detecting damage to the liver, muscles and blood cells 26

Enzyme Organ Distribution Comments

GOT Widespread but little in red cells Analysis of these enzyme started clinical enzymology

CPK Widespread but skeletal muscle is richest source

Monitoring skeletal & heart muscle disorder

Γ-GT Mainly liver Marker for hepatocellular diseases

LDH Widespread but has distinctive isoenzyme distribution

Monitor heart & liver disease

Acid phosphatase Specific activity in prostate gland Monitor prostatic cancer

Alkaline phosphatase

Widespread in tissues Diagnosis of bone disease

Some enzymes commonly assayed as part of clinical diagnosis

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Continuation…

CPK (Creatinine phosphokinase) & LDH1 (Isoenzyme of LDH) indicate amounts of myocardial infarct. If no further damage occurs, levels return to normal.

Congested liver can be due to inefficient pumping of the right side of the heart.

Most patients with metastatic prostatic carcinoma have elevated plasma phosphatase levels. RIA is used for the detection of this enzyme.

Enzyme assays may also reveal other organ involvement…

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Erythrocytes

Circulating erythrocytes are derived from erythropoietic cells (or erythron), the precursors of erythrocytes. RBCs arise from mesenchymal cells present in bone marrow

Major functions

Transport of oxygen from the lungs to the tissues Controls blood pH (CO2) is converted to

bicarbonate by carbonic anhydrase = major buffering

system) RBCs lack nucleus & other organelles;

utilizes anaerobic metabolism

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Structure & Composition

RBC s have a biconcave disc shape (6-9 µm in diameter; 1 µm thick; 2-2.25 µm at the periphery)

Most of the solid matter is hemoglobin ( the conjugated protein responsible for the red color of the blood)

Behaves like an osmometer

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The Erythrocyte Membrane

Composed largely of protein (49%) & lipid (43%) with a small amount of carbohydrate (8%)

Has a cytoskeleton which controls the shape of the membrane & limits the lateral mobility of some intrinsic proteins

Some of the protein is glycoprotein covalently linked to CHO (Sialic acid)

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Membrane Changes in Diseases

Mature RBCs synthesize very little lipids but:

SphingomyelinPhosphatidylcholine in the outer half of the bilayer those in plasma lipoproteins

Cholesterol exhanges freely with serum cholesterol

The important factor that affects this exchange is the activity of the plasma enzyme Lecithin-Choelsterol Acyl Transferase (LCAT) – responsible for the formation of majority of esterified cholesterol and is inhibited by bile acids 33

Erythropoiesis

During gestation, erythrocytes are formed in various tissues occurring successively in:

Yolk sac – main site for the 1st weeks of gestation

Liver & Spleen - from 6 weeks to 6-7 months & can continue to produce until about 2 weeks after birth

Lymph Nodes

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Continuation…

From 6-7 months of fetal life onwards… the bone marrow is the only source of new blood cells

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Continuation…

Erythroid cells in the bone marrow are called normoblast (a large cell with dark blue cytoplasm, a central nucleus with nucleoli & slightly clumped chromatin

Reticulocytes A reticulocyte stage results when the nucleus is

finally extruded from the late normoblast. In this stage it still contains some ribosomal RNA and can still synthesize Hb

Reticulocytes spends 1-2 days each in the circulation & bone marrow before it matures mainly in the spleen when RNA is completely lost

A single pronormoblast usually gives rise to 16 mature red cells

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Substances needed for erythropoiesis

The bone marrow requires many precursors to synthesize new cells: Metals: Iron, manganese, cobalt

Vitamins: B12, folate, ascorbic acid

Amino acids

Hormones

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Hemolysis

Maybe produced by substances that dissolve or change the state of membrane lipids (ether, chloroform, bile salts & soaps) .

Certain biological toxins (venomous snakes & hemolytic bacteria)

Physical forces (UV rays, freezing, thawing)

Aging – this is why whole citrated blood cannot be used after 5-7 days

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Red Cell Metabolism

The components required for these include:

1. ATP – maintenance of membrane function

2. 2,3 –diphosphoglycerate (2,3 – DPG) to modulate O2 affinity

3. NADPH – to prevent hemoglobin denaturation

4. NADH – to maintain the heme in the Fe(II) state

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Continuation…

The predominant metabolic fuel is glucose where they serve as gluconeogenic precursors

The 2 ATP molecules are utilized in the ion pump in the cell membrane

Failure to produce enough ATP results in an ability to maintain ionic balance leading to accumulation of Ca2+ and shape change

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Continuation…

2,3 DPG is a metabolite unique to the RBC. At a concentration of 4-5mM, it is almost equimolar to Hb

20-25% of 1, 3 DPG pass to 2, 3 DPG by mutase, therefore ATP yield decreases from glucose

2,3 DPG depends on the relative rates of the mutase& phosphatase reactions

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Glutathione

Can be used for the removal of H202. This reaction protects the membrane from

oxidative damage. A deficiency of any enzyme of the glycolytic,

phosphogluconate or GSH-GSSG pathway may seriously compromise the energy dependent maintenance of membrane integrity.

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Hereditary Hemolytic Anemias

Have been associated with deficiencies of the following enzymes:

Enolase enzyme – deficiency leads to decreased ATP required to maintain the biconcave shape of RBC

Glucose-6-P-Dehydrogenase – deficiency may result in increased hemolysis & severe hemolytic anemia

Pyruvate Kinase - deficiency may lead to bizarre model which is extremely fragile and readily hemolyzed

Other enzymes such as hexokinase,glucose –phosphoisomerase, phosphofructokinase, triose phosphate isomerase, 2-3 diphosphoglycerate dismutase

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Erythrocyte Destruction Senescent erythrocytes are

engulfed primarily in the reticuloendothelial cells of the spleen

Free hemoglobin is released and binds to plasma proteins (e.g. haptoglobin)

Complex is transported to liver where Hb portion is split

Heme portion is transported to plasma & converted to bilirubin; excreted in the bile

Iron is released & stored in the

liver for reuse45

Hemoglobin

1 liter of blood usually contains 150g of hemoglobin; each gram can combine with 1.34ml of oxygen

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Continuation…

1 liter of blood can carry 200ml of oxygen, 87 times higher than plasma alone.

Each RBC contains ≈ 640 million Hb molecules

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Hemoglobin Structure

The 4 chains are held together by non-covalent bonds

There are 4 binding sites for oxygen

The Hb molecule is nearly spherical; packed together in a tetrahedrical way

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Continuation…

The amino acid sequence of hemoglobin is known for 20 species. However there are 9 positions in the sequence that contain the same amino acid in nearly or all species studied. These conserved positions are especially important for the function of hemoglobin:

1. Some of them are involved in oxygen binding sites

2. Stabilizing the molecule via forming H-bond between the helix

3. Some (e.g. GLY) for easy contact between the chains

4. Some (e.g. PRO) to terminate the elix

5. The non-polar residues (Alanine, Isoleucine) are important because reversible oxygenation of heme group depends on its location where it is protected from water.

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Continuation…

Normal hemoglobin is of several types containing 4 sub-units made up of various combinations of 4-5 different related peptide chains

Hemoglobin Structure Stage of Life % in Adult % in Newborn

Gower I ζ2ε2 0-5 weeks embryo None Up to 40

Gower II α2ε2 4-13 weeks embryo

None Up to 35

Portland ζ2γ2 4-13 weeks embryo

None Up to 35

Fetal (F) α2γ2 Newborn & adult < 1.0 80

A1 α2β2 Newborn & adult 97 20

A2 α2δ2 Newborn & adult 2.5 < 0.5 50

Biosynthesis of Hemoglobin

It has been estimated that there are 30 trillion erythrocytes in the circulating blood & ≈ 3 million/sec are destroyed

Globin moiety is formed from amino acids from the body pool in amounts of about 8g/day in the normal adult

14% of the amino acids from an average daily protein intake are used for globin formation

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Availability of Fe++

Total body content of iron is about 2-6g & is not excreted in this form

Found in porphyrin ring of the heme complexThe first type of compounds (Hb, myoglobin,

cytochromes,catalase) are associated with the physiology

The second type is concerned with absorption, transport & storage of iron

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Iron Absorption No iron absorption takes place in

the stomach Stomach acid is essential for iron

reduction Duodenum contains "apoferritin"

(converts Fe ++ to Fe+++) Ferritin may then act as an iron

store or transported to the serosal side where it is released in the ferrous form

In the blood, iron is bound by specific α-1 Globulin (transferrin)

54Iron is stored in the liver & bone marrow in 2 forms: Ferritin & Hemosiderin (agglomeration of ferritin molecules)

Transport of Oxygen

If arterial blood is analyzed for its oxygen content, it is found to contain 18-20% volume

Hb is an allosteric protein: the binding of additional O2 Hb enters the binding of additional O2 to the same Hb molecule

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Continuation…

The sigmoidal property of the curve is believed to be due to heme-heme interactions

Heme-heme interaction means binding at one heme facilitates the binding of oxygen at the other hemes on the same tetramer & vice versa

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Cooperative Property

When environmental oxygen levels are high, partially saturated hemoglobin molecules exhibit enhanced affinity for binding additional oxygen molecules, a specialized behavior referred to as cooperativity.

Hb has the capacity to bind between 1 and 4 O2 molecules, ranging from fully "desaturated" Hb (deoxyHb) to fully "saturated" Hb (oxyHb).

As part of this process, Hb also serves to replenish the "oxygen stores" maintained by myoglobin (Mb), the O2-binding protein in muscle which releases its oxygen in response to high levels of muscle activity.

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2, 3 Bispospoglycerate (2,3 –DPG)

Is very important for long-term regulation of Hb affinity to O2

2,3 BPG shunt is a pathway derived from glycolysis. Competition with oxygen for binding site on ß-subunits Hypoxia stimulates 2,3 BPG synthesis, i.e. improve O2

release 2,3 DPG binds electrostatically to β- subunits through Lys

82, His 143 & N-term. The juxtaposition of these groups is favorable for 2,3-DPG binding only in the T-state.

DPG stabilizes the deoxyHb by cross-linking the β chains. In other words, DPG shifts the equilibrium to tense form.

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Clinical Significance of DPG

It has been approved that DPG levels decrease from 4.5mM to 5.0mM Ill patients may take longer time to regain DPG when blood is

transfused Inosine can be converted to DPG inside RBC. Inosine can now be used

to preserve integrity of stored blood In hypoxia (e.g. emphysema), airflow in the bronchioles is blocked so

the pressure increases as DPG increases. DPG levels lead to 27% increase in the amount of oxygen due to pressure changes. (Also in high altitude adaptations)

Fetal Hb has high affinity to transfer oxygen from maternal to fetal circulation. HbF binds DPG less strongly than does HbA & consequently has higher oxygen affinity.

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Life span of RBCs RBCs have a limited life span of 120

days The "Red Cell Theory of

Aging" is based on observed Ca2+ that occurs in old erythrocytes

Ca2+ rises to 0.5mM, enough to activate transglutaminase present in cell membrane

Transglutaminase form cross-links by creating iso-peptide bonds

Red cells with cross-linked membrane proteins are less flexible & are removed in the circulation by the spleen

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RBC Destruction Hemoglobin from senescent

erythrocytes (phagocytosed in the reticuloendothelial cells of the spleen) are transported to the liver bound to the plasma protein haptoglobin

The globin portion is reused as amino acids & the heme moiety is converted to several steps to the bile pigments

Bilirubin, Urobilin & Stercobilin are colored (BILE PIGMENTS)

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Continuation…

The pigments (biliverdin & bilirubin) are extracted in bile

The iron of heme is removed & the process, bound to plasma transferrin & either recycled as new hemoglobin or stored in the liver as ferritin

In the liver, bilirubin, either from Hb or from other hemoporteins is transported bound or loosely associated with plasma albumin

In the small intestine… Conjugates with glucoronic acid to form

bilirubin diglucoronide which is water soluble & is readily excreted by means of the bile into the intestine

Hydrolysis in the intestine by a β-glucoronidase into bilirubin & glucoronic acid

Reduction of bilirubin by bacterial floral action to colorless D or L- Urobilinogen

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Urobilinogen

First part is reabsorbed & excreted in the urine as oxidized orange-yellow pigment L-Urobilin

Second part is reduced in the intestine to L-Stercobilinogen & excreted as an oxidized pigment L-Stercobilin in the feces (Faecal urobilinogen)

Urinary urobilin is increased if… Hemolysis is excessive when large amounts of bilirubin enter the

bowel & are converted to stercobilinogen There is liver damage (impairs re-excretion of normal amounts of

urobilinogen into the bile)

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Erythrocyte Abnormalities

A large number of human diseases are associated with abnormal function of the erythrocytes including

Altered rates of erythrocyte production & destruction

Defects in iron or heme metabolismCombination of these conditions

These diseases are called ANEMIAs…64

Nutritional Deficiency Anemia

Hematopoietic precursor cells are particularly sensitive to any insult that impairs DNA synthesis. This leads to appearance of characteristic megaloblasts - corresponds to normoblasts (characterized by increased ratio of RNA to DNA). The cause can be deficiencies in metal traces, folic acid & vitamin B12

Larger amount of hemoglobin than other proteins, & constant daily losses of Hb must be replaced by resynthesis

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Continuation…

Iron-deficiency – lack of dietary iron or excess blood loss (e.g menstruation)

Folic acid deficiency – its deficiency leads to megaloblastic anemia as it is a co-factor for a variety of reactions to 1-carbon metabolism (synthesis of purines & thymines)

Vitamin B12 deficiency – deficiency leads to pernicious anemia. Based on malabsorption of Vitamin B12 due to failure of the gastric mucosa to secrete adequate intrinsic factors

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Hemolytic anemias

Anemias associated with increased destruction of erythrocytes, characterized by shortened life span of cells

Isoimmune hemolytic disease – in newborns; caused by transplacental transfer of maternal blood-group Abs capable of reacting with fetal erythrocytes

Hereditary spherocytosis - associated with the presence of spherical erythrocytes that are more fragile to hypotonic solutions; nature of defect unknown

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Continuation…

Paroxysmal nocturnal hemoglobinuria – erythrocytes are abnormally sensitive to lysis by complement

Sickle Cell anemia – abnormal Hb, HbS aggregates on deoxygenation & the aggregates deform the shape of the cell, rendering susceptible to lysis

Thalassemias – caused by defective synthesis of α & β globin chains

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Sickle Cell Anemia

1. Characterized by the sickle-cell or crescent shape of the erythrocytes when the oxy HBs is converted to deoxy HbS at low PO2.

2. At intracellular concentrations, molecules of deoxy HbS aggregate to form filaments on tubules of indeterminately high molecular weight

3. The sickle-cell causes severe anemia since they have increased mechanical fragility

4. Sickle cells also impede blood flow through capillaries

5. It is genetically transmitted

6. Vigorous physical activity at high altitude, air travel in unpressurized plane, & anesthesia can be potentially hazardous to a person with this disease

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Characterization of HbS

HbS has between 2 & 4 more net + charges per molecule than net HbA

Non-polar residue on the outside of HbS (due to Val) causing low solubility

Sticky patch on the outside of its β chains & are present on both deoxy HbS & oxy HbS but not on HbA

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A S

pI of Oxy Hb 6.87 7.09 = 0.22

pI of deoxy Hb 6.88 6.91 = 0.23

Thalassemias

Normally the rates of synthesis of the α & β chains of Hb must be virtually identical.

α-Thalassemia

In α-Thalassemia, there is deficiency in α chains & hence β chains precipitate; β- thalassemia is the reverse

Results from deletion of the α-globulin gene (Homozygotes with α-Thalassemia exhibit a syndrome known as hydrops fetalis)

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Continuation…

β- thalassemia β- thalassemia are heterogenous β- globin gene is deleted β- globin gene remains intact & β- globin

mRNA is synthesized but not translated In many β- thalassemia, the β- globin gene is

present but very little β- globin mRNA is produced

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Blood Groups Isoagglutinins (blood group substances) are found on the erythrocyte

surfaces & are responsible for the major immunological reactions of erythrocytes (Blood Types)

Surface of RBCs carry antigens (agglutinogens), the plasma carry agglutinins

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Continuation…

ABO system is more complicated than the outline given. The ABO groups actually have 6 groups:

A1 A2 B AB A2B O

The following rules must be observed in blood transfusion:

• If the recipient's ABO group is known, give blood of the same group if possible

• Give Blood group O if the ABO group is unknown

• If the recipient's blood group is AB, neither antibodies are found are found in plasma so any red cells can be given

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Rhesus Groups The term Rhesus (Rh) blood group system refers to the 5 main Rhesus

antigens (C, c, D, E and e) as well as the many other less frequent Rhesus antigens. The terms Rhesus factor and Rh factor are equivalent and refer to the Rh D antigen only. Rh + gene is the dominant gene; Rh – is the recessive gene

Genotype Symbol Rh(D) status

cde/cde rr -

CDe/cde R1r +

CDe/CDe R1R1 +

cDE/cde R2r +

CDe/cDE R1R2 +

cDE/cDE R2R2 +75

Blood Coagulation Injury to a blood vessel initiates a

series of reaction involving 3 separate processes:

1. The damage end contracts

2. Platelets begin to adhere to the injured endothelium & form a plug

3. Blood clot formation

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Clotting

When the vessel is punctured or cut, the endothelium brings blood into contact with sub-endothelial collagen

Platelets at the site of injury are influenced to stick

As the platelets aggregate they release vasoactive amines (serotonin & epinephrine) & prostaglandin metabolites (thromboxane A2) which stimulate vasoconstriction

The plug is called thrombus & it is a major chemical defense against blood loss

The actual blood clotting processes that lead to a proper clot are set into motion by 2 mechanisms: intrinsic & extrinsic pathways: 77

Must be initiated rapidly when the vascular system is damaged but must occur when the circulatory system is intact

The Coagulation Cascade

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The Intrinsic (Intravascular system)

So termed because all factors involved are present in the vascular system The 3 factors involved lead to the activation of factor X & in turn to the

conversion of prothrombin to thrombin

A. The Hageman factor binds to collagen or to vasoactive peptide such as Kallikrein, resulting to a proteolytically active form XIIa

B. XIIa activates XI by hydrolyzing an internal peptide bond

C. In the presence of Ca2+, IX is activated to IXa. This activation is vitamin K dependent.

D. In the final step factor X is converted to Xa by IXa in the presence of VIII (hemophilia A factor), platelet phospholipids and Ca2+ ions

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The Extrinsic (Extravascular system)

The factors involved are supplements of the intrinsic to ensure more rapid coagulation

Factor VII is converted to active form VIIa by factor III in the presence of Ca 2+

80

Conversion of factor II (Prothrombin) to factor IIa (Thrombin)

The rest of the reactions are common in both patrhways

Factor Va, platelets, phospholipids & Ca 2+ to promote the reaction

81

Conversion of Factor I (Fibrinogen) to factor Ia (Fibrin) By thrombin, ARG-GLY in α-A & β-B chains of fibrinogen is

released in a form of fibrinopeptide A & B from NH2 terminal ends of the chains.

The gamma chain is not affected Factor VIIIa (the fibrin-stabilizing factors FSF is present in

human platelets & in plasma Factor VIIIa (fibrinoligase) is a trans glutaminase that catalyzes

the formation of cross-linked peptide bonds

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Continuation…

Once the clotting cascade is initiated, mechanisms must operate to prevent clotting from spreading throughout the intravascular system.

Plasmin & fibrinolysin prevent such spread & dissolve any clots that do form.

Plasmin is derived from inactive plasminogen in a reaction that requires blood & tissue factors including factor XIIa & Kallikrein

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ANTICOAGULANTS

In vitro… A number of substances prevent coagulation [oxalate,

fluoride, citrate, EDTA – they precipitate Ca 2+ and bind to it]

Bile salts are inhibitors of thromboplastin Dicumarol – is an antagonist of vitamin K by impairing

its biosynthesis Heparin – is a complex polysaccharide β- diglucoronic

acid & α-D-glucosamine

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