Blood BiochemistryBlood BiochemistryBlood BiochemistryBlood Biochemistry

Composition of Blood Blood is the body’s only fluid tissue It is composed of liquid plasma and formed el

ements Formed elements include:

Erythrocytes, or red blood cells (RBCs) Leukocytes, or white blood cells (WBCs) Platelets

Hematocrit – the percentage of RBCs out of the total blood volume

Components of Whole Blood

Withdraw blood and place in tube

1 2 Centrifuge

Buffy coat:leukocyctes and platelets(<1% of whole blood)

Plasma(55% of whole blood)

Erythrocytes(45% of whole blood)

Formed elements

Plasma

The blood fraction obtained after removal of the cell

ular components

About 77%-81% in the total blood values

Hydrometer is 1.050-1.060, pH is 7.35-7.45, osmotic

pressure is 770kPa (37°C) in the normal human

relevant to coagulation factors, immunoglobulins an

d complements

Serum

The blood fraction after separation of the protein fibrinogen from plasma

Generally obtained by allowing the blood to clot In this process, fibrinogen is converted to an

insoluble protein, fibrin, which is easily removed Serum does contain some degradation products of

clotting factors

Plasma composition

plasma

Clotting factors

Serum

Liquid: water

solids

Gases:O2, CO2

protein

Nonprotein nitrogen (NPN)

Low-molecular-weight organic substances such as Serum solids glucose, lipids, vitamins, hormones and so on

electrolytesNa+, K+, Ca2+, Mg2+

CI-, HCO32-, HPO4

2-

Non-Protein Notrogen (NPN) Non-protein nitrogenous compounds

urea, uric acid, creatinine, creatine, nucleotides, amino acids, bilirubin, polypeptides, glutathione and many others

The Concentration of NPN

14.28~24.99 mmol/L, 50% of NPN is blood urea nitrogen (BUN)

Source of NPN

derived from the metabolism of nucleic acid and proteins

Excretion of NPN

transported to the kidneys fro excretion from the urin

Significance

act as an index of renal function

Male versus female Hematocrit (% volume that is red

cells)40-50% in males35-45% in females

Function of Blood Blood as a transport system

transport nutrients and oxygen to the cells and carri

es away cellular waster products

Blood as a regulative system

maintaining normal acid-base balance in the body; R

egulating the water balance and body temperature

Blood as a defense system

white blood cells and the circulating antibodies

Coagulation and fibrinolysis

Section 1

Plasma Proteins

Plasma Proteins More than 200 Most abundant

Albumin - 4-5 g/100 mL g-glubulins - ~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

Zone Electrophoresis of Plasma Proteins

- +

pI6.0 5.6 5.1 4.7

globulins albumin

Protein Separation Size Exclusion Chromatography (SEC)

Porous matrix (sephadex)

Affinity chromatography

molecule attached to a column that

specifically binds the protein of interestCoenzyme / enzymeAntigen / Antibody

SDS-PAGE (polyacrylamide gel electrophoresis) Separates by size Proteins are complexed with SDS to give the sa

me charge density

Two Dimensional ElectrophoresisDecreasing Mr

Decreasing pI

Characteristics of Plasma Proteins Most plasma proteins are synthesized in the liver, howev

er, certain proteins are synthesized in other sides Generally synthesized on membrane –bound polyriboso

mes With the exception of albumin, almost all plasma protein

s are glycoproteins Many plasma proteins exhibit polymorphism Each plasma protein has a characteristic half-life in the c

irculation The levels of certain proteins in plasma increase during a

cute inflammatory states or secondary to certain types of tissue damage

(1) Functional enzymes of the plasma

Have catalysis in the plasma, such as

thrombin, lipoprotein lipase, LCAT etc

Functions of Plasma Proteins

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

COP relates to blood volume

Proteinsol’n

Water

P =

(2) non-functional enzymes of the plasma

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 prot

eins) Defense against infection (antibodies, comple

ment proteins)

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:

AlbuminAlbumin

30 Å

80 Å

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 Maintaining colloid osmotic pressure of blood (8

0% due to albumin)Colloid osmotic pressure is generated by plasma prot

einsThe most abundant of the plasma proteinsThe lowest molecular weight of the major protein mol

ecules in the plasmaHigh negative chargeRegulates water distribution

Transportation

Albumin can act as a carrier molecule for bilirubin, fatty acids, trace elements and many drugs

Section 3

Metabolism of the Blood Cells

Red cells 40 - 50% of blood volume 5 x 106 cells /mL Composed of a membrane surrounding a solution of

hemoglobinnon-nucleated, no intracellular organellesno proliferationcell membrane in excess so that deformation doe

s not rupture Shape

Biconcave disc8 mm in diameter, 2.7 mm thick, volume ~ 90 m

m3, area ~ 160 mm2

Cellular Elements of BloodCellular Elements of Blood

Scanning Electron Micrograph of Red Blood Cells

Why this shape?

Area to volume ratio is high Facilitates diffusion of O2 and CO2

minimal distance of contents from surfaceOriginates in bone marrow (hematopoiesi

s)

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

Production of Erythrocytes

Hematopoiesis – blood cell formation Hematopoiesis occurs in the red bone marro

w of the: Axial skeleton and girdles Epiphyses of the humerus and femur

Hemocytoblasts give rise to all formed elements

Production of Erythrocytes: Erythropoiesis

A hemocytoblast is transformed into a committed cell called the proerythroblast

Proerythroblasts develop into early erythroblasts The developmental pathway consists of three phase

s Phase 1 – ribosome synthesis in early erythroblasts Phase 2 – hemoglobin accumulation in late erythroblasts a

nd normoblasts Phase 3 – ejection of the nucleus from normoblasts and fo

rmation of reticulocytes Reticulocytes then become mature erythrocytes

Production of Erythrocytes: Erythropoiesis

Figure 17.5

The major function of the red cells

Delivering oxygen to the tissues, helping in

the disposal of carbon dioxide and protons

formed by tissue metabolism

Normal red cell breakdown

haemoglobin

haem

protoporphyriniron

Bilirubin(free)

COExpired airtransferrin

erythroblastBilirubin glucuronides

Stercobilin(ogen)Urobilin(ogen)

Urine

Liverconjugation

faeces

globin

Amino acids

Hemoglobin synthesis Heme synthesis starts with the condensation of glycine and suc

cinyl coenzyme A under the action of a rate limiting enzyme δ-aminolevulinic acid (ALA) synthase.

δ -ALA will be formed. Pyridoxal phosphate (vit. B6) is a coenzyme for this reaction.

+HSCoA + CO2

ALA synthase

（ Pyridoxal phosphate ）

This step takes place in the mitochondria

COOH

H2C

CH2

C¡«SCoA

O

CH2NH2

COOH

COOH

H2C

CH2

C

CH2NH2

O

ALA dehydratase

2H2ONH

OH

O

OH

O

NH2

COOH

CH2

CH2

C

C

N

O

H H

H H

This step occurs in the cytoplasm

A series of biochemical reactions will follow.

Two molecules of δ-ALA condense to form a pyrrole c

alled porphobilinogen (PBG)

Four PBG

Linear tetrapyrrole

uroporphyrinogen IIIcoproporphyrinogen Ⅲ

Deaminase

UPG III isomeiase

UPG III decarboxylase

Four PBG condense to form a tetrapyrrole uroporphyrinogen III.

UPG III is then converted to coproporphyrinogen.

This step occurs in the cytoplasm

Haemoglobin synthesis CPG then changes to pro

toporphyrin which ultimately combines with iron in the ferrous state (Fe2+) to form haem.

Iron is brought to the developing red cells by a carrier protein ( transferrin) which attaches to special binding sites on the surface of these cells.

Transferrin releases iron and returns back to circulation.

Haemoglobin synthesis

Each molecule of haem combines with a globin chain.

A tetramer of four globin chains each with its own haem group in a pocket is formed to make up a haemoglobin molecule.

Haemoglobin structure

Haem consists of a protoporphyrin ring with an iron atom at its centre.

The protoporphyrin ring consists of four pyrrole groups which are united by methane bridges (=C-).

The hydrogen atoms in the pyrrole groups are replaced by four methylene (CH3-), two vinyl (-C=CH2) and two propionic acid (-CH2-CH2-COOH) groups.

Metabolic Characteristics of Mature Erythrocytes

Can not carry out synthesis of nucleic acid and proteins

Can not obtain energy by oxidative phosphorylation of the mitochondria

ATP is synthesized from glycolysis and is important in process that help the red blood cell maintain its biconcave shape and also in the regulation of the transport of ions and of water in and out of the cell

The principal modes of glucose metabolism are anaerobic glycolysis and the pentose-phosphate pathway

Glycolysis

Obtain energy by glycolysis of glucose

Utilize 2ATP moleculars, produces 4ATP moleculars with a net gain of 2ATP

- The function of ATP

To maintain the correct ion balance, brought about by the pumping out of sodium in exchange for potassium

To maintain the correct conformation of the cell

To protect against the formation of methaemoglobin

To synthesize NAD+ and glutathione

The pathway of 2,3-bisphosphoglycerate (2,3-BPG)

Formation of 2,3-BPG

Glucose

1, 3-BPG

Glycerate 3-phosphate

2, 3-BPG

Diphosphoglyceromutase

Phosphoglycerate kinase

Lactate

Diphosphoglycerate phosphatase

The role of 2,3-BPG

Plays an important role in the binding of oxygen to hemoglobin in erythrocytes

Combine with hemoglobin, causing a decrease affinity of hemoglobin for oxygen

Hemoglobin(Hb)

pO2 2,3-DPG (lungs)

pO2 2,3-DPG (tissues)

Oxyhemoglobin

(HbO2)

The role of the pentose phosphate pathway

Produce the NADH which is essential for the regeneration of reduced glutathione from oxidized glutathione

NADP+

NADP++H+

2GSH

GSSG

Glutathione reductase

Pentose phosphate pathway

The role of glutathione are as follows

The role in the destruction of hydrogen peroxide (H2O2) in erythrocytes

NADP+

NADP++H+

2GSH

GSSG

H2O2

2H2O

Glutathione reductase

Glutathione peroxidase

Reduction of methemoglobin Methemoglobin does not combine with molecular ox

ygen and does not have the function of transporting oxygen

Normally, methemoglobin is reduced to the ferrous state by the NADH-dependent methrmoglobin reductase

MHb (Fe3+)Methrmoglobin reductase

Hb (Fe2+)

NADH+H+ NAD H2O½ O2

Genetic abnormality-deficiency of glucose-6-phosphate dehydrogenase

glucose-6-phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway

A deficiency of this enzyme will lead to failure of restoring GSSG to GSH in the erythrocytes, a step essential for the removel of H2O2

Cell damage is likely to result from oxidation of the membranes by the H2O2, leading to hemolytic anemia

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

White Blood Cells (Leukocytes)White Blood Cells (Leukocytes)

granulocytes

Function Defense against foreign invaders

bacteriavirusesforeign materials (including biomaterial

s) Phagocytosis

Neutrophils, macrophages Move to foreign particle by chemtaxis

Chemicals induce migrationToxins, products of inflamed tissues, co

mplement 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 immu

nity B cells responsible for production of antibodie

s 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)SwellRelease toxins into cytoplasm

Helper T cellsMost numerousActivate B cells, killer T cellsStimulate activity by secretion of IL2Stimulate macrophages

Suppressor T cellsRegulate activities of other cell types

Erythropoietin Mechanism

Figure 17.6

Imbalance

Reduces O2 levels in blood

Erythropoietin stimulates red bone marrow

Enhanced erythropoiesis increases RBC count

Normal blood oxygen levels Stimulus: Hypoxia due to decreased RBC count, decreased availability of O2 to blood, or increased tissue demands for O2

Imbalance

Start

Kidney (and liver to a smaller extent) releases erythropoietin

Increases O2-carrying ability of blood

Haemoglobin catabolism*normal red cell destruction*

Red cell destruction usually occurs after a mean life span of 120 days.

The cells are removed extravascularly by macrophages of the reticuloendothelial system (RES), specially in the bone marrow but also in the liver and spleen.

Red cell metabolism gradually deteriorates as enzymes are degraded and not replaced, until the cells become non viable, but the exact reason why the red cells die is obscure.

Haemoglobin catabolism*normal red cell destruction*

The breakdown of red cells liberates

1- iron for recirculation via plasma transferrin to marrow erythroblasts

2- protoporphyrin which is broken down to bilirubin.

3- globins which are converted to amino acids.

Normal red cell destruction

- The bilirubin circulates to the liver where it is conjugated to glucuronides which are excreted into the gut via bile and converted to stercobilinogen and stercobilin(excreted in faeces).

- Stercobilinogen and stercobilin are partly reabsorbed and excreted in urine as urobilinogen and urobilin.

Normal red cell destruction

A small fraction of protoporphyrin is converted to carbon monoxide (CO) and excreted via the lungs.

Globin chains are broken down to amino acids which are reutilized for general protein synthesis in the body.

Normal red cell breakdown

haemoglobin

haem

protoporphyriniron

Bilirubin(free)

COExpired airtransferrin

erythroblastBilirubin glucuronides

Stercobilin(ogen)Urobilin(ogen)

Urine

Liverconjugation

faeces

globin

Amino acids

Haemoglobin abnormalities

There are mainly two types of abnormalities, these are :

Quantitative abnormalities: where there is reduction in the production of certain types of globins e.g. thalassaemia

thalassaemia Qualitative abnormalities: where there is pro

duction of abnormal haemoglobin e.g. sickle cell anaemia.

Composition and Function of Blood

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 (cellular fraction)

> Plasma - 55% by volume (non-cellular fraction)

Cells Red cells (erythrocytes) 5x106/mL

White cells (leukocytes) 7x103/mL Platelets (thrombocytes) 3x105/mL

Blood must carry 600 L of O2 from lungs to tissues each day Very little carried in plasma since O2 only sp

aringly soluble Nearly all bound and transported by Hb of R

BC Possible for Hb to carry four O2 molecules, o

ne on each chain, one on each chain

Oxygen Binding of HbOxygen Binding of Hb

O2 depleted Hb solution placed in contact with O2(g)

Equilibrium reaction Fraction (s) of Hb converted to oxyhemoglobi

n